i!LAS:Y"LE E'A ATPase OF THE ,AIZ SCUTSLL .M
IHETJIA LEE WHEELER
A DISSEiRTATION PRESENTED TO THE GRADUATE COU'JCII. OF
THE UNIVERSITY OF FLORIDA
I N P-ARTIAL FULFPI L],MENT OF TIIE REQUI.REtEN S 'FO TiE'
,DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
ACKN OWLCDGEME;: 'ES
I want to thank Dr. T. E. Hunphr eys for his guidance,
encouragement, and friendship throughout all phases of this
I als. c want to thank the other members of my supervi.scry.
committee: Dr. R. H. Biggs, Dr. G. Bowes, Dr. M. Orif.fith,
snLQ DPr. H. Aldrich.
I want to express special thanks to Dr. H. Bli-oi.:! aind
Dr. V. Gracen for their graciousu Essistance with the electron
r.icrcoscopy pfhasc of the resee.rch.
'iABLE OF CONTENTS
ACKNCO '.LEDGEMENTS i
LIST O' TABLES . .
LIST OF FIGURES .* vi
LIST 0T SYMBOLS v
TnHTODUCTION . * 1
LITEHATURE REVIEW 2
The Role of ATFases in Membrane Transport . . 2
ATiases in Whole Cells or Tissues . . . 5
ATPases from Plant Cellular Fractions . . 9
Bacterial Membrane ATPases . . . . 15
Animal Cell ATPases 19
Chloroplast Membrane ATPase 22
Mitochondrial ATPase . . . 4
Effect of DNP on ATPase 27
Inhibitors of ATPase 23
RESULTS . B.35
Cell-surface Phosphatase Activity of Fresh
Scutellum Slices .. . .. 35
Phosphatase Activity of Frozen Scutellum Slicer 38
Substrate Specificity 45
Stoichiometry of the Phosphatase Reaction
w~th ATP as the Substrate . ...
? ';sv!e Kinetics . . . 5
Ef:'Fcti of Divalent Cations on the Phosphatase
Activity ... i3
E['f-ct of Na* and -iC on the Phosphatase
Activity ........ . 62
1iTaot of DHP on the Phosphatase Activity 66
Cyto.!ogical Studies 70
ijt ochondrial ATPase 73
DISCUSSION . .
Fresh Scutellum Slices .
Frozen Scutellum Slices .
Effect of DNP .
Enzyme Localization .
EXPERIMENTAL . . .
Preparation of the Scutellim Slices
HC1- or H20-Treatment
Analysis of Pi .
ATP Analysis .
ADP and AMP Analysis .
Preparation of Na+- and K+-free ATP
Total Nitrogen and Phosphorus .
ATP Product Localization . .
Phosphotungstic-Chromic Oxide Stain for
Plasmalemma of Frozen S-utellum Slices
BIOGRAPHICAL SKETCH .
S . 86
S . 88
LIST OF TABLES
1. Effect of H20 or HC3 treatment on phosphatase
activity of frozen-thawed scutellurr slices . 39
2. Pi leakage and uptake 43
3. Total Pi, nitrogen, and lipid 44
4. Substrate preference S8
5. Stoichionetry with fresh and frozen HC1-treated
tissue . . . 52
6. Effect of Na+ and K+ on phosphatase activity . 6
7. Inhibitors . 69
LIST OF FIGURES
1. Phosphatase activity of fresh scutellum slices 37
2. Phosphatase activity of frozen scutellum slices .41
3. Phosphotungstic acid stain for plant plasmalemma .47
4. Time-course stoichiometry with fresh and frozen
HCl-treated tissue 51
5. Pi formation in fresh and frozen scutellum slices 55
6. Rate of phosphatase activity with increasing
ATP concentration 57
7. Kinetics studies with ATP and ADP . 59
8. Effect of Mg2+ on phosphatase activity . 61
9. Effect of divalent cations on phosphatase
activity . . 64
10. Effect of DNP on phosphatase activity of fresh
scutellum slices . 68
11. Electron micrograph of ATPase localization in
fresh scutellum slices . 72
LIST 0" SYMBOLS
?DP adenosine 5'--diphosphate
AMP adenosine 5'-monophosphate
AMP--PNP adenylyl imidodipihsphate
ATP adenosine 5'-Triphosphate
CDP cytidine 5'-diphosphate
CFl cytidine 5'-monophosphate
CTP cytidine 5'-triphosphate
DCCD N-N '-dicyclohexylcarb odiimide
DES diethyl stilbesterol
DNP 2, 4-dinitrophenol
EDCD l-ethyl-3 (3-dimethyl-amino propyl)-carbodlinide
SMP guanosine 5'-monophosphate
GTP guanosine 5'-triphosphate
IAA indole-3-acetic acid
MES 2 (N-morpholino) ethane sulfonic acid
HIOPS rorpholinopropane sulfonic acid
NEM n-ethyl maleimide
PCM!, p-chloromercuribenzoic acid
PCIMS? p-chJ oromercurinhenyl sulfonic acid
PEP phopphoenol pyriivate
TNPP pn-nitrophenyl phosphate
TMP thymiidine 5 '-monophosphate
TPT triphenyltin chloride
TT'P thymidine 5 --t-riphosp3hate
UDP uridine 5'--diphosphate
UDPG uridine 5'-diphosphoglucose
UTP uridine 5'-triphosphate
Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
PLASMALEMMA AT'Pase 0? THE IMAIZE SCUTELLUM
IHeijia Lee Wheeler
Chairman: Thomas E. Humphreys
Major Department: Botany
An ATPase in the plasmalemma of the maize scutollumn cell
was characterized. This enzyme had a pH optimum of 6.5, was
stimuldated by Mn2+ > i g2+ > Ca2+ > Co2+, and was further
stimulated by Na' and K'. The Km for ATP was 0.65 mAM. Nu-
cl side 5'-triphosphate was the preferred substrate.
