Effects of electrical stimulation and postmortem aging on selected properties of myofibrillar proteins as related to ten...


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Effects of electrical stimulation and postmortem aging on selected properties of myofibrillar proteins as related to tenderness
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xiii, 226 leaves : ill. ; 29 cm.
Hooshyar, Parvin
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Thesis (Ph. D.)--University of Florida, 1989.
Includes bibliographical references (leaves 203-225).
Statement of Responsibility:
by Parvin Hooshyar.
General Note:
General Note:

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University of Florida
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Full Text







To my father,
in memory of my mother.


My thanks go to Dr. R.L. West, committee chairman, and

the people of the department of animal science, especially

meats section, for giving me refuge and accepting me as one

of their own. Their priceless support included the

unlimited use of their facilities, procuring specific pieces

of equipment and financing this research.

Gratitude and appreciation also are offered to my

graduate committee: Dr. R.L. West for his patience and

guidance; Dr. R.C. Littell for his generous assistance and

concern throughout my graduate studies; and Drs. D.M. Janky,

J.W. Lamkey and M.R. Marshall for their support.

To Drs. H.C. Aldrich and G.W. Erdos, special thanks are

expressed. This dissertation would have remained incomplete

without their invaluable assistance and instructions in

electron microscopy and without having free access to the

facilities of the IFAS electron microscopy laboratory.

The assistance received from L.E. Eubanks with the

slaughter of the bulls, A.T. Zometa with the sarcomere

length determinations and W.M. Jones with the art work is

gratefully acknowledged.


Special gratitude is offered to Bridget Walker and

Janet Eastridge for their assistance and friendship.

Deepest appreciation is extended to my "adopted" parents

Dot and James Cowart for their help, kindness and moral

support during the course of this study.

Finally, to my father, Mr. Mohammad Sherif Hooshyar, my

affection and appreciation are expressed for his immense

love, patience, financial and moral support.


ACKNOWLEDGMENTS...................................... iii

LIST OF TABLES.......... .............................. vii

LIST OF FIGURES........................................ x

ABSTRACT...................................... ........ xii

INTRODUCTION.... .................................... .. 1

LITERATURE REVIEW.................. .... ............ 6

Myofibrillar Proteins........................... 6
Proteins of Thick Filaments................... 7
Proteins of Thin Filaments...................... 12
Muscle Contraction............................. 16
Actomyosin..................................... 17
The Z Line and Alpha Actinin.................... 17
Cytoskeletal Proteins.......................... 18
Factors Affecting Meat Tenderness................ 26
Nonproteolytic Alterations..................... 27
Enzymology of Meat Aging...................... 40
Postmortem Protein Degradation................ 48
Electrical Stimulation........................... 58
Effects of Electrical Stimulation
on Postrigor Muscle.......................... 60
Applications of Electrical Stimulation......... 62
Effects of Electrical Stimulation on
Biochemistry of Muscle...................... 64
Effects Organelles of Muscle Syncytia.......... 65
Mechanisms of Tenderization................... 66
Effects of Electrical Stimulation on
Myofibrillar Proteins........................ 73

MATERIALS AND METHODS ............................... 77

Experimental Design .............................. 77
Sample Preparation............................... 77
Temperature Determination........................ 78
Sample Collection................................ 78
Determination of pH.............. ............... 78
Sarcomere Length Determination.................. 79
Water Holding Capacity........................... 79

Warner-Bratzler Shear Force Determination........ 80
Actomyosin Extraction............................ 81
Protein Determination............................ 82
Protein-Protein Interaction Determination........ 83
Dissociation of Actomyosin by ATP................. 85
Sulfhydryl Groups Determination................... 85
Determination of ATPase Activity.................. 86
Titin and Nebulin Detection....................... 87
Denaturing Gel Electrophoresis.................. 88
Reagents and Buffers........................... 88
Acrylamide Gel Preparation..................... 90
Sample Preparation for SDS-PAGE................ 91
Protocol for SDS-PAGE ......................... 91
Gel Densitometry................................ 92
Transmission Electron Microscopy................. 94
Statistical Analyses............................. 95

RESULTS AND DISCUSSIONS............................... 96

General Studies................................. 96
Temperature and pH Determinations.............. 96
Shear Force.................................... 99
Thaw and Cook Loss............................. 103
Water Holding Capacity......................... 103
Sarcomere Length............................... 107
Actomyosin Study................................. 110
Actomyosin Extraction and Electrophoresis....... 111
Sensitivity of Actomyosin to ATP................ 136
Myofibrillar ATPase Activity .................. 143
Sulfhydryl Group Contnet....................... 145
Protein-Protein Interaction in
Actomyosin Solutions......................... 148
Myofibril Degrdation Study....................... 165
Contractile Proteins Degradation............... 170
Titin................................... .. ..... 173
Nebulin......................................... 182
Ultrastructural Studies.......................... 183

SUMMARY AND CONCLUSIONS............................... 200

REFERENCES.............................................. 203

BIOGRAPHICAL SKETCH................................... 226


Table Page

1 Mean values for thaw loss (%), cook loss (%) and
shear force (kg) of strip loin steaks from
Angus bulls..................................... 102

2 Mean values for sarcomere lengths (Mm) of
longissimus dorsi muscles of Angus bulls........ 108

3 Protein concentrations (mg/ml) in actomyosin
solutions extracted from longissimus dorsi
muscles.......................................... 116

4 Mean values for percentage of protein bands
exhibiting nonsignificant treatment by day
interaction (P>0.05) in actomyosin solutions
obtained from electrically stimulated (ES) and
control (NS) longissimus dorsi muscles as
determined by the densitometry of SDS-PAGE
gels............................................ 118

5 Mean values for percentages of myosin heavy
chain bands in actomyosin solutions as
determined by SDS-PAGE.......................... 120

6 Mean values for percentages of myosin light
chain 2 bands in actomyosin solutions
as determined by SDS-PAGE....................... 122

7 Mean values for percentages of C protein
bands in actomyosin solutions as determined
by SDS-PAGE...................................... 123

8 Mean values for percentages of alpha
actinin bands in actomyosin solutions as
determined by SDS-PAGE.......................... 125

9 Mean values for percentages of 55,000 dalton
protein bands in actomyosin solutions as
determined by SDS-PAGE.......................... 126

10 Mean values for percentages of actin bands
in actomyosin solutions as determined
by SDS-PAGE.................................... 128


11 The ratio of percentages of myosin to actin
bands as determined by SDS-PAGE of actomyosin
solutions........................................ 131

12 Mean values for percentages of troponin T bands
in actomyosin solutions as determined
by SDS-PAGE..................................... 132

13 Mean values for percentages of tropomyosin
bands in actomyosin solutions as determined
by SDS-PAGE....................................... 133

14 Mean values for percentages of 30,000 dalton
protein bands in actomyosin solutions as
determined by SDS-PAGE.......................... 135

15 Mean values for Mg+2 and Ca+2 ATPase activities
(Pi Ag/min/mg protein) in actomyosin
extracted from longissimus dorsi of Angus
bulls.................................... ....... 144

16 Mean values for sulfhydryl group (SH) content
(moles/105) in actomyosin solutions extracted
from longissimus dorsi muscles.................. 146

17 Activation energies (Ea) for protein-protein
interaction in actomyosin solutions from
longissimus dorsi muscles obtained at 12 hr
postmortem ................................... 160

18 Activation energies (Ea) for protein-protein
interaction in actomyosin solutions from
longissimus dorsi muscles obtained on day 1..... 161

19 Activation energies (Ea) for protein-protein
interaction in actomyosin solutions from
longissimus dorsi muscles obtained on day 3..... 163

20 Activation energies (Ea) for protein-protein
interaction in actomyosin solutions from
longissimus dorsi muscles obtained on day 7..... 164

21 Mean values for percentage of protein
composition as determined by densitometry of
SDS-PAGE gels of myofibrillar fractions from
electrically stimulated (ES) and control (NS)
longissimus dorsi muscles by treatment........... 171




22 Mean values for percentage of protein
composition as determined by densitometry of
SDS-PAGE gels of myofibrillar fractions by
postmortem storage day........................... 172

23 Mean values for titin1 (%) and titin2 (%) in
titin doublet obtained from SDS-PAGE gels of
myofibrils from electrically stimulated (ES) and
control (NS) longissimus dorsi muscles of Angus
bulls .................... ........................ 179


Figure Page

1 Postmortem temperatures of electrically
stimulated and control sides..................... 98

2 Postmortem pH values in longissimus dorsi
muscles obtained from electrically stimulated
and control sides............................... 101

3 Water holding capacity of longissimus dorsi
muscles of electrically stimulated and
control sides................................... 106

4 Electrophoretic separations of proteins in
actomyosin solutions extracted from
electrically stimulated and control
sides stored at -1 C for twelve hr (Day 0)
and one day postmortem.......................... 113

5 Electrophoretic separations of proteins in
actomyosin solutions extracted from electrically
stimulated and control sides stored at -1 C
for three and seven days postmortem............. 115

6 The ATP sensitivity of actomyosin extracted
from electrically stimulated and control samples
on days 0 and 1.................................. 138

7 The ATP sensitivity of actomyosin extracted from
electrically stimulated and control samples on
days 3 and 7.................................... 140

8 Arrhenius plots of protein-protein interaction
rates in actomyosin solutions extracted from
electrically stimulated and control samples
at 12 hr (Day 0) postmortem..................... 152

9 Arrhenius plots of protein-protein interaction
rates in actomyosin solutions extracted from
electrically stimulated and control samples
on day 1........................................ 154


10 Arrhenius plots of protein-protein interaction
rates in actomyosin solutions extracted from
electrically stimulated and control
samples on day 3................................. 156

11 Arrhenius plots of protein-protein interaction
rates in actomyosin solutions extracted from
electrically stimulated and control
samples on day 7................................. 158

12 Electrophoretic separations of myofibrillar
proteins of myofibrils prepared from
electrically stimulated and control
muscles on days 0 and 1.......................... 167

13 Electrophoretic separations of myofibrillar
proteins myofibrils prepared from
electrically stimulated and control muscles
on days 3 and 7................................... 169

14 Electrophoretic pattern of migration of titin
doublet and nebulin from electrically stimulated
and control muscles on days 0 and 1............. 176

15 Electrophoretic pattern of migration of titin
doublet and nebulin from electrically
stimulated and control muscles
on days 3 and 7................................. 178

16 Micrographs of control (NES) and electrically
stimulated (ES) beef longissimus dorsi at 2 hr
postmortem (Bar = 1 Am; X 24,000)................ 186

17 Micrographs of control (NES) and electrically
stimulated (ES) beef longissimus dorsi at 12 hr
postmortem (Bar = 1 Am; X 22,500) ................ 189

18 Micrographs of control (NES) and electrically
stimulated (ES) beef longissimus dorsi stored
at -1 C for one day (Bar = 1 Am; X 22,500) ...... 191

19 Micrographs of control (NES) and electrically
stimulated (ES) beef longissimus dorsi stored
at -1 C for three days (Bar = 1 Mm; X 27,000)... 193

20 Micrographs of control (NES) and electrically
stimulated (ES) beef longissimus dorsi
stored at -1 C for seven days
(Bar = 1 Mm; X 27,000) ........................ 195

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of The
Requirements for the Degree of Doctor of Philosophy



Parvin Hooshyar

December, 1989

Chairman: R. L. West
Major Department: Food Science and Human Nutrition

Alterations in the behavior of myofibrillar proteins

were investigated with respect to improvement in tenderness

due to high voltage electrical stimulation and postmortem

aging at -1 C for 7 days. Electrical stimulation and

postmortem aging significantly (P<0.05) enhanced the

tenderness of strip loin steaks obtained from Angus bull

carcass sides. Sarcomere lengths were longer in

electrically stimulated muscles than in the controls which

showed signs of cold shortening.

The determinations of myofibrillar ATPase, sulfhydryl

group content and ATP sensitivity did not exhibit signs of

weakening of crossbridges formed by myosin heads and actin

monomers. Electrical stimulation enhanced significantly

(P<0.05) the extraction of actomyosin early postmortem.

Electrophoretic studies indicated the presence of greater


percentages of actin, troponin T and tropomyosin in

actomyosin solutions from electrically stimulated muscle

than from the control at 12 hr postmortem.

The presence of alpha actinin, C protein and 30,000

dalton protein was exclusively detected in actomyosin

solutions from electrically stimulated samples at 12 hr.

The differences in protein components of actomyosin

solutions from electrically stimulated and control muscles

vanished at days 1, 3 and 7 of storage at -1 C. The

Arrhenius kinetic studies indicated the existence of

different mechanisms of protein-protein interaction in

actomyosin solutions from electrically stimulated and

control muscles.

Electrical stimulation and/or postmortem aging

significantly resulted in the degradation of titin and

nebulin. Electrical stimulation significantly enhanced the

conversion of titin 1 to titin 2 in the doublet. Electron

micrographs indicated accelerated loss of myofibril

structural integrity including the disintegration of the Z

disks and the I band due to electrical stimulation and

aging. Effects of electrical stimulation and aging on

contractile and regulatory proteins were limited to 12 hr

postmortem. The significant degradation of titin and

nebulin by electrical stimulation and/or postmortem aging

was indicative of the essential role of these cytoskeletal

proteins in the improvement of meat tenderness.



The traditional practice in the meat industry in the

United States has been to fatten the cattle in the feed lot.

This is due to the knowledge that subcutaneous fat acts as

an insulator and thus, prevents cold shortening and meat

toughness. Recent interest in health and fitness has made

consumers demand lean meat. Thus, the meat industry may be

forced to change its practice and produce lean animals.

This can result in increased incidence of cold shortening

and meat toughness (Marsh, 1977).

Electrical stimulation is the method of choice in

prevention of cold shortening. In addition, electrical

stimulation improves carcass lean quality and reduces aging

time. McKeith et al. (1980) reported that electrically

stimulated carcasses and sides had brighter, more youthful

colored lean, less "heat ring" and produced more palatable

rib steaks than did the controls.

The use of electrical stimulation is also beneficial on

subpopulations of cattle that have an inherent tendency to

have tough meat if not treated with some form of postmortem

conditioning (Savell, 1982). Electrically stimulated

carcasses from forage fed steers had lower shear values than

their nonstimulated counterparts for up to 14 days


postmortem. However, electrically stimulated carcasses from

grain fed steers had more desirable tenderness and lower

shear values than the controls only for 8 days postmortem

(Savell et al., 1981).

