EFFECTS OF ELECTRICAL STIMULATION AND POSTMORTEM
AGING ON SELECTED PROPERTIES OF MYOFIBRILLAR
PROTEINS AS RELATED TO TENDERNESS
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
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
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.
TABLE OF CONTENTS
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
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
Ultrastructural Studies.......................... 183
SUMMARY AND CONCLUSIONS............................... 200
BIOGRAPHICAL SKETCH................................... 226
LIST OF TABLES
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
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
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
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
LIST OF FIGURES
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
EFFECTS OF ELECTRICAL STIMULATION AND POSTMORTEM AGING
ON SELECTED PROPERTIES OF MYOFIBRILLAR
PROTEINS AS RELATED TO TENDERNESS
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
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
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
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
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
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 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
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
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
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 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
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 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 (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
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,
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).
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
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
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
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
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
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.
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
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).
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
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 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
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
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
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
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
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
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
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
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.
MATERIALS AND METHODS
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.
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.
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.
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
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
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 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).
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
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