This enzyme was capable of recognizing the substrate
from the outside of the cell, and the products of the reac-
tion remained in the bathing solution. It was stimulated by
DNP at pH's below 5.0.
The inhibitors DCCD (10 uM), PCMiB (0.5 vM) and NEM
(10 ,!M) ini'bited the enzyme from 0-20%, and PCMBS (5 pM) and
ET)C' (I10 iM) gave 32% and. 33 inhibition respectively.
Fresh and frozen ]ICf-treated slices gave nearly identi-
cal values fcr K,. pH optimra, stoichiometry, cation stimula-
tion, kinctics, inhibition. and substrate specificity.
Cytolgi cal Inc.lizatior. showed clear ATPase activity in
the pla nalemma.
:Ther are well-characterized ATPases dE.C. 126.96.36.199) in.
bacteria , mitochondria , chloroplasts , and animal
cels [3,1,5. Higher plant plasmalemma ATPases have also
been r reported [6,7,8,9], but they have not been as well char-
Plant plasmalemma ATPases have been implicated inr the
transport of various mineral ions, ariino acids and sugars
into the cell [10,11.,12,13,14]. Studies with higher plant
plasmalemma ATPaces have been done primarily with nartfa.lly
purified membrane fractions [6,7,8,9]. and do not necessar-
ily give much information about how the enzymes behave in
situ. Since ATPases are thought to be vectorial enzymes,
their biological role cannot be clearly understood from
studies with cell fractions or isolated membrane vesiclesp.
Attempts to isolate intact right-side-out vesicles by osmot-
ic lysis of spheroplasts have resulted in vehicles which
aVe not comparable to intact cell plasmalemr.a in the orien-
tcijon cf certain plasmalenmma-associated enzymes [15,16].
This investigation was undertaken to determine the
existence and location of an ATPase cn the surface of the
ntact maize scutellumn coll, and to determine some of the
.r':.acteristics of the enzyme.
The Role of ATPases in Membrane Transport
The properties and functional characteristics of ATPases
from animal cells and various subcellular organelles such as
mitochondria and chloroplasts are well known . There is
al-o a large amount of information about bacterial-membrane
Mitchell  proposed the "chemiosmotic hypothesis" as
a model in which ATP production is linked to oxicative phos-
phorylatilon in the mitochondria. The model proposes an en-
zyme (ATPase) situated in the inner mitochondrial mem'brene
with two functional portions. The catalytic portion is shown
as protruding from the membrane into the matrix and is pro-
posed to be responsible for ATP production. The other por-
ticn of the enzyme is embedded in the membrane and is pro-
posed to function as a proton pump. Such a pump would be
ariven by the energy from oxidative phosphorylationand thus
create the potential gradient across the inner mitochondrial
rnemi'- ane. This gradient has been implicated in the transport
of various mineral ions, amino acids and sugars [10,11,12,13,
Giaquinta  suggested that the uptake of sucrose from
the apoplast into the phloem sieve tube element was coupled
to o'i active influx of protons, and the efflux of K'. The
phiioeo, content had a relatively high K+ concentration and a
low1 H0 concentration (pH 8-8.5), and a high sucrose concen-
tration. If the apoplast pHl was between 5 and 5.5, a sub-
stantial electrochemical potential could exist across the
phloem plasmalemma, which could then be coupled to sugar
Proton uptake in yeast cells was studied at pE 4.5 to
5.5 [It]. There was good evidence that certain amino acids
(glycine, leucine, lysine, and phenylalanine) were co-trans-
portedi with the H+ into the cells.
West and Mitchell  found that the rate of lactose
inflow into E. coli cells which were kept in an anaerobic,
nonmetabolizing state was correlated with the rate of simul-
taneous effective inflow of Hn. They postulated that the
lactose and H+ pass together via a 6-galactoside symporter,
and that the pH difference that is consequently built up
across the membrane causes an effective outward leakage of
H+. The two transport processes, therefore, seemed to be
strictly coupled with a 1:1 stoichiometry initially, but
then there was a progressive drop in the observed ratio of
effective net H+ translocation to lactose translocation with
time. Since the cells were obligate aerobes, the conditions
of their growth prevented the production of the needed ATP.
There had to be a source of ATP within the cell to pump out
the H+ as they entered in order to keep up the initial stoi-
Harold et al.  measured the internal pH of Strepto-
coccus faecalis, undergoing glycolysis. Tney found that
those cells maintained an internal pH which was more alkal.ne
.han that of the medium by 0.5 to 1.0 pH units. This pH dif-
ference disappeared upon exha;ustion of glucose. The pH gra-
dient could also be abolished by DCCD and chlorhexidine. It
was inhibited by tetrachlorosalicylanilide, carbonyl-cyanide-
r.-chlo.ophenylhydrazone, nigericin, and monensin, which are
all proton conductors. The antibiotics, valinonycin and mon-
eatin, which facilitated the K" diffusion did not affect t!e
r.-i: gradient as long as the external K+ concentration was kept
high. At low K0 concentrations, these antibiotics lowered
the internal pH. They concluded that pH gradients are formed
by an energy-dependent extrusion of protons from the cell,
-hus creating a membrane potential. K+ accumulation then re-
sulted with the movement of K+ down an electrochemical gradi-
Beffagna et al.  reported work on a compound (fusi-
coccin or FC) which is a diterpene glucoside, and is active
in stimulating the H+/K+ exchange in plants, and also in-
creases in vitro K+-Mg2+ stimulation of the ATPase activity
from plasmalemma-enriched preparation of maize coleoptile and
spinach leaves. Their results were consistent with the hy-
nothesis that FC stimulated the proton extrusion and cation
uptake by being able to activate, at the cell membrane, a
H+/K+ exchange system which depends on ATP as an energy
sojrce. The evidence suggests that the plasmalemma-bound,
cation-dependent ATPase mediated the utilization of metabolic
energy for K transport.