Bulls gain weight more rapidly, utilize feed more

efficiently and produce higher yielding carcasses with less

fat than steers. However, carcasses of bulls can have a

coarse texture and dark lean color. Cooked steaks from

bulls are often less tender than those from steers.

Therefore, electrical stimulation has the potential for

enhancing the marketability of beef from young bulls

(Savell, 1982).

Hopkinson et al. (1985) investigated the effect of

electrical stimulation of carcasses from bulls and steers.

Tenderness of steaks was improved (P<0.01) by castration of

bulls, electrical stimulation of the carcasses and aging of

steaks. Effects of electrical stimulation on tenderness

were most marked in the nonaged samples from bull carcasses.

Steaks that had been electrically stimulated and aged were

comparable in tenderness to aged steaks from steers.

Controversy surrounds the exact mechanisms involved in

tenderness improvement by electrical stimulation. The

tenderness mechanisms most commonly cited are as follows:

(1) cold shortening reduction (Nichols and Cross, 1980); (2)

increased activity of acid protease (Dutson et al., 1980);

and, (3) physical disruption of myofibrils (Marsh et al.,


Myofibrils occupy 83 percent of the volume of the

muscle fiber and provide a majority of the structural

proteins (Offer et al., 1988). Myofibrillar proteins are

responsible for 50 to 100 percent of the variation in meat

tenderness depending on connective tissue content and the

state of the muscle being examined (Purchas, 1972).

Therefore, the state of myofibrillar proteins in muscle may

reveal the reason for tenderness caused by postmortem aging

and electrical stimulation.

Increased solubility of myofibrillar proteins and

degradation of troponin T (TnT) with concurrent appearance

of 30,000 dalton protein have been attributed to improvement

in meat tenderness (Fujimaki et al., 1965a,b; Cheng and

Parrish, 1978). McKeith et al. (1980) did not find any

measurable difference in the solubility of myofibrillar

protein of muscle from electrically stimulated and

nonstimulated steer carcasses. However, they noted a

significant decrease in the proportion of troponin T in

stimulated muscles from cows but none in the case of steers.

Salm et al. (1983) reported that electrical stimulation

enhanced degradation of the myofibrillar proteins, alpha

actinin and troponin T and increased the amount of 30,000

dalton protein.


In contrast, Sonaiya et al. (1982) reported that

electrical stimulation did not affect the appearance of the

30,000 dalton protein. George et al. (1980) examined the

electrophoretic pattern of myofibrillar proteins of

longissimus dorsi muscle from electrically stimulated and

nonstimulated carcasses at different intervals of time and

followed the changes in TnT content and shear force. They

did not find any relationship between the decay of shear

force and composition of TnT. Therefore, they concluded

that a large difference in shear force between electrically

stimulated and nonstimulated carcasses at day one postmortem

(8.0 versus 13.3 kg, respectively) was due to some other

changes in muscle brought about by electrical stimulation.

Degradation of the cytoskeletal network which maintains

sarcomere integrity and contributes to the tensile strength

may be essential in tenderness improvement (Locker, 1982).

Titin, nebulin and desmin are constituents of this

cytoskeletal network (Robson et al., 1984). Kasang (1984)

reported that electrical stimulation accelerated breakdown

of desmin.

Understanding the nature of alterations in myofibrillar

proteins as a result of electrical stimulation and aging is

important in choosing the appropriate parameters and

conditions to optimize meat tenderness. The objective of


this study was to determine the class of myofibrillar

protein affected extensively by electrical stimulation and

postmortem aging.

This goal was pursued by performing the following

experiments: (1) actomyosin study which represented changes

in contractile and regulatory proteins due to electrical

stimulation and aging; (2) degradation of titin and nebulin

which were indicative of disintegration of cytoskeletal

network; and, (3) ultrastructural study to determine

morphological changes in the myofibrils.


Myofibrillar Proteins

Myofibrillar proteins which constitute the myofibrils

are the largest fraction of proteins in muscle tissue.

Myofibrils are the long thin contractile elements inside the

muscle cell. They manifest along their length a structural

pattern that repeats approximately every 2.5 Am. These

repeated units, called sarcomeres, are in transverse

register. The characteristic cross striation across the

muscle cell is due to the alternation of light and dark

transverse bands. The light bands are called isotropic or I

bands and the dark bands are anisotropic or A bands. The I

bands contain only thin filaments. The A bands constitute

both thin and thick filaments (Huxley, 1969).

In the resting muscle, the dark A bands extend along

the axis of contraction and are about 1.5 Am long. The I

bands are about 1.0 Am long. The I bands are bisected by a

dense transverse line about 80 nm thick called the Z line.

This Z line serves as a link for the transmission of tension

along myofibrils and for the coordination of movement of

adjacent myofibrils. The central portion (0.5 gm) of the A

band is called the H zone. The H zone is less dense and is


bisected by a dense transverse line which is called the M

line (Lehninger, 1978).

In addition to Z and M lines, two faintly stained

transverse structures N1 and N2 lines have been located

within the I band by electron microscopy. These lines

frequently appear as rows of bead like thickenings along

thin filaments on either side of and parallel to the Z

lines. The N1 line is 0.05 Am wide and fixed in position

about 0.1 to 0.2 Am from the Z line center. In contrast,

the N2 line is wider (up to 0.15 Am) and varies in position

depending on the sarcomere length (Wang and Williamson,

1980). The location of N1 and N2 lines in the I band may

indicate that they are involved in the regulation or

maintenance of the changing spatial arrangement of thin

filaments from a square lattice at the Z line to a hexagonal

array near the A-I junction. The unique movement of N2

lines suggests that these lines can not be rigidly attached

to either thin or thick filaments. The N2 line may be

attached to longitudinal elastic filaments that have been

proposed to exist in myofibrils (Robson et al., 1984).

Proteins of Thick Filaments


Myosin molecule from mammalian skeletal muscle is about

150 nm in length and consists of a long alpha helical rod

shaped region that has two globular portions attached to one


end (Lowey et al., 1969). The two globular regions are

referred to as the myosin heads. Myosin heads contain both

actin and ATP binding sites. The rod regions of myosin

molecules bind to one another resulting in the formation of

backbone of the thick filaments.

Skeletal muscle myosin has a molecular weight of

480,000 and is composed of two large subunits called heavy

chains and four small subunits or light chains. Each heavy

chain has a molecular weight of 200,000. The light chains

have molecular weights that range approximately 16,000 to

27,500 depending on the type of subunit and source of

myosin. One pair of light chains is required for ATPase

activity. The second pair of light chains undergoes

reversible covalent phosphorylation which plays a regulatory

role in actin and myosin interaction (Adelstein and

Eisenberg, 1980).

Myosin consists of a number of proteolytic fragments.

When myosin is digested with trypsin, two major fragments

are formed. Heavy meromyosin, the largest fragment,

contains both of the myosin heads and a portion of the

myosin rod. Heavy meromyosin has a molecular weight of

350,000. It is soluble in low ionic strength and contains

both the actin binding and the ATPase activity. The other

fragment is called light meromyosin. It has a molecular

weight of 150,000 and it is not soluble at low ionic

strength (Lowey et al., 1969).


Heavy meromyosin can further be digested to form S-1

and S-2 fragments. The S-1 fragments have a molecular

weight of 115,000 and are the individual myosin heads that

contain the actin binding and ATPase sites. Each globular

unit is 7 nm in diameter and has a 45 percent alpha helical

content. The S-2 fragment is a small portion of myosin rod

but it is soluble at low ionic strength (Weeds and Pope,


Adenosine triphosphatase activity of myosin. Adenosine

triphosphatase (ATPase) activity of myosin is stimulated by

calcium (Ca+2) and inhibited by magnesium (Mg+2). The

ATPase activity resides entirely in the head region. There

are two catalytic sites (S-1 fractions), each containing an

inhibitory and a catalytic sulfhydryl (SH) group. The light

chains are involved in ATP binding and ATPase activity

(Squire, 1975).

Sulfhydrvl groups. Myosin contains greater than 40

thiol groups. Class 1 thiol group is essential for the K+1

dependent ATPase. Blockage of these thiol groups activates

the Ca+2 ATPase. Class 2 is essential for ATPase but when

blocked subsequently to the blocking of the class 1

residues, the Ca+2 ATPase is inactivated. Both have one

thiol per active center of myosin. Class 3 can be blocked

without affecting the ATPase activity. Changes in

activities of SH groups occur when ADP, ADP-Pi or ATP


analogues induce different conformation of myosin (Mannherz

and Goody, 1976).

The M proteins

The M band of striated muscle is found at the center of

the thick filament. The model of sarcomere suggests that

the M line consists of two structural components: the M

filaments which run parallel to the myosin rods and the M

bridges which are arranged at right angles to myosin and

interconnect the M filaments with myosin at the center of

the M line (Etlinger et al., 1976).

Trinick and Lowey (1977) have verified the presence of

two M line polypeptides. They reported that one of the two

components is creatine kinase, a dimer with subunit

molecular weight of 42,000. Creatine kinase could have two

functional roles, one as a structural component of the M

line and another as the enzyme involved in the reaction that

forms ATP and creatine from creatine phosphate and ADP.

The other protein associated with the M line is a

doublet corresponding to polypeptides with molecular weights

of 193,000 and 182,000. The two bands are evident in

polyacrylamide gels of purified myofibrils indicating that

these proteins represent intrinsic M line structural

peptides. This high molecular weight protein binds to both

myosin and creatine kinase. The M line of skeletal muscle


is thought to join together the thick filaments of the

myofibril (Porzio et al., 1979).

The C protein

The C protein is part of the thick filament of both

skeletal and cardiac muscle. This protein has a molecular

weight of about 140,000 by SDS polyacrylamide gel

electrophoresis (SDS-PAGE). The C protein forms a series of

seven transverse stripes on each side of the thick filament.

The stripes are 7 nm in width and are spaced at 43 nm

intervals. It constitutes about 2 percent of total

myofibrillar proteins (Moos et al., 1975). Offer et al.

(1973) suggested that C protein has a structural role in the

formation of thick filament.

Thick filament structure

The cigar-shaped thick filaments from mammalian

skeletal muscles have a length of 15,000 nm and a diameter

of 14 nm. There are 200 to 400 molecules of myosin per

thick filament. The thick filaments are 45 nm apart and

arranged in a hexagonal pattern. Each thick filament is

surrounded by six thin filaments, also in hexagonal array.

Cross bridges, composed of the head regions of the myosin

molecules, project from the filament in a helical

arrangement with an axial spacing of 14.5 nm. There also is


a zone devoid of cross bridges in the middle of the thick

filament (Lehninger, 1978).

Proteins of Thin Filaments


Actin is the main constituent of the thin filament.

This protein has a molecular weight of 42,000. Globular

actin, or G actin, is stable in water. It is a single

polypeptide chain and contains 376 amino acid residues.

The G actin polymerizes into F actin (fibrous). Actin

contains nucleotide binding sites and a high affinity site

for divalent metal ions (Mg+2 or Ca+2). Therefore, the G

actin monomers are linked through Mg+2 or Ca+2 bridges and a

nucleotide prosthetic group (Elzinga et al., 1973).

Fibrous actin is 6 nm wide and is a double stranded

right handed helix. Fibrous actin forms the backbone of the

thin filament and also provides binding sites for both

troponin and tropomyosin. In muscle, when Ca+2 is present,

F actin comes into contact with the myosin head of thick

filaments and there is a rapid breakdown of ATP, ultimately

resulting in muscle contraction. Thus, the role of actin is

both structural and enzymatic through activation of myosin

ATPase (Zechel and Weber, 1978).


Tropomyosin (MW = 70,000) is a rod shaped protein, 40

nm in length, and represents 10 to 11 percent of total

contractile proteins. When crystallized, it forms square

net lattices (Lehninger, 1978). It is composed of two 100

percent alpha helical polypeptide chains which are twisted

around each other to form a coiled-coil structure. There

are two tropomyosin subunit isoforms found in skeletal

muscles, which are referred to as alpha and beta chains.

Alpha tropomyosin has a molecular weight of 34,000 and beta

tropomyosin has a molecular weight of 36,000 (Mannherz and

Goody, 1976).

Tropomyosin is involved in regulating skeletal muscle

contraction in conjunction with troponin. In muscle,

tropomyosin is in close association with thin filaments. It

extends through the grooves of the actin helix. When Ca+2

concentrations are elevated, tropomyosin may move such that

F actin can bind to myosin, resulting in muscle contraction

(Asghar and Pearson, 1980).

Troponin complex

Troponin is present in association with the thin

filaments. It connects the long chains of tropomyosin into

the grooves of actin at regular intervals of 38 nm. It has

a molecular weight of 76,000 and is composed of three

nonidentical subunits: troponin T (TnT), troponin C (TnC)


and troponin I (TnI). Troponin in concert with tropomyosin

regulates the interaction of actin and myosin (Mannherz and

Goody, 1976).

Calcium binding subunit. Calcium binding subunit (TnC)

is a single peptide with 159 amino acid residues and has a

molecular weight of 17,000 to 18,000. It is the most acidic

subunit (pI=4.1). Troponin C binds four Ca ions and forms a

complex with both TnI and TnT. Tropnin C does not bind

actin or tropomyosin. The complex formation with TnC is

Ca+2 dependent (Potter and Gergely, 1974).

Troponin inhibitory subunit. The troponin inhibitory

subunit (TnI) has a molecular weight of 21,000 and is

insoluble at low ionic strength (<0.2). This polypeptide

(pI = 5.5) with 178 amino acid residues interacts directly

with actin. It inhibits actin-tropomyosin cofactor activity

when added in 1:7 molar ratio. High concentrations of TnI

can inhibit the interaction of actin with myosin. Low

concentrations of TnI plus tropomyosin result in complete

inhibition of actin and myosin interaction. Troponin I

binds to TnC in the presence of Ca+2. The TnI-TnC complex

is soluble at low ionic strength (Ohtsuki et al.,1986).

Tropomyosin binding subunit. Tropomyosin binding

subunit (TnT) is the largest troponin subunit having a

molecular weight that ranges from 37,000 to 45,000 depending

on the source of the skeletal muscle (Greaser et al., 1967).

It is the most basic subunit (pi=8.8) and is insoluble at


low ionic strength. A single protein of 256 amino acids,

troponin T binds strongly to isolated tropomyosin and F-

actin-tropomyosin complex independently of Ca+2. It does

not combine with TnI and interacts weakly with pure actin.