Walker and Smith [211 measured t'.he intracellular pH in
Chara. The pH difference across the olasmra membrane arose
from an ATPase which extruded 2 protcns for each ATP hydro-
lyzed. The electric potential difference was determined by
the ATF/ADP concentrations with the external pH lower than
the internal. This electric potential difference across the
clasmalemna of Chara was thought to be to a large extent a
result of H+ extrusion.
When Streptococcus lactis was depleted of metabolic en-
ergy, a loss of transport function occurred . Active
transport of sugar could be induced in these nonmetabolizing
cells when a proton motive force was artificially generated
either by a pH gradient or by inducing membrane potential
with a valinomycin-induced K+ diffusion.
ATPases in Whole Cells or Tissues
Electron microscopy of ATPase localization in sieve
cells of Pinus showed prominent ATPase activity in the plasma
membrane at neutral pHl with ATP or UTP as substrates .
No such localization was observed with B-glycerol phosphate
as the substrate. The substrates were introduced from the
outside of the cell, yet the cells showed localization in the
Bentwood and Cronshaw  reported similar results in
phloem of' ricotiana. The plasma membrane and endoplasmic
reticulum were free of reaction product when B-glycerol phos-
phate was used as the substrate. Acid phosphatase activity
was very high on the vacuolar membrane with this substrate.
Acid pospphatase activity showed :to i.2t or Ca2+ stimulation.
Gilder and Cronshaw [21; reported ATPase localization in
the phloem of Cucurbita in the petiole and minor veins. The
enzyme activity was localized in the sieve elements, compan-
ion cells, and parenchyma cells. The activity was found at
the cells' surfaces and associated with the dispersed P--pro-
teins of mature sieve tube elements. They concluded that the
activity of the ATPase on the plasmalemma was directly relat-
ed to the ion assimilation, and those in the P-proteins to
callose deposition. They found activity in the mitochondria
and dici.yosomes and regions of the phloem cell walls. They
also looked at the distribution of ATPase activity in differ-
entiati.ng and mature phloem cells of tobacco and its rela-
tionship to phloem transport . In a later paper ,
they ran substrate specificity and inhibition studies with
fresh tobacco leaf homogenates. They found no inhibition by
NEM, but 96% inhibition with PCMB. Substrate specificity,
when expressed as a percent of ATP activity, gave 132% with
GTP, 90% with ITP, 82% with CTP, 83% with ADP, 77% with UTP
and 1% with AMP. They suggested a possible role of the ATP-
ase as the energy source for phloem transport.
Hall  reported the cytochemical localization of ATP-
sse activity in corn root cells by using the lead phosphate
precipitation procedure. The activity was associated with
the plasma membrane, nucleus, mitochcndria, golgi, vacuole,
and endoplasmic reticulum. I.Most cells examined showed high
AT'fase activity at the root surface, plasma membrane, and
pltasmloesmiata. The ATPase activity seemed especially to be
assocLated with vesicles close to the surface of the cell,
which suggested that ion transport may involve a process of
pinocytosis, using ATP as the energy source. He suggested in
another paper  that the ion transport mechanism may be by
way of invagination and uptake by the formation of small ves-
icles. Surface B-glycerol phosphatase activity had a differ-
ent localization pattern than did the ATPase. The activity
of the former was localized predominantly in the cell walls.
Biochemical studies  confirmed this finding.
In experiments with isolated barley roots, oligomycin at
5 ug/ml did not show any effect on the rate of hydrolysis of
ATP in the presence of Mg2+ and K+, indicating that specific
ATFase-iriven transport on the outer surface of the roots
must be absent, and that hydrolysis of ATP on this surface is
accomplished by nonspecific phosphatases . They were
able to show that the use of ATP as a qualitative substrate
for determining the presence of ATPase in plant cells is in-
appropriate since the phosphatase, which is widely distrib-
uted in these cells, can usually readily hydrolyze the same
spectrum of phosphoorgano substrates in which ATP is found.
Hanson and Kylin  reported a Na+-K+-activated ATPase
in sugar beet roots. Kylin and Gee  related the Na+-K+
ATPase activity to salt tolerance in Avicennia. They found
that the ATPase activity with salt stimulation which occurred
with peaks at three different Na+-K+ ratios was greatest at
pH 6.75. Salt stimulation of ATPase was found in acid as
we'l as slightly alkaline regions. In general, plant ATPases
seem to have a pH optimum of about 7.0, ard a strong require-
ment for divalent cations . There are indications that
ATPase may have many isoenzymes .
Hodges et al.  reported the absorption of inorganic
ions by plant root cells to be an energy-requiring process,
dependent on aerobic respiration with ATP as the energy
source. Ion uptake was inhibited by DNP, arsenate, and oli-
gomycir. There was a high correlation between KC1- and RbCl-
activated ATPase and K+ and Rb absorption by root tissues.
The kinetics of monovalent ion transport and monovalent ion-
stimiulated ATPase activity were similar.
FIsher and Hodges  and Kylin and Kahr  have re-
ported Ca2+- and Mg2+-activated ATPase from oat and wheat
roots. Much of the work is done with plant root tissues,
since there is a great interest in the role of the ATPase ac-
tivity in ion uptake . Sexton and Sutcliffe  looked
at ATPase activity in various tissues of the pea root. They
found a peak activity in the region of the apical meristem
vith a maximum at 1.5 mm behind the apex. The activity fell
rapidly from this point and leveled off until about 24 mm
from the tip, when it again increased in the zone of lateral
root for'matioen. On a per cell basis, activity reached maxi-
mum at about 10 to 12 mm from the apex. The ATPase activity
in the cortex appeared to be associated with the plasmalemma
cr cell wall. Such work implicated the ATPase with the role
of ion uptake in the root. Ratner and Jacoby  studied
the nonsDecific salt effect on Mg2+-dependent ATPase from
grass roots. They found KCI, NaCI, ethanolamine chloride,
and choline chloride were all stimulators of the ATPase ac-
tivity in corn root microsomal fraction. They concluded that
ther cation activation of ATPase from grass roots was rather
nonspecific and not necessarily related to the capability of
the roots to absorb cations.