The function of TnT is to connect TnI-TnC complex to the

thin filament. In connecting the TnC-TnI complex to F-

actin-tropomyosin, TnT provides at least one additional Ca+2

insensitive link. It also increases binding of TnC-TnI to

pure F actin.

Thin filament structure

Thin filaments are 1 Am in length and 7 to 8 nm in

diameter (Huxley, 1972). The three most abundant proteins

of the thin filaments are actin, tropomyosin and troponin,

which are present in a 7:1:1 molar ratio (Murray and Weber,

1974). The F actin helix has a repeat of 37 nm. This helix

appears as two chains of actin that are wound around one

another. The long rod shaped tropomyosin molecules are

located in the grooves between the two actin strands and are

arranged in the grooves such that there is a very small

amount of overlap from the end of one tropomyosin to the

start of the next. Troponin is attached to both actin and

tropomyosin and is bound to a region of the tropomyosin

molecule approximately one third of the distance from the C-

terminus (Squire, 1975).

Muscle Contraction

Muscle contraction consists of the cyclic attachment

and detachment of the globular portion of the myosin

molecule to the actin filament. The attachment is followed

by a change in the angle of myosin-actin attachment, so that

myosin and actin filaments slide past each other. The

energy for the process is provided by ATP and is released by

the interaction of actin with myosin which activates the

myosin ATPase activity (Adelstein and Eisenberg, 1980).

Muscle contraction and relaxation are regulated by the

action of the tropomyosin and troponin complex.

Conformation of TnC is changed on binding with Ca ions.

This change is transmitted to TnI and TnT which leads to a

strong binding of TnC with TnT. Thus, the inhibitory effect

of TnI is removed by disengagement from actin. This results

in movement of tropomyosin which then allows the actin and

myosin to interact (Taylor, 1979).

In postmortem muscle, the thick and thin filaments are

able to interact strongly and persistently. This is due to

the termination of ATP resynthesis and prevalence of ATP

hydrolysis. This interaction which forms actomyosin confers

upon the muscle the high stiffness that is characteristic of

the rigor state (Jeacocke, 1984).


Actomyosin is produced from interaction of actin with

myosin. Each F actin filament can bind several myosin

molecules. The molar ratio of G actin monomers to myosin in

actomyosin complexes may be as high as 1.7 (Herring et al.,

1969). Actomyosin is soluble at ionic strengths greater

than 0.6. The ratio of myosin to actin and the particle

weight of actomyosin depend on experimental conditions (pH,

KCl and MgC12 concentrations) and protein concentration

(Noda and Maruyama, 1960).

Upon extraction, there is a large increase in viscosity

and flow birefringence (Mueller, 1960). When actomyosin

undergoes dissociation in the presence of ATP and Mg+2,

there is a large and rapid decrease in the viscosity of

actomyosin solution. Following dissociation, hydrolysis of

ATP occurs. When ATP is completely hydrolysed to ADP, the

actin and myosin reaggregate (Muhlrad et al., 1965).

The Z Line and Alpha Actinin

Structure of the Z line

The basket weave (woven) lattice represents the

fundamental en face pattern of the vertebrate Z disc

(Ullrick et al., 1977). It is postulated that the Z band is

made of two Z disc halves. Sjostrand (1962) presented

micrographs of the split Z lines indicating the presence of

thin connecting strands between the two halves.

When muscle is stretched, the two halves do not

separate. They also do not fall apart readily in the

myofibrillar preparations. This may indicate that

"connecting filaments" between the two halves are of tightly

coiled "elastic" structures which could "uncoil" when

subjected to high degree of mechanical stress or during the

process of sarcomerogenesis (Wang and Williamson, 1983).

Alpha actinin

Alpha actinin (two percent of total myofibrillar

proteins) is located in the Z line. This protein has a

molecular weight of 206,000 dalton and is composed of two

100,000 molecular weight subunits. At 40 to 50 nm length

and 4 nm diameter (Suzuki et al., 1976), this protein has an

alpha helix content of about 74 percent. Granger and

Lazaride (1978) showed that alpha actinin is uniformly

present in the interior of isolated Z disks. It has a

supporting role in the attachment of actin filaments to Z


Cytoskeletal Proteins

Myofibrils are the major cytoskeletal elements in the

striated muscle cell (Goll et al., 1977). The proteins

desmin (O'Shea et al., 1979), connection (Maruyama et al.,

1977a,b), titin (Wang et al., 1979) and nebulin (Wang and

Williamson, 1980) have been discovered and isolated from


muscle cells. These proteins have distinct cytoskeletal

roles and are important in maintenance of muscle integrity

(Robson et al., 1984).


Animal cells contain a class of cytoplasmic filaments

with diameter of 10 nm. Ishikawa et al. (1968) observed the

presence of these 10 nm or "intermediate size" filaments in

striated myofibrils in developing myogenic cells in culture.

Desmin, a 55,000 dalton protein, may be a component of the

10 nm filament (Lazaride and Hubbard, 1976). Desmin makes

up about 0.18 to 0.35 percent of the total protein in

homogenized skeletal muscle. Desmin is composed of two

major isoelectric variants: alpha (acidic) and beta (basic)

isoforms (Robson et al., 1981).

Immunoferritin labeling of ultrathin frozen sections of

intact fixed sartorius muscle showed the presence of desmin

between adjacent Z bands and as strands peripheral to Z

bands, forming apparent connections of the Z bands with

adjacent sarcolemma, mitochondria and nuclei. No desmin

labeling was observed in the vicinity of the T tubules.

Desmin serves to interconnect myofibrils at the level of

their Z bands and to connect Z bands with other specific

structures and organelles in the myotube but not the T

tubule system (Tokuyasu et al., 1983).


Connectin is a highly insoluble elastic protein

responsible for the elasticity and mechanical continuity of

myofibrils of striated muscle (Maruyama et al., 1980).

Connectin consists of doublet bands, alpha and beta

connections. It appears that beta connection is a proteolytic

product of alpha connection. Beta connection is extracted

together with myosin in a salt solution leaving alpha

connection in the residue (Maruyama et al., 1981a).

Molecular weight of alpha connection is 2.8 megadalton and

2.1 megadalton for beta connection. The absence of evidence

for subunits or more than one chain indicates an exceedingly

long molecule (Maruyama et al., 1984).

Maruyama et al. (1981b) showed that connection is

hydrolyzed by trypsin, chymotrypsin, papain and serine

protease but not by calcium activated factor (CAF).

Connectin forms a three dimensional network covering the

entire region of the myofibril between Z lines. The

presence of the net-like structure extending through Z lines

of myofibrils in cardiac muscle was clearly demonstrated by

electron microscopy after the removal of myosin and actin.

The diameter of the very thin filament forming the net was

approximately 2 nm (Toyoda and Maruyama, 1978).


Titin, designating a pair of titanic proteins

(MW=1,000,000), composes approximately 10 percent of the

myofibrillar mass (Wang, 1982). Titin1 is insoluble in all

but powerful denaturing substances and tends to lose a small

fragment, giving a derived protein (titin2). "Native" or

"beta connection" and "soluble titin" are equivalent to

titin2 (Wang et al., 1979; Maruyama et al., 1984; Trinick et

al., 1984). There are 21,500 amino acid residues in titin2

with a mean residue weight of 130 dalton. Thus a fully

extended chain of titin1 is over 7.8 Am assuming

36.2 nm per residue. This span is far beyond any sarcomere

length but corresponds with a maximum extension obtainable

in beef fibers (Locker, 1987).

Electron micrographs of shadowed titin2 molecules

suggest a beaded structure and a spectrum of lengths that

may exceed a micron (Wang et al., 1984; Trinick et al.,

1984; Maruyama, 1986). Negative staining shows a regular

beaded structure of period 4.0 nm and diameter 3.5 nm

(Trinick et al., 1984). Tertiary structure of titin is made

of beta structure and random coil, and alpha helix is

lacking (Trinick et al., 1984 and Maruyama, 1986).

The polypeptide chain contains long "fold" sequences

alternating with short "binder" sequences which interact in

antiparallel array. This allows extensibility and recovery

by reversible unravelling. The titin filaments have a


zigzag knobby structure of period about 6 nm and knob

diameter of about 3 nm. The sudden violent stretch and

release of a molecule may cause a tangle in the backbone by

bringing together binder sequences which were not neighbors.

The molecular folding would then occur in a disordered way,

producing the nodules (Locker, 1987).

Chemical and immunological studies indicated that titin

is distinct from myosin, actin and filamin (Wang et al.,

1979). Titin has been localized by antibody staining at the

A-I junction and under certain conditions at the Z line, the

H zone and throughout the A band (Wang et al., 1984).

Maruyama (1986) proposed that six titin strands pass along

the A filament from near the M line, fuse in the I band and

terminate in the Z line. Titin may be a component of the

putative elastic filaments and/or the gap filaments (Locker,


Gap filaments

Small bundles of muscle fibers can be stretched to five

times their resting length. Consequently, a gap of up to 2

Am opens between the A and I band. This is spanned by thin

gap filaments (Locker and Leet, 1975). Gap filaments are

highly elastic in live muscle and remain somewhat elastic

and strong after heat denaturation. They are resistant to a

variety of powerful protein solvents but are vulnerable to

proteolytic enzymes (Locker and Leet, 1976a).


Tensile strength is the dominant factor in shear force.

Gap filaments are the only tension resisting unit of

myofibrils to survive cooking with any integrity. Locker

(1982) suggested that gap filaments form a core to an A

filament, emerging at one end only, passing between the I

filaments, through the Z line between the I filaments of the

next sarcomere and into a second A filament, again

terminating as a core. Thus they are continuous through the

Z line but not the sarcomere (Locker, 1982).

Gap filaments function as a series of elastic

components in muscle. As a core protein, they may be

involved in organizing the laying down of A and I filaments

in the differentiation of muscle. Gap filaments support the

N2 line which may act as a perforated spacer, marshalling

the I filaments from a square array at the Z line into

hexagonal array at the edge of A band. This arrangement may

also guide I filaments back into the A band when they have

been fully withdrawn (Locker and Leet, 1976b).

The gap filaments arise from the coalescence of T

filaments in the I band. Their extreme extensibility is

from an unravelling of a beaded structure in the titin

molecule (Locker, 1987). Electron micrographs of shadowed

titin show six parallel beaded filaments of the same period

emerging from the A filament tip. Single beaded filaments,

up to 0.5 pm long, can be seen lying loosely against the

myosin heads (Wang et al., 1984). Such filament clusters


are seen at both ends of an A filament, thus, challenging

the claim that gap filaments emerge at one end only (Locker

and Leet, 1976a).

Locker (1987) proposed a new model for the "gap" or

"third" filaments of muscle which were renamed "T

filaments". These consist of single titin molecules,

spanning the half sarcomere from M line to Z line. He

hypothesized that six T filaments lie longitudinally on the

surface of the A filament, one against each peripheral

subfilament of myosin. The C protein molecules overlie the

T filaments in transverse and axial rows, with their long

axis following a helix. Each C protein molecule helps to

bind a pair of T filaments to one A strand, but not to bind

A strands together.


Nebulin (MW = 600,000 to 800,000) represents 3 to 5

percent of the total myofibrillar proteins. Nebulin is more

prone to proteolytic degradation than titin (Wang et al.,

1979). Wang and Wright (1988) reported the amino acid

composition of nebulin is distinct from titin, especially in

its lower proline content (5.9 vs. 7.4 percent in titin),

and a higher total content of lysine and arginine (41.1 vs.

33.9 percent in titin).

Wang and Wright (1988) reported that nebulin is an

integral component within the skeletal muscle sarcomere that


spans a minimum of 1 Am between the Z line and the distal

region of thin filaments. Furthermore, it constitutes a

set of discontinuous, parallel, inextensible filaments

attached at one end to the Z line.

The inextensibility of nebulin filaments as reflected

by the stretch resistance of nebulin epitopes, contrasts

sharply with the elastic stretch-dependence of the

coexisting I band domain of titin filaments. These distinct

behaviors indicate that nebulin and titin must each

represent a separate set of parallel filaments and can not

be serially connected components of the same longitudinal

filaments (Wang and Wright, 1988).

Wang and Wright (1988) proposed a composite filament

model for the sarcomere of skeletal muscle. A sarcomere

consists of four biochemically distinct filaments: myosin,

actin, titin and nebulin, all of which are directly actinn,

titin and nebulin) or indirectly myosinn via titin) linked

to the Z line. Titin and nebulin may interact with and

adhere to myosin and actin, respectively, to form composite

filaments, and that these composite filaments may be in fact

the morphological structures identified as thick and thin

filaments in electron microscopy.

This composite filament model has possible implications

in the assembly, length regulation, stability and function

of actin filaments. In view of the fact that actin, even in

the presence of accessory or capping proteins, is incapable


of assembling into filaments of predetermined and uniform

length such as those found in skeletal muscle sarcomeres,

the idea of a long protein template that directs and

regulates assembly of actin becomes increasingly more

attractive (Wang and Wright, 1988).

Factors Affecting Meat Tenderness

Tenderness is probably the most important quality

factor of meat to a consumer. The molecular basis for

differences in muscle tenderness remains unknown. The

amount of connective tissue and the sarcomere length in the

muscle are correlated with meat tenderness (Herring et al.,

1965; Cross et al., 1973; Marsh, 1977; McKeith et al.,

1985). These relationships explain only a small proportion

of the total variations (MacBride and Parrish, 1977).

Tenderness and toughness depend not only on the tensile

strength of the individual longitudinal elements of muscle

but also on the adhesive strength of the linkages between

them (Davey and Gilbert, 1977). Toughness is induced (1) by

cell components being interlocked into a tighter structural

continuum through muscle shortening; (2) by heat coagulation

and denaturation of cellular proteins; and, (3) by heat

shrinkage of connective tissue. Tenderness is induced (1)

by meat aging which breaks down various structural

components within the muscle cell and (2) by prolonged


cooking which gelatinizes collagen of the connective tissue

(Davey and Gilbert, 1968; Davey, 1983).

Nonproteolytic Alterations

Postmortem tenderness originates from proteolytic

and/or nonproteolytic changes. The nonproteolytic

alterations in the contractile apparatus consist of changes

in conformation of actin and myosin, dissociation of

actomyosin or inhibition of actin and myosin interaction.

They occur during the first 72 hr postmortem when the

largest changes in tenderness happen and are temperature

dependent (Locker, 1960; Davey et al., 1966).

Postmortem muscle looses the ability to maintain

isometric tension after 24 to 48 hr postmortem. This loss

of tension development could result from either

"depolymerization" of actin or a conformational change in

myosin (Busch et al., 1967; Jungk et al., 1967). Levy et

al. (1962) suggested a conformational change in myosin could

occur during ATP hydrolysis.

Fujimaki et al. (1965a) indicated that actomyosin

extracted at various postmortem times contained variable

concentrations of myosin and actin. They observed a maximum

content of actin in actomyosin extracted from the muscle at

2 days after slaughter. Davey and Gilbert (1968) reported

that when compared to actomyosin from unaged muscle, a 10

percent increase in the content of actin in the actomyosin


fraction extracted from rabbit muscle aged for 7 days at

4 C. They concluded that the I filaments were being

disintegrated and/or freed from the Z band.

The breakdown of F actin may originate from loss of

forces that bind G actin subunits together (Wierbicki et

al., 1956; King, 1966). Johnson and Rowe (1961), in

ultracentrifugal studies, concluded that upon dissociation

of the actomyosin, actin was not normally liberated into

solution as F actin. The actin moiety appeared in three

forms: depolymerized actin, G actomyosin and gel actin.

Besides reacting to form actomyosin, actin contributes

to the structure of the myofibrils by associating either

with itself or with some other proteins to form filaments.

The strength of this association varies along the length of

the myofibrils. A decrease in the strength of this

association permits a greater extraction of actin from

postrigor than from prerigor muscle. Such release of actin

would render the muscle more tender by weakening the muscle

fibers and would account for the fiber breakages observed by

histological means (Weinberg and Rose, 1960).

Dissociation of the actomyosin complex

Actomyosin formation contributes to the development of

the rigid and inflexible state of rigor in postmortem

muscle. Thus, it was assumed that the resolution of rigor

may be caused by the slow dissociation of actomyosin into


actin and myosin (Wierbicki et al., 1954; Partmann, 1963;

Takahashi et al., 1965, 1967). Weinberg and Rose (1960)

indicated that actomyosin was dissociated into actin and

myosin during postmortem storage.

Partmann (1963) demonstrated that fiber fragments of

aged muscle contracted upon addition of ATP. This suggested

the presence of some dissociation of actomyosin during

aging. They stated that postmortem tenderization originated

from either a dissociation or an inhibition of the actin-

myosin interaction. When an actomyosin solution was stored

at lower temperature, lower pH or higher ionic strength, the

interaction between myosin and actin became less strong and

further storage brought about an irreversible dissociation

(Okitani et al., 1967).

The fact that postmortem muscle never fully regains its

original macroscopic extensibility argues against a complete

dissociation of actomyosin during aging (Asghar and Yeates,

1978). However, Fujimaki et al. (1965a,b) have indicated

that the actomyosin complex in postmortem muscle can be

dissociated by less ATP than that required for the muscle

just after death. On this basis, it was suggested that the

weakening of actin-myosin interaction occurred in postmortem



Weinberg and Rose (1960) studied the interaction of

actin and myosin in the region of thick and thin filament

overlap. They suggested that meat became more tender if

rigor linkages between the two sets of proteins were

gradually weakened. Chaudhry et al. (1969) and Robson et

al. (1967) have reported pronounced changes in the

physicochemical properties of actomyosin from postmortem

muscle. It was concluded that slippage or partial

dissociation at the point of interaction of myosin cross

bridges with the actin filament may occur during aging.

Determination of changes in actin-myosin interaction

The modification of actin-myosin interaction is

determined by (1) sarcomere length determination; (2)

increase in protein extractability; (3) changes in the Ca+2

and Mg+2 modified ATPase activity of actomyosin and of

myofibrils; (4) changes in sulfhydryl groups; and (5)

weakening of the actomyosin complex or increased sensitivity

to ATP.

Sarcomere length. Assuming the sarcomere length varies

inversely with the formation of actomyosin, the measurement

of sarcomere length at various time intervals after

slaughter would reflect the degree of actomyosin formation

or dissociation (Asghar and Yeates, 1978). Gothard et al.

(1966) found no significant difference between the inrigor

and the postrigor sarcomere length of the muscle fibers.


However, Takahashi et al. (1967) reported that sarcomeres

lengthened again during aging even in the absence of ATP.

Protein solubility. Toughening in stored frozen fish

is paralleled by loss in extractability of the myofibrillar

protein (Love, 1962). These changes are due in part to

denaturation and aggregation of myosin and to the formation

of new actin-myosin links (Connell, 1962).

Weinberg and Rose (1960) reported that the amount of

protein extracted in KCl solutions of various ionic strength

from chicken muscle was greater 24 hr postmortem than it was

immediately after the death of the animal. The extra

protein was considered to consist of actin and actomyosin.

This extensive release of myofibrillar proteins was

contributed to progressive weakening of the linkages of

these proteins with relatively insoluble components of the

muscle cell.

Davey and Gilbert (1968) investigated changes occurring

during aging in the extractability of the myofibrillar

proteins of meat from beef and rabbit carcasses.

Approximately 52 percent of myofibrillar proteins of unaged

meat was extracted in 40 min at 2 C; whereas, from aged meat

as much as 78 percent was extracted. They postulated that

the increase in the percentage of myofibrillar protein

extracted during aging resulted from either a progressive

weakening of the fibrous protein linkages with the insoluble


components in the muscle cell, or from a disintegration of

the insoluble stroma itself.

Davey and Dickson (1970) hypothesized that changes

occurring during aging are possibly due to a weakening

rather than to a breaking of structural attachment. Thus,

distinct and convincing histological changes may not be very

obvious. As aging proceeds, the muscle cell apparently

suffers a weakening of its lateral attachments and becomes

more succeptable to breakdown during homogenization. This

is shown in the first instance by the loss of precise

ordering of myofibrils and shown after prolong aging by a

complete breakdown to single myofibrils. Such weakening of

lateral attachments, probably at the Z line, is the most

distinctive physical change of aging. This weakening alone

can be invoked to explain the low stretching property of

aged meat.

Myofibrillar protein solubility of both rabbit and beef

muscle in 0.5 M KC1, 0.1 M phosphate, pH 7.4 increased

markedly with increasing postmortem storage at temperatures

up to 25 C. Time and temperature of postmortem storage

caused appreciable alteration in protein solubility. These

alterations could not be directly related to changes in

tenderness or sarcomere length or to species differences in

the effects of temperature on postmortem shortening

(Chaudhry et al., 1969).


The ATPase activity of myofibrils. It is known that

myofibrillar ATPase activity of muscle initially increases

during postmortem storage (Goodno et al., 1978). The reason

for the increased ATPase activity is due to enhancement of

actin and myosin interaction (Ito et al., 1978). Postmortem

degradation of troponin also enhances the myofibrillar

ATPase activity by removal of the inhibitory effect of this

protein on the actin-myosin interaction and Mg+2 modified

ATPase activity.

The Mg+2 modified ATPase activity indicates changes in

the integrity of actin-myosin interaction and the

tropomyosin-troponin complex. The Ca+2 modified ATPase

activity is an indication of the integrity of myosin (Suzuki

and Goll, 1974). The Mg+2 and Ca+2 modified ATPase

activities were investigated during storage at 0, 1 and 7

days postmortem. The Mg+2 modified activity increased after

24 hr and then declined with increased storage time. The

Ca+2 modified ATPase activity decreased gradually during

storage. The more denatured actomyosin was, the more rapid

the increase in the Mg+2 modified ATPase activity at the

initial stage of storage (Okitani et al., 1967).

Goll and Robson (1967) noticed that Ca+2 and Mg+2

induced ATPase activity of myofibrils prepared from muscle

24 hr postmortem at 2 and 16 C increased significantly from

the corresponding activities of muscle at death. On

treatment of myofibrils with trypsin, the Mg+2 modified


activity decreased and the Ca+2 induced activity remained

approximately the same. It was concluded that a very

specific and limited proteolysis of myosin, actin or one of

the regulatory proteins or weakening of the actin-myosin

interaction occurred.

Fujimaki et al. (1965a) showed that actomyosin from

muscle 12 and 24 hr postmortem had higher Mg+2 ATPase

activity than that from 0 hr and 5 or 10 day aged muscle.

The increase in ATPase activity of actomyosin extracted 12

to 24 hr postmortem was attributed to: (1) stronger binding

between myosin and actin; (2) increased F actin content;

and, (3) conformational changes in actomyosin. They

suggested that changes in ATPase activity were possibly

associated with tenderization process in muscle.

Ikeuchi et al. (1978) showed an increase in

myofibrillar ATPase activity during postmortem storage of

muscle and an increase in Ca+2 sensitivity of myofibrils.

Ito et al. (1978) concluded that the cause of this

phenomenon was a change of the actin-myosin interaction

during postmortem storage. This was due to either (1) an

increase in the affinity of actin to myosin which activates

myofibrillar ATPase, resulting in the increase in Ca+2

sensitivity of myofibrils or (2) to the degradation of

regulatory proteins, probably due to a Ca+2 activated factor

(Dayton et al., 1976).


The effect of regulatory proteins on the actin-myosin

interaction during postmortem storage of muscle was

investigated by using a reconstituted complex of actin,

heavy meromyosin (HMM), tropomyosin and troponin. Little

difference was found in Ca+2 sensitivity of the regulatory

proteins between at death and 168 hr postmortem muscles.

The electrophoretic patterns showed that there was a slight

change of myosin structure and a noticeable degradation of

troponin from 168 hr postmortem muscle compared to at death

muscle. These results suggested the possibility that

increase in myofibrillar ATPase activity during postmortem

storage of muscle was mainly due to the increase in actin-

myosin interaction allowed by the degradation of troponin

(Ikeuchi et al., 1980).

In contrast, Hay et al. (1973) observed no change in

the Ca+2 and Mg+2 induced ATPase activity of muscle during

aging. Jones (1972), Hay et al. (1972) and Wolfe and

Samejima (1976) found no change in ATPase activity and

reduced viscosity in actomyosin extracts from aged meat.

The assumption was that changes in viscosity or ATPase

activity was completely dependent on the association and

dissociation of actomyosin. These experimental results

contradicted the contention that aging affected the

dissociation of actomyosin. The results were also

incompatible with the hypothesis that the actomyosin


interaction undergoes weakening during postmortem aging

(Asghar and Yeates, 1978).

Oxidoreductive changes in the sulfhydryl groups.

Myosin contains 8 to 9 moles of sulfhydryl (SH) groups per

105 g of protein (Kielley and Barnett, 1961). Sulfhydryl

groups of myosin are essential for the binding of myosin to

actin. Sulfhydryl groups of myosin are masked with actin in

actomyosin molecule. They are revealed with the addition of

pyrophosphate to dissociate actomyosin into myosin and

actin. Therefore, the increase in SH content during storage

of actomyosin can be attributed to the change in the binding

site of myosin to actin in actomyosin molecule. It also

suggests that the interaction between myosin and actin

becomes less strong during the storage of actomyosin

(Okitani et al., 1967).

Okitani et al. (1967) reported that the amount of SH

groups in actomyosin solution stored in 0.6 M KC1 (pH 5.7)

at 3 C increased with storage time. Strandberg et al.

(1973) reported partial involvement of the SH groups in the

actin and myosin interaction of postmortem muscle. They

stated that modifications of SH groups contributed to

postmortem alterations in ATPase activity and rate of

turbidity development in actomyosin.

Chajuss and Spencer (1962) ascribed the improvement in

tenderness during aging to cleavage or reorientation of

inter- and/or intramolecular disulfide bonds. Such cleavage


of disulfide bonds in aging meat was possibly caused by

redox enzymes naturally present in muscle. The probable end

products of the reaction might be sulfonates with a small

amount of sulfinates due to sulfhydryl exchange.

Weakening of actomyosin complex. Superprecipitation or

flocculation of actomyosin in the presence of Mg+2 and ATP

has been used as a measure of contractibility. Actomyosin

from 12 and 24 hr postmortem superprecipitated faster than

that from 0 hr muscle. However, actomyosin from 5 and 10

day aged muscle superprecipitated less rapidly than that

from 12 and 24 hr postmortem muscle. Superprecipitation was

more rapid in actomyosin from tough than tender muscle at

low KCl concentrations. This observati on suggested that

actomyosin from tough muscle had a stronger interaction or

higher amounts of some protein factors such as alpha actinin

than did the tender muscle (Okitani et al., 1967).

When ATP was added to a suspension of actomyosin gel,

superprecipitation took place, with the subsequent clearing

phase. The Mg+2 ATPase activity during superprecipitation

was greater than the one during clearing. Actomyosin from

tough muscle underwent a more rapid turbidity change than

the tender muscle in 50 mM KC1. There was a delayed rate of

superprecipitation in actomyosin from the tough muscle

compared to tender muscle in 100 mM KC1. At low KC1

concentrations, alpha actinin enhanced superprecipitation,


while at high KC1, alpha actinin promoted clearing (Ebashi

and Ebashi, 1965; Seraydaria et al., 1962).

Fujimaki et al. (1965a) investigated the strength of

actin and myosin interaction in actomyosin solutions during

aging. This was achieved by additions of various

concentrations of ATP. The protein concentration in the

supernatant was measured after ultracentrifugation.

Increased protein content in the supernatant was indicative

of weakening of actin and myosin interaction.

Actin-myosin complex in postmortem muscle was

dissociated by ATP levels about one sixth to one third of

those required for at-death muscle (Fujimaki et al., 1965a).

Okitani et al. (1967) concluded that the minimum

concentration of ATP needed to dissociate actomyosin fully

to myosin and actin became less after storage of actomyosin

at pH 5.7 and 3 C. This indicated that the interaction

between myosin and actin became less strong with the elapse

of the storage period.

Protein-protein interaction

A protein molecule in its natural state usually

consists of a chain of amino acids folded together in a

"native" structure which is particular to that protein type.

The native structure of a protein molecule is held together

by internal molecular forces. Protein-protein interaction

leads to changes in the secondary and tertiary structure of


the protein molecule. Protein-protein interaction is also

called association, aggregation and polymerization (Waugh,

1954; Reithel, 1963).

The internal molecular forces in most cases are

weakened as the temperature increases. This leads to

"thermal denaturation" above certain temperatures. Protein-

protein interaction is also referred to as denaturation,

because many of the reversible and irreversible denaturation

phenomena lead to aggregation. Denaturation of proteins

involves unfolding and aggregation. The amino acid chain is

more or less unfolded, whereby parts of the interior of the

previously native molecule is exposed at the molecular

surface. This causes a change in the affinity of the

protein molecule for other molecules (Deng et al., 1976;

Martens et al., 1982).