ATPases from Plant Cellular Fractions
A common technique in work dealing with plant enzymes is
to quick-freeze the tissue, perchloric-acid extract the pro-
tein, and neutralize with KOII. Such a procedure yielded,
from several plant tissues, an acid-resistant ATP-hydrolyzing
enzyme with a pH optimum at about 5 and no activity below 3
or above 9 . This enzyme did not hydrolyze sugar phos-
phates, nucleoside monophosphates, PEP, or UDPG.
Another approach in the characterization of plant ATP-
ases has been to homogenize the tissue, separate the various
fractions with differential centrifugation, and test each
fraction for enzyme activity . Leonard et al.  have
identified at least five membrane-associated ATPases, as well
as a soluble ATPase which was most likely a nonspecific acid
phosphatase. They found a plasmalemma fraction with an ATP-
ase which was more effective at pH 6.0 than at 9.0, and was
KCl-sensitive. The mitochondrial fraction ATPase had greater
activity at pH 9.0.
Jolliot et al.  looked at the three ATPases associ-
ated with membrane fractions from potato tubers: the plasma
membrane, the mitochondrial membrane and the microsomal mem-
brane. The microsomal ATPase seemed to be insensitive to the
presence of' Nar and K+. Otheti workers [41 ,42,4 3,44], however,
found that the microsomal fraction ATPase was indeed stimu-
lated by Na4 and K+. Jolliot et al  found that the plas-
ma-memb'rane ATPase activity was stimulated by Mg2+ at pH 6.75,
whereas rhe mitochondrial ATPase activity was stimulated by
Mg2' at pH 9.5 or above. The mitochondrial ATPase was also
found to be oligomycin-sensitive, whereas the plasma-membrane
enzyme was not. The stimulation of the mitochondrial ATPase
was about 10 times more effective when stimulated with mono-
valent cations than that of the plasma-membrane ATPase.
Kasamo and Yamaki  found that IAA promoted the ac-
tivity of Mg2+ ATPase in mung bean hypocoty1cell homogenates
enriched for plasmalemma. The IAA seemed to hind to the ATP-
ase and supply the additional protons to the cell wall. They
confirmed that Mg2+ ATPase was associated with the plasmalem-
ma, and that IAA binds to the ATPase and promotes its activ-
ity in vitro.
Tikhaya et al.  isolated a membrane fraction enriched
with plasmalemma fragments by differential centrifugation in
a sucrose density gradient. The fraction contained ATPase
ortivity which was increased by Mg2+ but not by Na+-K+ or by
Na+ and K+ alone. Addition of both Na+ and K' in the pres-
ence of Mg2+ resulted in a considerable activation, with a
maximum at a ratio of Na+/K+ = 2.0 to 2.5. Ouabain (0.1 mM)
completely inhibited the Na+-K -stimulated activity and
decreased the activity to that with Ig2+ alone. Ouabain had
no effect on the enzyme in the presence of .g2+ alone, Mg2+
and K+, or Kg2+ and Na+. The pH optimum of ouabain-inhibited
ATPase was 8.0. They see a role of the plant ATPase in the
transport of ions analogous to that in animal systems.
Lai and Thompson  were able to obtain purified plasma
membrane from bean plants. Large levels of ATPase activity
were found in the membrane preparation. The purified plasma
membrane had from 3 to 6 times the ATPase activity of the
crude homogenate on a specific activity basis. The levels of
contamination were determined by glucose-6-phosphate succin-
ate dehydrogenase and 5'-nucleosidase activities. All were
low or not detectable.
Leonard and Hotchkiss  reported the properties of an
isolated plasma-membrane ATPase from corn root. Maximum ac-
tivity was observed at pH 7.0 with Mg2+ alone and 6.5 with
Mg2+ and K+. When no ion was present, pH had no effect over
a range of 6.0 to 9.0. Other cations, such as Ca2+ and La+,
were potent inhibitors, but only in the presence of Mg2+.
Oligomycin did not inhibit the ATPase activity but DCCD was a
good inhibitor. Several cations, K+, HbF, "a+, and Co2+
were all good stimulators. The best substrate proved to be
ATP, followed by GDP and UDP. Nurminen et al. L46] reported
various enzymes located on the cell wall and plasma membrane
of baker's yeast. The wall was digested enzymatically and
the released enzymes were assayed. During the enzymatic di-
gestion, saccharases and acid phosphatases were released to
the medium. The Mg2+-ccpendent A'T'Pase was not. It appeared,
therefore, that the Mg2+-dependent ATPase was bound to the
Hendrix and Kennedy  reported an ATPase activity in
plamsma-raembrane-enriched fraction of soybean root and callus
tissue. The enzyme from both sources was activated by diva-
lent caicons, Mg2+, > Mn2+, > Zn2+, > Ca2+, > Sr2, a fur-
ther stimulated by monovalent salts, K+, > Rb+, > Cl-, > Na ,
> Li', > NH4-, > Cs+, > Tris. No synergistic effects between
Na- and K1 were seen. The pH optimum for ATP hydrolysis was
6.5 and the substrate preference was ATP >> ADP > GTP > CTP >
Hodges and Leonard  reported ATPase activity in iso-
lated plant root plasmalemma, which was Mg2+-dependent, and
was further stimulated by monovalent cations. The enzyme did
not require the simultaneous presence of Na" and K+ for maxi-
mrum activity and was not inhibited by ouabain. They felt
that the monovalent ion stimulations of the ATPase were as-
sociated with the plasma-membrane fraction. Since there were
many membrane-associated ATPases, the separation and purifi-
cation procedures were difficult. The authors used discon-
tinuous density gradients to separate the various membrane
fractions. They verified the plasma-membrane fraction by
staining with phosphotungstic acid stain and found that 75%
of the membrane in this fraction was plasma membrane.