If this affinity increases, the degree of aggregation

between protein molecules will increase. This leads to the

formation of continuous and strong gels or precipitates

(Hegg et al., 1979). If affinity decreases, a

solubilization of the native aggregate may follow instead.

The two processes, thermal denaturation and change in degree

of aggregation may have quite different reaction rates.

Depending on the strength of the native versus the denatured

aggregates, the denaturation of a protein type in meat may

lead to a softening or toughening of the sample (Martens et

al., 1982).


Arrhenius kinetic is utilized to determine the effect

of temperature on the rate of protein denaturation. It

provides thermodynamic and kinetic data to determine the

effects of different environmental factors on the

thermolability of myofibrillar proteins (Penny, 1967). The

energy of activation is determined from Arrhenius plot. The

energy of activation is the energy required to convert a

normal reactant into a molecule ready to undergo structural

changes necessary for product formation. The large

magnitude of activation energy is indicative of slow

reaction rate or vice versa (Berry, 1978).

Okitani et al. (1967) proposed that actomyosin is

denatured in two different ways. One is the irreversible

dissociation to myosin and actin. The other is spontaneous

aggregation. Deng et al. (1976) applied Arrhenius kinetic

in investigating protein-protein interaction in actomyosin

solutions. They showed that if the pH remained constant,

the reaction rate and extent of interaction increased with

increasing temperature. In beef, the increase in rate of

protein-protein interaction was correlated to a decrease in

water holding capacity and an increase in toughness (Deng,


Enzvmology of Meat Aging

Bird and Carter (1980) hypothesized that postmortem

changes occurring in meat texture are most likely due to the


action of endogenous proteolytic enzymes. The increase in

meat tenderness that accompanies postmortem aging is assumed

to be the result of breaking, fragmentation or, at least,

weakening of the myofibrillar structure at or near the Z

line (Parrish et al., 1973; Lawrie, 1980, 1983; Robson et

al., 1980, 1981, 1984). Therefore, the action of an enzyme

that is affective in postmortem muscle should be to catalyze

the hydrolysis of one or more proteins at a structurally

important site that results in weakening of the myofibril

(Goll et al., 1983).

Current evidence indicates that of the 13 known

lysosomal peptide hydrolases, only seven, cathepsins A, B,

C, D, H, L and lysosomal carboxylase B are located inside

skeletal muscle cells. Three of the seven muscle lysosomal

peptide hydrolases are exopeptidases, two are

carboxypeptidases and one is a dipeptidyl amino peptidase.

Cathepsin H is both an aminopeptidase and the remaining

three muscle cathepsins B, D and L are all endopeptidase

(Goll et al., 1983).

Only one of the reported neutral and alkaline protease

is located inside skeletal muscle cells. This neutral

protease is the Ca+2 dependent proteinase (CAF). With the

possible exception of cathepsin N, which can degrade


collagen, any protease that contributes to the postmortem

tenderization needs to be located inside muscle cells (Goll

et al., 1983).

Lysosomal enzymes

Schwartz and Bird (1977) reported that cathepsin D

maximally degraded purified native myosin and actin at pH

4.0. Myosin hydrolyzed by cathepsin D activity initially

showed heavy chain fragments in the 175,000 to 150,000

dalton region. These large fragments were then further

broken down to many fragments of molecular weights less than

100,000 dalton. They also reported that the rate of actin

hydrolysis was only about 10 percent of that observed for


Ogunro et al. (1979) have shown that cathepsin D

isolated from cardiac muscle had activities toward myosin

and actin similar to those of the skeletal muscle enzyme.

It produced the gross degradation of actin and myosin.

Furthermore, myosin was degraded into specific polypeptides

which were relatively resistant to further hydrolysis.

Matsumomoto et al. (1978) reported that the addition of

cathepsin D to purified muscle proteins resulted in

degradation of myosin heavy chains, tropomyosin, troponin T,

troponin I, and alpha actinin but not of actin. Scott and

Pearson (1978) reported that cathepsin D hydrolyzed soluble

collagen, or its cross linked peptides to a limited degree.


Purified cathepsin D was incubated with bovine skeletal

muscle myofibrils under in vitro conditions resembling those

found in postmortem muscle. Electrophoretic analysis of

myofibrils treated at pH 5.5 and held at 37 C showed

degradation of myosin heavy chains and titin. A small

amount of actin, tropomyosin, troponins T and I, and myosin

light chains were also hydrolyzed (Zeece et al., 1986a,b).

Cathepsin B degrades myosin, actin and TnT at low pH

and high temperature. Schwartz and Bird (1977) found that

at pH 5.2, cathepsin B hydrolyzed myosin into a major

fragment of 150,000 dalton that seemed resistant to further

degradation and into a heterogeneous group of fragments of

10,000 to 50,000 dalton.

Cathepsin D at pH 4.0 degraded myosin more rapidly and

more extensively than cathepsin B, forming two major

fragments of 110,000 and 107,500 dalton and several smaller

fragments (Goll et al., 1983). Both cathepsin B and D break

down actin at pH 5.0 with cathepsin D being the most active

forming fragments of 35,000 and 12,000 dalton. Neither

cathepsin B or D have any appreciable effect on myosin at pH

values above 6.0.

Cathepsin D is the main protease in the muscle.

However, Okitani and Fujimaki (1972) did not consider their

action to be significant in tenderization. Cathepsin D

treated myofibrils were not fragmented to any greater extent

than untreated myofibrils. Raising the pH and/or lowering


the temperature greatly reduced the effectiveness of

cathepsin suggesting that the enzyme does not play a

principal role in the tenderization process occurring in

muscle postmortem (Zeece et al., 1986a,b).

Cathepsin L degraded both the heavy and light chains of

myosin completely after 22 hr at pH 5.0 (Goll et al., 1983).

Cathepsin L removed the Ca+2 sensitivity of myofibrillar

ATPase activity. The specific activity of cathepsin L

against myosin was 10 times greater than that of cathepsin B

(Okitani et al., 1980). Cathepsin G and N could degrade

collagen (Goll et al, 1983; Etherington, 1984). However,

cathepsin L had greater specific activity on collagen than

cathepsin N.

Penny and Dransfield (1979) emphasized that the pH

range within which these enzymes were active did not

preclude them from being operative in postmortem muscle,

especially at high temperatures. For example, cathepsin B

and D exhibited optimum activity at pH 5.2 and 4.0,

respectively. At postmortem muscle pH (5.5 to 5.6),

cathepsin B and D still had 50 percent and 30 percent of

their optimum activity, respectively.

The low pH and high temperature conditions of muscle

have been found to be quite favorable for certain other

lysosomal enzymes which catabolize mucopolysaccharides of

the ground substance and some cross linkages of collagen in

the nonhelical region (Etherington, 1976). The activity of


beta galactosidase, beta glucuronidase and acid ribonuclease

have also been reported to increase in the soluble fraction

of meat during aging. Ono (1971) reported that beta

galactosidase played a more important role than beta

glucuronidase in the meat conditioning process.

Calcium activated factor (CAF)

The location of CAF is inside of striated muscle cells

at the level of the Z disk (Ishiura et al., 1980; Dayton and

Schollmyer, 1981) and adjacent to the cytoplasmic face of

the plasma membrane. It is maximally active between pH 6.5

and 8.8 and needs 1 to 2 mM Ca+2 for maximal activity and

has very little activity below 0.1 mM Ca+2. It is not

activated by most other divalent cations. It requires a SH

group for activity (Dayton et al., 1976). The activity of

CAF is optimal at pH 7.0. Its activity reduces to 80

percent of optimal at pH 6.0, to 40 percent at pH 5.5 and is

almost zero at pH 5.0.

Dayton et al. (1976) reported the presence of a low

calcium requiring CAF (uM CAF). Low calcium requiring CAF

and CAF give identical banding pattern on SDS-PAGE with both

patterns showing bands at 80,000 and 30,000 dalton.

Furthermore, the anti-80K IgG which is specific for CAF,

cross reacts strongly with purified AM CAF.

Low Ca requiring CAF is also activated to varying

degrees by several other divalent cations including Mg+2 and

Mn+2. It showed maximal activity between pH 7.5 and 8.0

(Dayton et al., 1981). Koohmaraie et al. (1986) reported

that at conditions similar to those of postmortem storage

(pH 5.5 to 5.8 and 5 C), AM CAF retained 24 to 28 percent of

its maximum activity.

Busch et al. (1972) reported that CAF was capable of

degrading Z disk of myofibrils from rabbit skeletal muscle.

Dayton et al. (1976) reported that the CAF degraded

troponins T and I, tropomyosin and C protein but not

troponin C, myosin, actin and alpha actinin. Olson et al.

(1976) and Olson and Parrish (1977) associated the

degradation of troponin T to a 30,000 dalton protein with

CAF. Penny and Dransfield (1979) reported that CAF digested

myofibrils in the region of C protein and degraded unknown

polypeptides with molecular weight of 80,000 and 76,000.

Calcium activated factor may be involved in the degradation

of desmin which is associated with the Z disk and in

digesting the gap filamemts (Dayton et al., 1981).

Myofibrillar ATPase and CAF. The alteration of the

Mg+2 modified ATPase activity of myofibrils was studied

(Suzuki and Goll, 1974). Treatment with CAF for 30 min

caused a 20 to 25 percent increase in Mg+2 modified ATPase

activity. The CAF treatment for 360 min under the same

conditions caused a decrease in Mg+2 modified ATPase

activity at the highest ionic strength. The increase in

Mg+2 modified ATPase activity may have originated from CAF


degradation of troponin T, whereas the decrease in Mg+2

modified activity may be due to CAF destruction or the

release of alpha actinin from myofibrils.

Action of CAF on Z bands. Suzuki and Goll (1974)

reported that the extent of Z line degradation caused by CAF

in postmortem muscle depended directly on the relative loss

of Ca+2 sequestering ability by the sarcoplasmic reticular

membrane and on pH decline. The Z lines remaining in

myofibrils after short periods of CAF treatment always

appeared fainter under phase microscope than Z lines in

untreated muscle. This suggested that CAF may very quickly

weaken Z lines before its removal entirely.

The postmortem role of CAF and cathepsins. As muscle

pH drops below 5.5, catheptic enzymes begin to act, mainly

on the sarcoplasmic proteins, but possibly also on

sarcomeres that have been previously attacked and freed from

the Z line by CAF (Suzuki and Goll, 1974). Calcium

activated factor and lysosomal enzymes may be working on the

principal of division of labor in the postmortem aging

process, whereas each become ineffective at different stages

of the process, with the pH drop. Thus, in the early stages

of the meat conditioning process when pH is high, CAF may

play a major role. As the muscle pH drops below 6.0, the

cathepsins may be more active to affect the ripening changes

(Goll et al., 1983).


Postmortem Protein Degradation

Myofibrillar proteins degradation

Ikeuchi et al. (1980) demonstrated that minor bands

appeared between heavy and light chains of myosin after 168

hr postmortem. This may be due to breakdown of myosin or

some other myofibrillar proteins. A significant breakdown

of troponins was evident. No change in actin and

tropomyosin was observed.

In bovine longissimus muscles with postmortem pH in the

range of 5.5 to 7.0, degradation of the myosin heavy chain

was most obvious in low pH muscle, followed by intermediate

and high pH muscles. On the other hand, degradation of

troponins, tropomyosin and alpha actinin, all components of

thin filaments and Z line, were most pronounced with high pH

muscle. Intermediate pH muscles did not show much

degradation of muscle proteins, resulting in tougher meat

than either low or high pH muscles (Yu and Lee, 1986).

Davey and Graafhuis (1976) demonstrated specific

breaking at the A-I junction. Yu and Lee (1986) indicated

extensive degradation of thick and thin filaments,

particularly myosin heavy chains and M lines for the low pH

muscles. Therefore, they concluded that tenderization of

low pH meat appeared primarily due to the proteolytic action

of acidic proteases.


Degradation of troponin and tropomyosin complex

The degradation of these proteins may contribute to the

weakening of the actin-myosin interaction (Samejima and

Wolfe, 1976). Troponin T has a stabilization effect on

actin filaments. Thus, its degradation may weaken the

myofibrils to the point where actin filaments are released

from the Z band (Olson and Parrish, 1977).

Olson and Parrish (1977) have demonstrated that

myofibrillar fragmentation is strongly related to beef

tenderness. Olson et al. (1977) have shown that a 30,000

dalton component is a degradation product of Troponin T.

The appearance of the 30,000 dalton component seems to be

related to postmortem tenderization and myofibril

fragmentation index. MacBride and Parrish (1977) observed

that the 30,000 dalton component was present in tender but

absent in tough bovine longissimus muscle.

Cheng and Parrish (1978, 1979) reported that the most

prominent and distinguishable change occurring during

postmortem storage of muscle at 2 C was the gradual

degradation of TnT and concurrent appearance of 30,000

dalton component. Parrish (1977) reported that the 30,000

dalton component was a degradation product of TnT and was

related to beef steak tenderness.

Koohmaraie et al. (1984a) investigated changes in

myofibrils isolated from the longissimus muscle of control

and cold shortened muscles after 0, 1, 3, 7, and 10 days of


postmortem storage at 2 C. The myofibrillar proteins of the

cold shortened muscles were affected by postmortem aging in

a similar manner to that of the normally chilled muscle.

The primary changes in the muscles were the gradual

appearance of 110,000, 95,000 and 30,000 dalton components

and the disappearance of desmin and TnT components of

myofibrils. A gradual increase in the intensity of a

protein around 55,000 dalton was observed in both control

and cold shortened muscle. They concluded that the

myofibrillar proteins of cold shortened muscles are affected

by postmortem aging in a manner similar to that of the

normally chilled muscles.

Changes in myofibrillar proteins of bovine longissimus

and semitendinosus muscles were examined during 14 days of

postmortem storage at 2 C by SDS-PAGE. Major changes in

both muscles were: (1) appearance of 95,000 dalton

component; (2) gradual disappearance of troponin T and

gradual appearance of 30,000 dalton component; (3) gradual

increase in the intensity of a protein around 55,000 dalton;

and, (4) gradual appearance of 110,000 component (Koohmaraie

et al., 1984b).

Disorganization of Z band (alpha actinin)

Myofibrillar breakage produced by stretching aged

muscle strips occur predominantly at thin filament insertion

with Z disc (Davey and Dickson, 1970; Dayton et al., 1981).