Scarborough  reported the presence of a plasma-mem-
brane ATPase in Neurospora. He used thiocyanate, which
penetrates biomrembranes and becomes asymmetrically iistribu-
ted across the membrane in the presence of a membrane poten-
tia. difference. The uptake of Mg2+--ATPase-dependent 14C-SCN-
was used as a measure of ATP-hydrolysis-dependent generation
of an electrical potential across isolated plasma-membrane
veSicles. He concluded that the ATPase of this plasmalernma
co-ordinated the movement of ions in one direction, thus pro-
ducing an electro-potential gradient across the membrane.
Adenosine 5'-triphosphatase containing plasma membrane
of oat roots formed vehicles in isolation . It was Mg2+
activated, but high concentrations of 4g2+ and ATP were in-
hibitory. The Km was between 0.64 and 1.24 mM, due to the
variable amounts of Mg2+ ATP complex and free ATP present.
The true substrate for the ATFase was believed to be Mg2+ ATP.
Primary roots of Zea were ground and the plasma-membrane
vehicles obtained with sucrose density gradient . They
found a K+-stimulated ATPase, with a pH optimum of 6.5. The
mitochondrial ATPase fraction had its maximum activity at
Leonard and Hodges  reported an ATPase from the
plasma membrane of oat roots. This enzyme was activated by
g2+, > 2+, > Zn2+, > Fe2+, > Ca2+, and further activated
by K The p1l optimum with Mg2+ activation was 7.5 and 6.5
for Mg2+ and K+. The Km for Mg2+ activation was 0.84 mM, and
for K' activation it was 0.72 mM at pH 6.0.
Maize root homogenates were found to contain two dis-
tinct KC1-stimulated ATPases [45,51]. One was associated
with the plasma-membrane fraction and the other with a frac-
tijn of the smooth intracellular membrane which could not be
in preparation of plasma-membrane vesicles from Avena
rcots, Sze and Hodges  fou-nd that some were closed, some
partially closed and others completely unsealed. The sealed
ones were presumably inside out as well as right side out.
These isolated membrane vesicles were found to be similar in
their response to the plasma -embrane in situ as far as pas-
sive influx and efflux of inorganic ions were concerned.
Washing excised corn roo- resulted in an increase in the
Mg2+-K+ ATPase . This was correlated with ion accumula-
tion. The increase in activity was limited to the microsomal
fraction. There seemed to be a close relation between the
increase in the ATPase activity and phosphate absorption.
Washing seems to augment or activate the membrane transport
mechanism by way of protein synthesis.
The ATPase from plasma membrane of marine diatoms re-
quired Mg2+ for basal activity and was further stimulated by
Na+ and K- . Divalent ions Mn2+ and Co2+ were able to be
partially substituted forMg2+, but Ca2+ inhibited the enzyme.
The preferred substrate was ATP. The apparent Km was 0.8 mM.
The enzyme was insensitive to ouabain, but PCMB and NEM were
Adenosine 5'-triphosphatase in plasma membrane has also
oeen implicated in the opening and closing of the guard cells
. Epidermal cell extracts of Commelina benghalensis had
two isoenyrmes of ATPase. The firn had a pH optimum of 5.5
and was activated by Ca2+. The second, with a pH optimuri of
7.5, vi.as activated by K+. The authors associated the stomat--
al movement with the activity of these enzymes, primarily by
regulating the K+ influx and efflux. The first form of the
enzyme was associated with closure and the second with the
opening of the stomata.
Cassagne et al.  reported a Na+-K+-stimulated Mg24
ATPase from the plasma membrane fraction of leek epidermal
cells. The rapid ion movement in the guard cells was associ-
ated with ATPase activity. The question of a HNa-K+ ATPase
occurring in plant membranes is not entirely settled, but
there seems to be support for its existence [6,56,57].
The presence of an ATPase in the plasma membrane does
not seem to be universal. Heinrich F58] looked at various
enzymes including ATPase in the nectaries of the Aloe plant.
He found ATPase activity in the ER, but not in the plasmalem-
ma. These cells are thought to be involved in the secretion
of sugars. He felt, therefore, that the sugar-secreting ac-
tivity was limited to the involvement of the ER in these
Bacterial Membrane ATPases
Alder and Rosen  reported that lysed membrane vesi-
cles from E. coli spheroplasts were right side out, and vari-
ous enzyme markers indicated this to be so. However, the
plasmalemma ATPase which is normally associated with only the
inner side of the membrane was found on both sides of the
vehicle in about 50-50 distribut-on. They postulated that
certain membrane proteins may be translocated from the inner
surface to the outer surface, creating a mosaic.
Rosen and McClees  reported that Ca2+ accumulation
in lysed E. coli vesicles was stimulated by ATP. These vesi-
cles were believed, therefore, to be inside out. The accumu-
lation of Ca2+ by these vesicles reflected a system which in
vivo nay be responsible for the active extrusion of Ca2+ from
Right-side-out membrane vesicles from E. coli were capa-
ble or ATP production  when the vesicles were loaded with
ADP and Pi, and an artificial proton gradient across the mem-
brane was produced with a more acid outside and a more basic
inside. The synthesis of ATP required, in addition, Mg2+
It was inhibited by DCCD and carbonyl cyanide. It was stimu-
lated by valinomycin in the presence of KC1. Altendorf and
Staehlin  obtained E. coli membranes via osmotic lysis.
This technique has been used widely to obtain right-side-out
vehicles. Such lysed vesicles, however, exhibited some 50%
to 60% of the total ATPase activity found in the whole cell
when the substrate was introduced from the outside.