Davey and Gilbert (1967) expressed the view that aging

caused disruption of myofibrils, especially the Z band. The

electron microscope studies have indicated that the site of

postmortem cleavage of myofibrils is at or near Z line.

This cleavage results in a marked change in the

characteristic pattern of cross striation or complete loss

of Z band (Fukazawa and Yasui, 1967; Stromer and Goll,


Davey and Dickson (1970) hypothesized that during meat

aging, the disintegration of the Z band to the point of its

disappearance is much slower than increases in tenderness.

They assumed that Z band first undergoes an unobservable

weakening. This is sufficient to affect the mechanical

strength, and further disorganization is merely an extension

into the observable range. This was supported by the

evidence that aged meat stretched quite readily by applying

a little force, resulting in the cleavage at the Z-I


Johnson and Bowers (1976) confirmed that Z lines lost

their prerigor density during aging. They suggested that

the decrease in shear value of meat was possibly due to the

loss of lateral attachment between fibrils and loss of Z

lines. The disintegration of Z bands of the sarcomere may

contribute to the loss of tensile strength of myofibrils and

increased tenderness (Busch et al., 1972).


The Z bands are composed of alpha actinin (Briskey et

al., 1967). Cheng and Parrish (1978) reported that upon

aging, alpha actinin disappeared from muscle extracts. The

bonding of alpha actinin to the Z disk is apparently

weakened during aging. Assuming that alpha actinin has a

key structural role in anchoring opposing thin filaments

from adjacent sarcomeres, its loss from the Z disk may lead

to a considerable reduction in myofibrillar tensile

strength. As postmortem aging increases, myofibrils become

shorter, more fragmented and Z disks are degraded (Locker,


Changes in cvtoskeletal proteins

Recent work has focused on the possible role of the

cytoskeletal proteins desmin, titin and nebulin in

tenderness. These proteins constitute the cytoskeletal

network in the myofibrils(Locker and Leet, 1976a,b; Locker,

1982, 1987; Wang and Ramirez-Mitchell, 1983; Wang and

Wright, 1988). The disintegration of the cytoskeletal

network can account for the postmortem changes in the

physical properties of muscle and for the increased

tenderness after cooking of stored meat (Young et al.,


Desmin degradation. Alterations in desmin may disrupt

muscle cell integrity, especially transversal (cross link)

elements and improve meat tenderness (Robson and Huiatt,


1983). Desmin is degraded in postmortem muscle at about the

same rate as is TnT. Storage of bovine skeletal muscle

under aseptic conditions for only one day at 15 C results in

a loss of 10 to 25 percent of desmin. After 7 days of

storage nearly all intact desmin has disappeared (Robson et

al., 1984). Purified desmin or desmin in intact myofibrils

is rapidly broken down by proteases including CAF (Goll et

al., 1977; Robson, 1980, 1981, 1984).

Connectin degeradation. The disappearance of the

connection filaments from the myofibrils during conditioning

of muscle has been documented (Takahashi and Saito, 1979).

The degradation of the connection has been implicated as

being responsible for increasing meat tenderness during

conditioning (Locker, 1982). It has been suggested that

degradation of titin by endogenous muscle enzymes might be

involved in the postmortem decrease in muscle cell integrity

and improvement in meat tenderness (Suzuki et al., 1985).

Maruyama et al. (1977a) indicated that when chicken muscle

from local market was used, the yield of extracted connection

was low (1 g/kg of muscle). The yield from freshly

slaughtered muscle was 5 to 6 g/kg; therefore, autolysis of

connection occurred during storage.

Ultrastructural studies indicated a network structure

between the Z disks vanished when the amount of connection

fell to zero. Connectin is responsible for 30 percent of

the total elasticity of living skeletal muscle (Maruyama et

al., 1977b). Takahashi and Saito (1979) reported that the

amount of connection decreased with increasing time of

postmortem storage, whereas the rate of decrease depended on

the source of the muscle. With chicken breast muscle, the

time needed to achieve a constant minimal value was only 1

day, whereas it required 7 days with rabbit back muscle.

The loss of elasticity of muscle coincided well with

decrease in the connection content.

King et al. (1981) also have demonstrated that

connection was degraded during the heating of meat. King et

al. (1981) suggested that carboxyl (aspartyl) protease may

be responsible for at least some of the degradation because

breakdown was inhibited by addition of pepstatin. King and

Harris (1982) showed that meat was significantly less tender

when proteolysis of connection was inhibited by pepstatin.

Several researchers have minimized the significance of

connection breakdown in postmortem tenderness. Suzuki et al.

(1985) investigated the influence of connection on meat

tenderization during conditioning using chicken myofibrils.

Significant differences in the content and the

electrophoretic pattern of the connection isolated were not

observed between the preparations from fresh and stored

muscle. They concluded that connection was unlikely to be

responsible for meat tenderization caused by conditioning.

Further evidence that connection contributes little to

meat toughness follows the known relationships between


tenderness and animal age (Bouton et al., 1978a,b). Fugii

et al. (1978) reported evidence for the presence in

connection of reducible crosslinks derived from lysine and

hydroxylsine aldehydes. Such crosslinks in collagen become

more resistant to heat as the animal ages (Shimokomaki et

al., 1972). Thus, it might be expected that connection would

be more stable in the muscle of older animals. However,

they observed an extensive degradation of connection in older

animals. This was attributed to increased activity of acid


Gap filaments degradation. Locker (1982) postulated

that the shortening-tenderness relationship in conditioned

beef muscle is due to gap filaments. Consequently, the

improvement in tenderness due to conditioning results from

the degradation of gap filaments. Experiments presented by

Dutson (1983) have demonstrated that the ultrastructural

appearance of gap filaments is disrupted by incubation at pH

5.4 and 37 C for 1 hr. Incubation at pH 5.4 and 2 C, at pH

7.4 and 37 C, and at pH 7.4 and 2 C also caused some

ultrastructural disruption of gap filaments. Although, less

disruption of the gap filaments occurred at 2 C or at pH

7.4, the gap filament disruption was sufficient to alter

tensile strength and to be a major component of muscle


Titin degradation. Titin is degraded in postmortem

muscle. Titin in myofibrils is rapidly broken down by

proteases. It seems that titin has an important

cytoskeketal role; therefore, degradation of titin may be

involved in postmortem decrease in muscle cell integrity and

improvement in meat tenderness and quality, but exact

relationships are not yet known (Robson and Huiatt, 1983).

Maruyama et al. (1981a) reported that titin was

degraded by several proteinases. Wang et al. (1979) have

characterized the upper titin band on SDS-PAGE gels as

titin1 (T1) and the lower titin band as titin2 (T2). They

have observed that the proteolytic degradation of titin is

always accompanied by the conversion of T1 to T2. This

finding indicates that the absence of T1 in tender muscle

occurs because proteolysis may be more prevalent in this


Ringkob et al. (1987) prepared myofibrils from bovine

psoas muscles removed from the carcass at 3 and 48 hr

postmortem and subsequently stained with monoclonal antibody

against titin. Titin antibody, in the 3 hr muscle sample,

stained two perpendicular bands per sarcomere which were

located in the I band immediately adjacent to the edges of

the A band. The staining pattern for many myofibrils

obtained at 48 hr postmortem was altered. In this case,

there were four anti-titin stained regions per sarcomere,

all located in the I band region. The results suggested

that the titin shape might be altered within the first 2

days postmortem and/or proteolysis of titin or a protein to

which it was attached occurred.

Lusby et al. (1983) investigated the fate of titin from

muscle at death and from samples stored at 2, 25 and 37 C

for 1, 3 and 7 days postmortem. Titin migrated as a closely

separated doublet of very high molecular weight (1 X 106

dalton) in myofibrils from at death muscle samples. With

increased storage time and temperature, the top band of the

titin doublet gradually disappeared. The lower doublet band

remained after 7 days storage at 2 or 25 C but disappeared

by day 3 at postmortem storage at 37 C. Thus, they

concluded that titin was degraded in postmortem muscle, and

the rate of degradation was enhanced by increases in storage

time and temperature.

Patterson and Parrish (1987) examined tough and tender

muscles for differences in myofibrillar protein degradation.

Purified myofibrils were prepared from infraspinatus

(tender) and rhomboideus (tough) muscles at 7 days

postmortem. Titin doublet was present in the tough sample,

whereas a single titin band existed in the tender sample.

Titin was degraded to a greater extent in myofibrils from

the infraspinatus than in myofibrils from the rhomboideus.

Very little nebulin was detected in either muscle.

Patterson and Parrish (1987) also detected an

unidentified protein band with an estimated molecular weight

of 240,000 dalton in tender sample and it was absent from


the tough sample. This 240,000 dalton protein may be the

cytoskeletal protein spectrin which functions in binding to

and cross linking actin filaments (Pearl et al., 1984). The

240,000 dalton protein was more prominent in tender samples.

Therefore, it might have also been the degradation product

of some higher molecular weight protein.

Nebulin degradation. Wang and Williamson (1980)

reported that nebulin was degraded more quickly in muscle

stored postmortem than was titin. Lusby et al. (1983)

demonstrated that the nebulin protein band which was evident

in the 0 day samples was faint in the 1 day sample at 2 C.

Locker and Wild (1985) reported that after aging, nebulin

was replaced by a triplet. They concluded the slowest band

that tended to disappear with time was probably nebulin


Nebulin in myofibrils is highly succeptable to

degradation by proteases including CAF. Thus, degradation

of nebulin by endogenous proteases may be involved in the

postmortem decrease in muscle integrity. However, the exact

relationships remain unknown (Robson and Huiatt, 1983).

Electrical Stimulation

Electrical stimulation involves the passage of

electricity through the carcass at some point in the

slaughter-dressing process. Electrical stimulation enhances

beef carcass lean quality and meat palatability traits


(Savell, 1982; West, 1982). McKeith et al. (1980) studied

the effect of electrical stimulation of carcasses from

steers and cows. Electrically stimulated carcasses and

sides had brighter, more youthful colored lean, less "heat

ring" and produced more palatable rib steaks than did


The meat from electrically stimulated carcasses was on

the average 10 to 30 percent more tender than nonstimulated

ones depending on species, grade and the nutritional status

of the animal. Rashid et al. (1983a) found about 11 percent

increase in tenderness in lamb meat that was electrically


Various muscles from a carcass tenderize to a different

degree by electrical stimulation. George et al. (1980)

indicated that electrical stimulation is most effective in

improving tenderness of the longissimus dorsi muscle. It is

partially effective in improving the tenderness of some of

the muscles of the sirloin and loin and relatively

ineffective in improving the tenderness of almost all

muscles of the chuck.

Calkins et al. (1983) studied the effect of electrical

stimulation on carcasses from steers. Nonstimulated sides

had the highest pH, lowest temperature, and produced steaks

that had the least sensory ratings compared to electrically

stimulated sides. Rashid et al. (1983b) reported that

electrically stimulated and slowly chilled lamb sides

exhibited more rapid pH decline, less cold shortening and

greater tenderness in longissimus dorsi and semitendinosus

muscles than sides chilled at 2 C.

Effects of Electrical Stimulation on Postrigor Muscle


Palatability ratings for tenderness of steaks from

electrically stimulated carcasses according to Savell (1979)

were improved by 21 percent when compared to ratings for

steaks from nonstimulated carcasses. In addition, flavor

scores for steaks from electrically stimulated carcasses

were improved about 10 percent when compared to the

controls. Calkins et al. (1982) attributed this difference

in flavor of electrically stimulated meat to differences in

the concentrations of adenine nucleosides and their


Effect of electrical stimulation on shear force

Pierce (1977) showed that steaks from electrically

stimulated sides of beef had significantly lower objective

shear force values and preferred ranking by panelists in

comparison to control steaks. George et al. (1980)

determined the influence of temperature/pH history on the

rate of tenderization of longissimus dorsi muscle from the

activation energy (61.5 KJ/mole) at different temperatures

during aging of meat. The shear force decay rate was higher


in stimulated than in nonstimulated carcasses (4.0 versus

2.3 kg/day).

Thaw and cook losses

Savell et al. (1978a) and Riley et al. (1980) observed

lower thaw losses in meat from electrically stimulated than

from nonstimulated meat. However, Savell et al. (1978b)

noted a higher cooking loss from electrically stimulated

meat. Elgasim et al. (1981) observed less cooking loss in

meat from electrically stimulated beef carcasses than from

nonstimulated controls.

Effects of electrical stimulation on water holding capacity

Electrical stimulation did not affect the amount of

drip in the package (Morgan, 1979). Bendall (1976) also

remarked that there are no appreciable differences between

muscles from control and electrically stimulated sides

either in the bag drip or the extracellular space. However,

the bag drip of the deep muscles of the round from the

stimulated carcasses tended to be slightly higher than that

from the controls.

Hamm (1977) reported that two thirds of the decrease in

water holding capacity (WHC) of postmortem muscle was

associated with the reduction in ATP content. Honikel et

al. (1981) indicated that neither shortening nor the

development of rigor had an immediate influence on WHC of


muscle. The small decrease in WHC was due only to pH fall

and independent of temperature. However, they demonstrated

that as much as two thirds decrease in WHC of salted meat

occurred due to rigor development and the remaining one

third was caused by decrease in pH.

Applications of Electrical Stimulation

Hot boning

Electrical stimulation accelerated postmortem

glycolysis in hot boned muscles, resulting in relatively low

pH values at high temperatures. Thus, the possibility of

cold induced prerigor excision toughening is alleviated

(Shivas et al., 1985). Lyon et al. (1983) reported that

electrically stimulated hot boned steaks had equal or

smaller shear force values than the control. The controls

did not receive electrical stimulation and were chilled at 2

to 4 C for 48 hr.

Meat grade improvement

Improvement in tenderness by electrical stimulation is

of greatest benefit on subpopulation of cattle that have an

inherent tendency to have tough meat if not treated with

some form of postmortem conditioning (Savell, 1982).

Electrically stimulated, forage fed steer carcasses had

lower shear values than their nonstimulated counter parts

for up to 14 days postmortem. However, the electrically


stimulated, grain fed steer carcasses had more desirable

tenderness and lower shear values than the controls for only

8 days postmortem (Savell et al., 1981).

Carcasses of bulls have a coarse texture and dark lean

color. Cooked steaks from bulls are often less tender than

those from steers. Therefore, electrical stimulation has

the potential for enhancing the marketability of beef from

young bulls. Savell et al. (1982) reported more youthful

lean color, firmer textured lean, higher marbling scores and

higher quality grades for electrically stimulated sides than

control from bulls.