Tn bacteria] membranes, inverted and right-side-out
vesicles showed Mg2+ ATPase activity and kept up a proton
gradient . In the absence of Mg2+ ATPase, little respi-
ration-driven Ca2+ transport could be observed. The results
suggested the presence of a Ca2+/H+ antiport.
When the EDTA-lysozyinm method of .embiane vesicle prepa-
ration was used, Hare et asl.  reported a 1:1 ratio of in-
verted and right-side-out vehicles.
Abra-ms and Smith  reviewed the general properties of"
bacterial membrane ATPase. There seemed to be good evidence
that the WATPase, by operating in reverse, acted as a coupling
enzyme in the oxidative phosphorylation of ADP to ATP. It
seems to mediate an ATP-driven transport of various solutes
against an electrochemical gradient under normal conditions.
The Km for ATP was found to be 2.5, 1.0, or 0.6 mM depending
on the species, and the pH optima ranged from 6.0 to 9.5.
The compound DCCD failed to inhibit solubilized enzyme, but
inhibited strongly when reattached to the membrane. The car-
bodiimide sensitivity factor was thought to reside in the
Riebeling and Jungermann  found an ATPase in the
membrane fraction from Clostridium pasteurianun. The enzyme
was Mg2+-dependent, but Co2+ and Mn2+ could replace Mg2+ in
the reaction. Ca2+ could not replace ;g2+. The effect of
Mg2+ was slightly antagonized by Ca2+. The monovalent cat-
ions Na+ and K+ had no stimulatory effect even in the pres-
ence of Mg24. The effect of ADP was inhibitory. The mem-
brane-bound ATPase was inhibited 80% by DCCD, but oligomycin,
ouabain, and sodium azide had no effect. The enzyme cculd be
solubilized by :2 M LiCI in the absence of Mg2+, and this re-
suJted in the instability of the enzyme. The ATPase -as
nearly completely released from the membrane by one washing
with 250 mM Tris HC1 at pH 7.5. The solubilized enzyme was
not stab-lo. The p1H optimum was between 7.0 and 8.5.
Kanson and Kennedy  found that ATPase from E. coli
reFrrane, purified via gel electrophoresis, was cold-labile
after release from its membrane. There was an 80% decline in
specific activity after 6 days at K. The best activators of
toe enzyme were Mg2+ and Ca?+. Purine nucleoside phosphates
were more effective than pyrinidines as substrates. The
Lineweaver-Burke plots of the enzyme activity were linear,
suggesting a single enzyme.
The use of a French press or sonication on E. coli cells
caused the membrane vesicles to be formed inside out. About
900 of the membrane vesicles formed with these techniques were
determined to be inside out . The assumption was made
that there were certain enzyme markers which determined sid-
eoness of the membrane. The ATPase was used as one such
mnrker. The author assumed that the hydrolytic portion of
the enzyme was directed internally, and that hydrolysis or
synthesis could only occur if the substrate could reach the
The ATPase from Paracoccus denitrificans  could be
solubilized from its membrane with washing in low salt con-
centrations. This ATPase resembled the coupling ATPase of
mitochondria, chloroplasts, and other bacteria. It was a
protein with a MW of about 3C3,000 and was negatively charged.
A. inhibitor protein was tightly bound to the enzyme in vivo
and could be destroyed by trypsin treatment. The ATP and ADP
were tightly bound to the enzyme and the ratio of ATP to ADP
was greater than 1.
Animal Cell ATPases
Shigekawa et al.  reported an ATPase in canine cardi-
ac sarcoplasmic reticulum which seems to be involved in Ca2+
transport. This enzyme had a Km of 0.18 mM and a pH optimum
of 6.8. It seemed to require both Ca2+ and ,g2 They pos-
tulated that Ca2+ was necessary for the enzyme substrate in-
termediate and Mg2+ was required for the decomposition of the
complex to release the enzyme and product. The concentration
of Ca2+ necessary for 1/2 maximal activation was 4.7 pM. The
same authors found  that this ATFase was further stimu-
lated by K > Na Rb > NH4+, > Cs+, > Li > Tris.
Ca2+ ATPase has been removed from sarcoplasmic reticulum
membrane, purified, and then incorporated into an artificial
lipid membrane . The enzyme so treated was then able to
catalyze ATP-dependent cation transport.
Two cell membrane fractions have been isolated from two
Erllch cell types. The two cell membrane fractions had non-
identical stimulatory responses to amino acids in their Mg2+
dependent activity to cleave ATP, despite the presence of
ouabain and the absence of Na+ and K4 . The first frac-
tion showed little Na+-K+ ATPase activity and the second
fraction showed a Na+-K+ enhancement.
ATPases from animal tissues, primarily muscle and micro-
somal fractions, were Mg2+_, Ca2+- or Na -K+-activated en-
zymes 169,70,71,72]. ATPase from sarcoplasnic reticulum [691
was Ca2+-g2+'-activated and could account for the large por-
tion of Uhe structure of the membrane itself. The ATPase
aggregate particles were about 90 A in diameter and had a MW
of about 102,000. Bovine brain microsomal fraction contained
a K'-activated ATPase which wds ouabain-sensitive . The
purified ATFase from membrane fraction of rectal glands of
the dogfish shark was stable at 0 for as many as 10 days,
wnereas chloroplast ATFase was cold-labile .
Grisham and Mildvan [731 reported on the properties of
ATPase obtained from sheep kidney medulla. They found Mn-+
and Ng2+ had the same binding site on the enzyme. The ATPase
required the presence of a divalent metal ion bound at the
active site. Their findings confirm the extrusion of protons
driven by ATP hydrolysis.
Sarcoplasmic reticulum had two distinct ATPases [71i].
The first was a Ca2+ ATPase, and the second a Na+-K+ ATrase.
The Ca2+ ATPase was an integral protein with a MW of approxi-
mately 102,000 and did not seem to have a subunit structure.
It comprised nearly 65% by weight of the total protein in the
sarcoplasmic reticulum. This enzyme hydrolyzed ATP in the
presence of Ca2+ and Kg2+. During the reaction, the enzyme
was transiently phosphorylated by the terminal phosphate
group of ATP and was Ca2+-dependent. Dephosphorylation was
This enzyme was thought to be surrounded by about 75 P-
lipid molecules. Its shape was unknown, but if it were spher-
ical it would be about C60 in diameter. ]t was thought to be
asymmetrically distributed with a greater portion in the cy-
tonlasmic leaflet. Calcium-ionATPase may also span the entire
thickness of the membrane, as does the Na-Kv ATPase .
This enzyme was involved in the transport of Ca2+ from the
sarcoplasmic reticulum to the'cytoplasm during muscle con-
traction. Under certain conditions, Ca2+-Mg2+ ATPase can
synthesize ATP from ADP and Pi.
Sodium-potassium-ion ATPase  required Na+, K+ and
Mg2+. The requirement for the Na+ was absolute, but other
monovalent cations could substitute for the K+. Its MW was
about 250,000. It was thought to be composed of 2 or more
subunits and was ouabain-sensitive. If the enzyme were
spherical, it would be about 85 A in diameter and capable of
spanning the membrane, but functional asymmetry was implied.
The ATPase from microsomal fraction of canine renal medulla
seemed to have two subunits, a large polypeptide and a small-
er polypeptide . Antibodies were produced against the
purified large chain of Na+-Kt ATPase and the antigenic site
was determined to be located on the inside surface of the
membrane. It was also found that this large chain was phos-
phorylated by MgATP from the inside.
Cardiac glycosides are known to bind to the ATPase only
at the outside surface of the cell. It was shown to bind to
a portion of the large chain of the Na+-K+ ATPase. Conse-
quently, the large chain must have had a surface exposed to
the outside of the cell and one exposed to the inside, and
therefore spanned the membrane. If it were spherical, it
would be about 75 A in diameter, and more than large enough
to span the membrane with adequate surface on each side.
Since the cardiac glycoside binding site was accessible from
the outside, and the phosphorylatiuo site from the inside,
and both sites are on the large polypeptide unit of the en-
zyme, the polypeptide was thought to have sequences exposed
to both sides of the membrane .
Although the ATPases normally function to hydrolyze the
ATP to Pi and ADP, the reaction can at times be reversed un-
der certain conditions. Lew and Glynn  reported the re-
versal of the ion pump which resulting in the synthesis rath-
er than hydrolysis of ATP in red blood cell ghosts. These
membranes were preincubated with Pi to load them up and then
incubated in a high-Na+ and K4-free medium. This led to an
increase in the ATP concentration which could be wholly or
partially prevented with ouabain. They felt that there was
strong evidence of a net synthesis of ATP as being linked
with the reversal of the pump.
Chloroplast Membrane ATPase
Ca2+ ATPase from lettuce chloroplast [771 seemed to be
composed of two parts: the portion embedded in the membrane
(CF ) and the portion protruding from the membrane (CF1).
The CF1 was also known as the coupling factor and was direct-
ly involved in the terminal steps of photophosphorylation,
and thus normally functioned in the synthesis of ATP. The
enzyme would also catalyze the hydrolysis of ATP under arti-
ficial conditions. The CF1 was shown to require heat activa-
tion and Ca2+. Other metal ions were inhibitory. The K values
for Ca2+, Mg2+ and Mn2+ stimulation were 2.7 mM, 2.0 mM and
4.0 mM respectively.
The chloroplast AITase CF7 seemed to be Mg2+-Ca2+-depen-
dent and required light .
Membrane-bound ATPase in chloroplasts of Euglena showed
a Ca2+- and Mg2+-dependent activity and could not be further
activated by other ions . This enzyme was highly specific
for purine nucleosides and was inhibited by DCCD, phlorizine,
ADP and Pi.
There were at least two distinct ATPases associated with
the chloroplast from spinach. One was membrane-bound, light-
activated, and sulfhydryl-dependent Mg2+ ATPase . The
other was a trypsin-activated Ca2+ ATPase. A chelating agent
(4,7 diphenyl-l,10-phenanthroline) was able to inhibit the
Mg2+ ATPase, but not the Ca2+ ATPase.
Certain properties of ATPase from spinach chloroplasts
were reported . It had a high specificity for ATP over
other nucleoside triphosphates and Ca2+ was the best activa-
tor. The Km at pH 6 was 0.11 mM. The enzyme was stimulated
by maleate, and the Km with 60 mM of maleate was 1.1 mM.
There seemed to be a necessity for the presence of lip-
ids (p-lipids in particular)  for the proper functioning
of the ATPase. Sulfolipids seemed to confer stability to the
ATPase activity of the chloroplast CFl, and the p-lipids were
important in the binding of CF1 to the membrane fragments,
which resulted in highly increased specific activity of the
ATPase from bacteria, mitochondria and chloroplasts
seemed to serve two major functions  and share many simi-
larities in the properties of the enzymes from the different
sources. The first was to function as a proton translocator
across the membrane. The second was to catalyze the ATP hy-
drolysis and synthesis and the regulation of these catalyses.