Hopkinson et al. (1985) investigated the effect of

electrical stimulation of carcasses from bulls and steers.

Tenderness of steaks was improved (P<0.01) by castration of

bulls, electrical stimulation of the carcasses and aging of

steaks. Effects of electrical stimulation on tenderness

were most marked in the nonaged samples. Bull steaks that

had been electrically stimulated and aged were comparable in

tenderness to aged steer steaks.

Stiffler et al. (1986) studied the effect of electrical

stimulation on Charolais crossbred bulls and steers of

similar background. Electrical stimulation reduced lean

maturity scores for bulls and steers. Electrical

stimulation was more effective in improving the tenderness

of steaks from bulls than those from steers.


Effects of Electrical Stimulation on Biochemistry of Muscle

Acceleration of postmortem glycolysis

Electrical stimulation accelerates the rate of

postmortem glycolysis in muscles to replenish the ATP which

is rapidly catabolized during the stimulation process

(Bowling et al., 1978). This amounts to an increase in the

biochemical reaction of the glycolytic pathway by 100 to 150

times due to mechanical activity of muscles. With the

acceleration of postmortem glycolysis, a rapid build up of

lactic acid occurs. In some cases, the pH of electrically

stimulated muscle can reach 6.0 in several hours instead of

the 12 to 16 hr that might be required for nonstimulated

muscles (Carse, 1973).

Hastening of rigor mortis

The overall process hastens the development of rigor

mortis. When all the potential sources of ATP are

exhausted, the ATP level is reduced to below 0.1 mol/g and

the extensibility of muscle is lost. Thus, the muscle locks

into rigor mortis at a much earlier postmortem time because

there is not enough ATP to break actomyosin bonds (Chrystall

and Devine, 1978).


Effects on the OrQanelles of Muscle Syncytia

Sarcoplasmic reticulum

Tume (1980) has shown that electrical stimulation

affected the Ca+2 dependent ATPase activity. This enzyme is

located in the phospholipid vesicles of the sarcoplasmic

reticulum and is responsible for driving Ca+2 ions pumped

across the membrane. Although the conformation and activity

of Ca+2 dependent ATPase was altered by electrical

stimulation, no change in the Ca+2 ion transport ability was



Dutson et al. (1980) reported an increase in free

activity of muscle lysosomal enzymes caused by electrical

stimulation of the ovine carcass. They suggested that the

lysosomal membrane is ruptured much earlier in muscle from

electrically stimulated carcasses than from nonstimulated


The Ca ion binding ability of the sarcoplasmic

reticulum starts decreasing with postmortem time. This is

possibly due to influence of lactic acid accumulation. The

low pH may be instrumental in releasing the cathepsins from

lysosomes when muscle temperature is still high. The

catheptic enzymes, in turn, may be impairing the ability of

sarcoplasmic reticulum. The whole system seems to operate

like a "feed back" mechanism (Asghar and Henrickson, 1982).


Will et al. (1980) reported the presence of swollen

mitochondria and T tubules in muscles from electrically

stimulated beef carcasses. Mitochondria swell in muscles

that show the most change in glycolytic rate on stimulation,

i.e., m. cutaneous trunci, m. longissimus and m.

sternomandibularis. However, they do not swell markedly in

muscles such as the m. masseter (Chrystall and Devine,


Mechanisms of Tenderization

Mechanisms by which electrical stimulation improves

tenderness are as follows: (1) cold shortening reduction;

(2) increased activity of acid protease; and, (3) physical

disruption of myofibrils (Cross, 1979).

Cold shortening reduction

Cold shortening is the shortening of prerigor muscle

due to stimulus of cold which results in a two to three fold

increase in toughness. Calcium ions and ATP contents are

the major factors which govern cold shortening of muscle.

The postmortem release of Ca ions from the sarcoplasmic

reticulum and/or from mitochondria at the time when the ATP

level in muscle is still high results in a significant level

of cold shortening (Cornforth et al., 1980).


The extent of cold shortening depends of the type of

muscle fiber. The cold shortening is directly associated

with the relative amount of mitochondria and inversely with

the amount of sarcoplasmic reticulum. Buege and Marsh

(1975) advanced the theory that the mitochondria release an

excessive amount of Ca ions at low temperatures and thus

overload the sarcoplasmic reticulum. Consequently, the

overload of free Ca ions initiates cold shortening. This

view was strengthened by the work of Cornforth et al. (1980)

who showed that the release of mitochondrial Ca ions flood

the saturated sarcoplasmic reticulum and initiates the


A minor amount of cold shortening will occur if the Ca

ions are released after some depletion of ATP from muscle

has taken place. The shortening in electrically stimulated

muscle is about 15 percent less than that in the controls

(Rashid et al., 1983a). The determination of sarcomere

length has been widely used to evaluate the degree of cold

shortening in muscles.

Muscles removed from electrically stimulated sides at

1 to 2 hr postmortem were found to have markedly longer

sarcomeres than those from nonstimulated sides. However,

muscle removed at 22 hr postmortem from stimulated and

control sides exhibit no difference in sarcomere length

(Bouton et al., 1978a). However, Will et al. (1980)

reported that sarcomere lengths of the muscles were not


significantly different between control and electrically

stimulated sides. This indicated no difference in the

amount of cold shortening.

Nichols and Cross (1980) and Whiting (1980) also noted

longer sarcomeres in fibers from muscles which were excised

from stimulated carcasses and immediately frozen than

similarly treated muscle from nonstimulated sides. The

longissimus dorsi muscle from electrically stimulated intact

carcasses was found to have longer sarcomeres than did

longissimus dorsi muscle from electrically stimulated sides.

However, the latter did not differ from the control sides.

Increased protease activity

Dutson et al. (1979) indicated that a greater amount of

lysosomal enzymes had been released from the lysosomes of

the electrically stimulated samples than those from the

controls. The electrically stimulated samples had a

significantly higher percent free activity and a

significantly lower amount of sedimentable specific activity

for both beta glucuronidase and cathepsin C.

Electrical stimulation causes a rapid drop in pH while

muscle temperature is still high. Therefore, conditions are

favorable for the action of the endogenous lysosomal enzymes

in muscle. These enzymes cleave certain linkages in the


myofibrillar proteins and possibly in connective tissue, and

thus, cause tenderization (Dutson et al., 1980).

Structural changes associated with electrical stimulation

Sarcomere integrity. Savell et al. (1978b) have shown

that the myofibril fragmentation indexes of electrically

stimulated and nonstimulated carcass were identical.

McKeith et al. (1980) found no evidence of structural damage

due to electrical stimulation of intact carcasses. However,

the electrically stimulated meat was more tender than meat

from the controls. George et al. (1980) showed no

indication of gross structural damage due to electrical


Contracture bands. Light and electron micrographs of

beef muscles from electrically stimulated and nonstimulated

carcasses reveal that structural damage occurs in the muscle

fibers of electrically stimulated samples. Contracture

bands are possibly caused by physical disturbance associated

with stimulation induced contractions. They are observed

with some electrically stimulated muscle fibers (Savell et

al., 1978a).

Will et al. (1980) observed contraction bands only in

the longissimus dorsi and supraspinatus muscles but none in

the psoas major and semitendinosus. However, George et

al.(1980) stated that their electron micrographs of

electrically stimulated samples revealed protein


precipitation, not structural damage. However, Marsh et al.

(1981) stated that electrical stimulation produced its

desirable tenderization effect mainly by fiber fracture.

Fabiansson et al. (1985) followed ultrastructural

change in electrically stimulated and nonstimulated dark

cutting beef at various times postmortem. In electrically

stimulated muscle, after 2 hr, less densely occurring thin

filaments and disorganized Z disk material resembling Z band

streaming were evident. At 3 hr, the I bands were reduced

in width and the Z disks were broadened. At 4 hr, there

were contractions of some sarcomeres with concomitant

tearing of neighboring sarcomeres with an altered appearance

of the Z disks.

At 6 hr, there were signs of heavy contractions which

resulted in a very dense appearance. This was accompanied

with a new banding pattern with condensed materials in the Z

disk region and the complete disappearance of the I band

region. The nonstimulated samples looked fairly normal at

2, 3 and 4 hr postmortem. However, some minor

irregularities in the Z disk could be seen. At 6 hr the Z

disks in some muscles were broadened, the I bands were

reduced in width and part of the muscle seemed to be in

rigor (Fabiansson et al., 1985).

Various researchers have occasionally expressed doubts

on the significance of contributions of cold shortening

prevention (Will et al., 1980), lysosomal protease activity


(Marsh, 1983; Takahashi et al., 1984) and fiber disruption

(George et al., 1980; Fabiansson et al., 1985) to the

tenderization effects of electrical stimulation. Therefore,

to assess the tenderizing ability of rapid acidification

alone, unconfounded by either length changes or fracture

effects, Takahashi et al. (1984) employed a mild cooling

routine to eliminate cold shortening and a low frequency

electrical stimulation technique to prevent

supraphysiological contraction and resulting structural


The fast glycolysing, but apparently unruptured tissue

was found to be significantly tougher than that from paired

nonstimulated sides. On the other hand, 60 Hz stimulation

caused a distinct tenderizing, even though the accompanying

pH decline was much smaller than that produced by the 2 Hz

treatment. The results indicated that, under slow cooling

conditions that elicit little or no contractile response in

the musculature, electrical stimulation exerts its

tenderizing action exclusively by rupturing the tissue.

Furthermore, rapid acidification per se may actually impede

tenderization (Takahashi et al., 1984).

Takahashi et al. (1987) performed a comparative

electron microscope study of the structural changes in

bovine muscle brought about 2 Hz and 60 Hz electrical

stimulation. Apart from the frequency difference, the two

treatments were identical. The treatments consisted of 500


volts a.c. with 600 pulses (2 Hz X 300 s and 60 Hz X 10 s)

and a time of application of about 40 min postmortem.

They reported that the sarcomeres of the controls were

uniform in appearance, transverse alignment and length. The

mean sarcomere length was 1.68 Am with extremes of 1.62 and

1.79 Am. The structure of tissues from low frequency (LF)

sides resembled that of the control, with no traces of

damage, nonalignment or uneven sarcomeres. The only noted

difference was that occasionally Z lines were more diffused

in LF tissue. The mean sarcomere length was 1.63 pm with

extremes of 1.51 and 1.76 Am (Takahashi et al., 1987).

The normal frequency (NF) samples exhibited an extreme

state of supercontraction of some sarcomeres while others

were stretched. The transition from a highly shortened

state to an unshortened and even a stretched condition was

often seen over a distance of only three or four sarcomeres.

Regardless of the state of contraction, transversely

adjacent units remained quite strictly in register. The

mean sarcomere length was 1.17 + 0.54 Am and extremes of

0.31 and 2.77 Mm (Takahashi et al., 1987).

The great difference in microstructural response

between the two treatments was explained by lack of

sufficient time between stimuli for relaxation to occur in

NF treatment. Thus, each successive contraction was added

to those preceding it to give a supraphysiological tetanus.

The 2 Hz current by contrast, permitted ample time between


stimuli for complete relaxation to take place. Therefore,

no cumulative effect could occur to provoke a tetanic

response. Thus, they concluded that fiber disruption

contributed significantly to the tenderizing effect of high

voltage electrical stimulation (Takahashi et al., 1987).

Effects of Electrical Stimulation on Myofibrillar Proteins

Effect on myofibrillar ATPase activity

Kang et al. (1983) studied changes in biochemical

properties of myofibrillar proteins of rabbit muscle which

had been stimulated soon after slaughter and stored at 0 C

for 7 days. Acto heavy meromyosin complex was also

reconstituted from actin and heavy meromyosin (HMM) which

had been prepared from at death and postmortem muscles.

Myofibrillar ATPase activity and the ATPase activity of acto

heavy meromyosin complex decreased at first and then

increased slightly during 7 days of storage.

In addition, the change of the dissociation constant of

acto-HMM complex of electrically stimulated muscle during

postmortem storage was quite small, i.e., 1.59 X 10-4 M for

at death muscle, 1.70 X 10-4 M for muscle stored for 1 day

and 1.49 X 10-4 M for muscle stored for 7 days. This

indicated that electrical stimulation treatment minimized

the postmortem change of actin-myosin interaction (Kang et

al., 1983).


Myofibrillar protein degradation

Sonaiya et al. (1982) investigated the effect of

electrical stimulation on the semimembranosus, longissimus

dorsi and triceps brachia muscles of cow carcasses. Only

one side of each carcass was stimulated (300 V a.c., 60 Hz).

In each muscle, temperature, pH and myofibril fragmentation

index (MFI) were measured and SDS-PAGE performed at 0, 3, 6,

24, 72 and 168 hr. Electrical stimulation resulted in

higher MFI, lower pH, higher temperature and lower shear

force compared to the control. However, the time of

appearance of the 30,000 dalton protein was not affected.

Therefore, they concluded that electrical stimulation

improves tenderness by myofiber disruption.

McKeith et al. (1980) found no measurable difference in

the solubility of myofibrillar protein of muscle from

electrically stimulated and nonstimulated steer carcasses.

However, they noted a significant decrease in the proportion

of troponin T in stimulated muscles from cows but none in

the case of steers.

George et al. (1980) examined the electrophoretic

pattern of myofibrillar proteins of longissimus dorsi muscle

from electrically stimulated and nonstimulated carcasses at

different intervals of time and followed the changes in TnT

content and shear force. They did not find any relationship

between the decay of shear force and composition of TnT.

Therefore, they concluded that a large difference in shear


force between electrically stimulated and nonstimulated

carcasses at day one postmortem (8.0 kg versus 13.3 kg) may

be due to some other changes in muscle brought about by

electrical stimulation.

Seideman et al. (1982) reported that the amount of

sarcoplasmic protein, M protein, C protein and alpha actinin

in hot boned (2 week conditioned) longissimus dorsi muscle

from electrically stimulated sides was significantly higher

than that from nonstimulated cold boned (2 week conditioned)

muscle. The total amount of myofibrillar proteins, myosin,

actin, troponin T, tropomyosin and myosin light chains were

not different between the treatments.

Studies on electrical stimulation of lamb carcasses

revealed no significant effect on the solubility of

different myofibrillar protein fractions. There was no

apparent difference in the SDS-PAGE profile of the

myofibrillar proteins extracted within 24 hr postmortem from

electrically stimulated and nonstimulated carcasses. The

different treatments had no effect on the extent of

actomyosin formation (Rashid et al., 1983a,b).

At a normal chilling rate, electrical stimulation

enhanced degradation of the myofibrillar proteins, alpha

actinin and troponin T and increased the amount of 30,000

dalton protein (Salm et al., 1983). Kang et al. (1983)

detected a noticeable change in electrically stimulated

muscle. It consisted of the appearance of 30,000 dalton and

27,000 dalton components during 7 and 21 days storage of

muscle. These proteins might be degradation products of

TnT. The intensity of an unidentified band between myosin

heavy chain and actin which might be derived from myosin

heavy chain, increased during postmortem storage.

Babiker (1985) reported that electrical stimulation

when accompanied by high temperature conditioning, lowered

the extractability of myofibrillar proteins and increased

their degradation, particularly that of the myosin

components. Troponin T and myosin light chain 2 were

similarly affected. Troponin T, I and C were degraded

earlier during aging in electrically stimulated muscles

incubated at high temperatures than that in conventionally

chilled muscle.

Kasang (1984) reported that electrical stimulation

accelerated solubilization from the cytoskeleton and

subsequent breakdown of desmin. A highly insoluble 22,000

dalton protein appeared in desmin enriched extracts

concurrent to desmin breakdown in all muscle samples. This

22,000 dalton protein was not related to any myofibrillar

proteins with known Z line location.


Experimental Design

A split plot experiment was conducted to compare the

effects of treatment (electrical stimulation vs no

electrical stimulation) and postmortem storage days (0, 1, 3

and 7) on selected properties of beef from Angus bulls. The

carcass sides were utilized as the split in order to control

between animal variations. Treatment was the main plot

effect and postmortem storage day was the subplot effect.

All analyses were performed in duplicate and the experiment

was replicated seven times.

Sample Preparation

Seven purebred 12 to 18 months old Angus bulls were

utilized in this study. The left side of each carcass was

electrically stimulated (ES) within 40 minutes after

bleeding. A Koch-Briton stimulator was used to apply 500

volts for 20 X 2 sec pulses to the left side. The right

side was not electrically stimulated (NES). The sides were

placed in a cooler (-1 C) within 1 hr postmortem.


Temperature Determination

Type T copper-constantan thermocouples were placed in

the center of the longissimus dorsi muscle at the 7th lumbar

vertebra region of each side. The temperature was monitored

for 20 hours with an Esterline Angus multi-point temperature

potentiometer model MRL-25. Carcass temperature values

versus time were plotted.

Sample Collection

Longissimus dorsi muscles were allowed to age on the

carcasses and samples were excised immediately prior to

analyses. Steak samples 2.5 cm thick were removed from each

side at 0, 1, 3 and 7 days storage. The first sample which

was removed after 2 hr postmortem was obtained from the 2nd

lumbar vertebra region. At each subsequent sampling period,

a 1 cm thick slice was removed before taking the actual

sample. The cut surfaces were covered with the unused

slices and the subcutaneous fat layer to prevent surface


Determination of pH

Five gram samples were wrapped in aluminum foil and

were frozen immediately and stored in liquid nitrogen.

Ssamples were pulverized with a hammer then mixed with 25 ml

of 5 mM iodoacetic acid solution which had had the pH


adjusted to 7.0 (Dutson, 1983). Muscle pH was measured with

a combination calomel electrode on a Corning model 130 pH


Sarcomere Length Determination

Sarcomere lengths were calculated using the equations

presented by Cross et al. (1981). Meat samples were cut

into small cubes then 3 to 5 g of the tissue was homogenized

in 20 ml of cold 0.25 M sucrose solution for 20 to 30 sec.

The homogenization was performed with a Virtis "23".

A drop of the homogenate was used to prepare a slide.

The slide was placed on the stage of a laser stand (Spectra

Physics model 155 He-Ne laser). The distance between the

origin and the first order diffraction band was measured.

The sarcomere length was calculated by the use of following


0.6328 X D X [(T/D)2 + 1]1/2
Sarcomere Length, Am =

D = distance in mm from specimen to the diffraction
screen. It was set at 100 mm.

T = distance in mm from the origin to the first order
diffraction band.

0.6328 = wavelength of the He-Ne laser light.

Water Holding Capacity

A modified procedure obtained from combination of

methods describe by Tarrant and Mothersill (1977) and Katoh


(1981) was utilized to determine the water holding capacity

(WHC). Ten g of glass beads (3 mm in diameter) were poured

into a 50 ml straight walled plastic centrifuge tube. Two

Kimwipes were placed on top of the glass beads. The lean

meat samples were cut into 0.5 cm thick discs. The discs

approximately had the same diameter (2.2 cm) and weight.

The discs also had the same fiber orientation and were

devoid of any visible fat and connective tissue. They were

weighed and placed flat on top of the Kimwipes in the

centrifuge tubes. Following centrifugation at 10 C for 30

min at 300 X G (Sorvall, RC 5, centrifuge), the discs were

then removed and reweighed. The WHC was represented by the

percentage of moisture loss.

Initial wt Centrifuged wt
Percent moisture loss = X 100
Initial wt

Warner-Bratzler Shear Force Determination

Two 2.5 cm steaks from each side were removed after

three and seven days of aging, wrapped and frozen at -18 C.

The steaks were thawed at 4 C for 18 hr before being broiled

on a Farberware Open Hearth electric broiler to an internal

temperature of 50 C. The steaks were then turned over and

broiling was completed when the internal temperature reached

70 C. The cooking temperature was monitored by the

insertion of copper-constantan thermocouple in the geometric


center of each steak. The temperature was recorded with a

Leeds & Northrup, Speedomax W, potentiometer.

Thawing and cooking losses were determined by weight

differences between frozen and thawed samples and thawed and

cooked steaks, respectively. Samples were allowed to

equilibrate to room temperature (22 C) prior to shearing.

Several 1.25 cm diameter cores, parallel to the muscle

fibers, were obtained from each steak. Each core was

sheared once perpendicular to muscle fibers with a Warner-

Brtzler shear device (AMSA, 1978).

Actomyosin Extraction

Actomyosin was extracted according to the procedure

described by Deng et al. (1976) which was modified. All

steps were performed at 4 C. Samples were trimmed of any

visible fat and connective tissue before grinding. One part

ground meat was mixed with six parts Weber-Edsall solution

(0.6 M KC1, 0.03 M KHCO3 and 0.01 M K2CO3). The mixture was

stirred with gentle mixing (150 rpm) on a Cole-Palmer multi-

point magnetic stirrer-H15 (Vario Mag) for 24 hr. The

suspension was centrifuged with a Sorvall RC 5 centrifuge at

9,000 X G for 1 hr. The supernatant containing natural

actomyosin was strained through a 12.5 cm diameter Ekco

brand stainless steel strainer. The natural actomyosin was

purified by dilution and precipitation method.

The dilution and precipitation method involved the

reduction of the ionic strength of the supernatant to 0.2.

The mixture was centrifuged at 7,500 X G for 20 min. The

resulting supernatant was discarded immediately and the

precipitate containing actomyosin was saved. The

precipitate was dissolved in 1 M KC1 (2 ml/g original sample

weight) with slow stirring. The mixture was centrifuged at

9,000 X G for 1 hr. The dilution and precipitation method

was repeated for two additional times.

The final precipitate was dissolved in 1.1 M KC1, 40 mM

tris-acetate buffer, pH 7.0 (2 ml/g original sample weight).

The mixture was centrifuged at 9,000 X G for 1 hr. The

supernatant was saved for 10 days at 2 Cas stock actomyosin

solution. The protein concentrations in stock actomyosin

solutions were determined by the Biuret method (Gornall et

al., 1949; Robson et al., 1968).

Protein Determination

The Biuret method (Gornall et al., 1949; Robson et al.,

1968) was used to determine protein content throughout the

entire research. Stock bovine serum albumin (BSA) solutions

in appropriate buffer were prepared (5.0 mg/ml). The stock

BSA was diluted with buffer to obtain protein concentrations

equal to 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mg/ml.

Four ml of biuret reagent was added to each test tube


containing 1 ml of protein solution. The reaction was

carried on for 20 min at room temperature.

The absorbance was read at 540 nm in a Perkin-Elmer,

Lamda 3, UV-VIS spectrophotometer. Standard curves for each

buffer systems were prepared by plotting the absorbance

values versus BSA concentrations. A 1:10 dilution of the

experimental protein solutions were utilized in performing

the Biuret procedure. The unknown protein concentration was

determined from the standard curve in the appropriate


Protein-Protein Interaction Determination

The protein-protein interaction in actomyosin solutions

as influenced by various temperatures and pH values were

studied as outlined by Deng et al. (1976). The stock

solution of actomyosin was diluted with 0.6 M KC1, 20 mM

tris-acetate buffer (pH 5.6, 5.8 and 6.0). Protein

concentration was adjusted to 0.5 mg/ml. The protein-

protein interaction was monitored by measuring the

absorbance changes versus time.

The experiments were carried out in a Perkin-Elmer,

Lamda 3, UV-VIS double beam spectrophotometer at a

wavelength of 320 nm. The spectrophotometer was equipped

with a continuous recorder, thermojacketed cell holders and

adaptors which allowed the sample compartment to be

maintained at the desired temperatures. The temperature


within the cuvettes was regulated through the cell jacket by

circulated water from a Cole Palmer model 1268 refrigerated


A Type T copper-constantan thermocouple was placed in

the center of actomyosin solution above the cell light path.

The temperature of the actomyosin solution was monitored

with a Leeds & Northrup Speedomax W recorder. The

temperatures used to study protein-protein interaction were

30, 40, 50, 60 and 70 C.

In order to minimize the initial extent of interaction,

maximum preparation time was kept within 30 sec. This

preparation time was the time necessary for filling the

cuvettes, actuating the recorder pen and adjusting the

spectrophotometer. Activation energies for heat induced

protein-protein interaction were calculated from Arrhenius

plots. The Arrhenius plots were based on the regression

coefficient at the maximum rate of absorbance change with

respect to temperature.

Activation energies were determined from Arrhenius


k = A e-Ea/RT

k = Slope at maximum rate

A = Preexponential factor

Ea = Activation energy (Kcal)

R = Ideal gas constant (Kcal mol-1 K-1)

T = Absolute temperature (K)

Dissociation of Actomyosin by ATP

The effects of electrical stimulation and aging on the

strength of actin-myosin interaction were determined with a

procedure described by Fujimaki et al. (1965a). The

dissociation of actomyosin was achieved by the addition of

varying amount of ATP (0 to 5,000 AM) to the suspension of

actomyosin (3 mg/ml) in 0.6 M KC1, 1 mM MgCI2, 0.02 M tris-

acetate buffer, pH 7.0.

This suspension was then centrifuged with a Beckman L5-

50B preparative ultracentrifuge at 100,000 X G for 3 hr at 2

C. The protein remaining in the supernatant was assumed to

be that dissociated by the added ATP. The protein

concentration in the supernatant was determined by the

biuret method.

Sulfhvdryl Groups Determination

Sulfhydryl groups were determined by the methods

outlined by Sedlak and Lindsay (1968) and Liu et al. (1982).

Two ml actomyosin solution (0.5 mg/ml) in 0.6 M KC1, 0.2 M

tris buffer, pH 8.0 was transferred to a test tube

containing 5,5-dithio-bis-2-nitrobenzoic acid (DTNB). The

volume of the mixture was brought up to 10 ml with 7.9 ml of

0.5 percent sodium dodecyl sulfate (SDS).

A reagent blank (without sample) and a sample blank

without DTNB were also prepared. The tubes were allowed to

react for 30 min with occasional shaking. The mixture was

filtered twice though Whatman No. 3 filter paper. The

absorbance readings were measured against appropriate blanks

at 412 nm. The calculations were made using the extinction

coefficient of 1.36 X 104 X M-1 cm-1.

Determination of ATPase Activity

The Mg+2 and Ca+2 ATPase activities of actomyosin

solutions were determined in 0.02 M KC1, 20mM Tris-acetate

buffer, pH 7.2 containing either 1mM MgC12 or ImM CaC12

(Wolfe and Samejima, 1976). Protein concentrations were

adjusted to 1 mg/ml. The assays were performed in the

presence of 2mM ATP at 25 C. The reactions were terminated

after 3 min by the addition of 50 percent TCA. Final

concentration of TCA in each test tube was 15 percent. Test

tubes were centrifuged at 14,000 X G at 15 C for 15 min in a

Sorvall RC5 centrifuge. Concentrations of orthophosphate in

the supernatant were determined by the procedure described

by Ames and Dubin (1960). A fresh mixture of ascorbate-

molybdate was prepared by combining one part of 10 percent

ascorbic acid and six parts of ammonium molybdate in 1 M

H2SO4 prior to the start of each assay. Seven parts of the

assay reagent was incubated with three parts of the

supernatant for 2 min. The absorbance was measured at 820

nm in a Perkin-Elmer, Lamda 3 UV-VIS spectrometer.


Titin and Nebulin Detection

Muscle myofibrils were prepared in order to study the

effects of electrical stimulation and aging on titin and

nebulin (Wang, 1982). All steps were carried out at 2 C.

Minced muscle (1 cm3 cubes) devoid of any visible fat and

connective tissue was homogenized with pyrophosphate

relaxing buffer (PRB). The homogenization was performed in

a blender at medium-high speed for three 15-sec bursts with

1 min rest intervals.

The PRB buffer was composed of low salt buffer (LSB)

supplemented with 2 mM sodium pyrophosphate. The LSB buffer

consisted of 0.1 M KC1, 2 mM MgC12, 2 mM EGTA, 10 mM tris-

maleate, 0.5 mM dithiotheritol, 0.1 mM phenylmethylsulfonyl

fluoride, pH 6.8. The PRB homogenate was centrifuged with a

Sorvall RC 5 centrifuge at 2,000 X G for 15 min.

The supernatant was decanted and the precipitate was

thoroughly resuspended in a minimum volume of LSB with a

plastic spatula. The suspension was poured through a 12.5

cm diameter Ekco brand stainless steel strainer into a large

beaker and the volume was brought up to eight times the

muscle weight with LSB. The suspension was centrifuged at

2,000 X G for 15 min. The washing steps were repeated


The precipitate was resuspended in triton 100-X buffer

(8 mg/g wet muscle) and stirred for 10 min. This buffer was

made of LSB supplemented with 0.5 percent (W/V) triton