Malyan and Makarov  looked at the mechanism of ATP
hydrolysis by soluble ATPases of chloroplasts (CF!) in the
presence of Mg2+ ions. A linear increase in reaction rate of
ATPase with ATP concentration up to 1 mM was observed at con-
stant Mg2+ concentrations. At high concentrations of MgC12
the dependence was more complex. At MgCl2 concentrations of
less than 0.1 mM, the reaction approached 2nd-order kinetics
with respect to Mg2+. An increase in MgClp concentration re-
sulted in a decrease in reaction order. It was assumed that
MgATP is the true substrate for ATP hydrolysis in which the
enzyme binds with a free Mg2+ to obtain its active conforma-
Mitochondrial ATPases and ATPases from sub-mitochondrial
particles of Jerusalem artichoke showed a maximum activity at
pH 9.0 9.3. It had a higher specificity for ATP than other
nucleoside triphosphates. Broken mitochondria also caused
the rapid hydrolysis of ATP. The enzyme was inhibited by oli-
Some general properties of mitochondrial ATPases were
reported by Penefsky . The isolated mitochondrial ATPase
was latent and was activated by the addition of DNP, which
uncoupled the oxidative phosphorylation, or by physical al-
teration of the membrane. There was an absolute requirement
for divalent cations, Mg2+, Co2+, iCn2+, Fe2+ or Ca24. The pH
optimum was about 8.0, and they were not inhibited by sulfhy-
dryl group reagents. They were highly sensitive to iodine,
DCCD and tetranitromethane. The sensitivity to DCCD depended
on the presence in the membrane of the oligomycin-sensitivity-
conferring protein or FC1 and a second protein FC2. The en-
zyme was stimulated 15% to 20% by methanol and by bicarbonate.
The activity of mitochondrial ATPase seemed to vary
somewhat from organism to organism. The ATPase activity from
dicotyledonous plants studied  closely resembled the ATP-
ase from animal and yeast mitochondria. They were inhibited
by oligomycin and had elevated molecular weights when separa-
ted from the organelle. The mitochondria from monocotyledon-
ous plants, on the other hand, had ATFases which behaved
quite differently. Monocot mitochondrial ATPases were unaf-
fected by oligomycin. Purified ATPases from monocots were
more stable to cold and had a lower molecular weight than
that obtained from dicots. Similar observations were made by
Jung and Hanson .
There seems to be general agreer.ent that mitochondrial
ATPases from various sources have a requirement for divalent
metal ions, and Mg2+, Co2+, Mn2+, Fe2+ and Co2+ were all ef-
fective . In beef heart nitochondria, the pH optimum was
reported to be 8.0 . This ATPase was not inhibited by
sulfhydryl reagents, had a high sensitivity to iodine and
tetranitromethane, and was virtually destroyed by DCCD. Jung
and Laties  found a potato mitochondrial ATPase that
showed a sharp optimum activity at pH 10.2.
Pullmen and Monroy  described a naturally occurring
inhibitor of mitochondrial ATPase from beef heart. A similar
phenomenon was reported by Jung and Laties  in potato mi-
tochondria. They found the endogenous ATPase activity of in-
tact potato mitochondria to be quite low and not stimulated
by Mg2+. Jung and Hanson [91,92] found that in cauliflower
mitochondrial ATPase, respiratory priming (the addition of
respiratory suostrate in the presence of Mg2+ and Pi) and
self priming (prolonged incubation with Mg2+ and ATP) caused
stimulation. In potato mitochondrial ATPase, however, such
treatments had no significant effect.
When membrane barriers w-re disrupted by sonication or
with detergents, such as Triton X-100 treatment, a slightly
Mg2+-dependent ATPase-activity increase occurred in the cau-
liflower mitochondrial ATPase , but such treatment caused
a ten- to fifteenfold increase in the activity of the potato
mitochondrial ATPase. They found that trypsin treatment
could induce ATPase activity in potato mitochondrial ATPase
when 2 mM MgCl2 were added. Cold liability seemed to be re-
pressed in mitochondrial ATPases if the enzyme was integrated
in the membrane . Oligorycin inhibition was not observed
in the isolated enzyme.
Boyer et al.  postulated a high-energy coupling sys-
tem in which ATPase acts in the hydrolysis or synthesis ca-
pacity depending on the situation and the site. For example,
the normal function in muscle contraction would be hydrolysis
and in mitochondria, synthesis. The release of the ATP syn-
thesized on the mitochondrial membrane requires an energy-
dependent conformational change at the catalytic site.
Penefsky  reported that an ATP analogue, AMP-PNP,
could not be hydrolyzed by mitochondrial ATPase. He found
that AMP-FNP was a potent competitive inhibitor of soluble
membrane-bound mitochondrial ATPase and could also inhibit
other ATP-dependent reactions. However, AMP-PNP did not in-
terfere with the synthesis of ATP from ADP and Pi. The con-
clusion from the data was that the hydrolytic site of the
ATPase was not identical to the catalytic site for ATP syn-
Beef heart mitochondrial ATPase had tightly bound ATP
and ADP in both membrane-bound and isolated forms . At
one site ADP was bound, from a second ATP was lost, and at a
third ADP was phosphorylated. There were four equivalent
sites in all and they passed through each of the four states
in turn in a rotating-wheel manner.
Effect of DNP on ATPase
The inhibitor DNP increased the permeability of the mi-
cochondrial and chloroplast membranes to H+ [97,98] and had
a great effect on sugar transport in the corn scutellum .
It increased sucrose transport out of the vacuole and inhib-
ited the uptake across the plasmalemma [100,101]. At the
plasma membrane, DNP was reported to hydrolyze sucrose, which
was thought to be a result of the compound's interference
with the normal function of the disaccharide transport system
. The compound was thought to cause these changes as a
direct result of its ability to increase the permeability of
the membrane to H+. Humphreys  showed rapid pH-depen-
dent H+ influx into slices induced by DNP. The H+ influx was
very slow at pH 5, but increased rapidly as the pH was low-
ered over the range of 5 to 3.5. The influx of H+ was accom-
panied by a nearly equal K+ efflux. He concluded that the H+
influx was induced by DNP, but the H+ carrier might be an
Lambeth and Hardy  reported that DNP decreases the
activity of purified rat liver mitochondrial ATPase. Mitchell
 reported that DNP may be acting as an uncoupler of the
ATPase by specifically equilibrating the electrochemical po-
tential of H+ across the coupling membrane. He postulated
the existence of a proton pump which was separate from the
redox and ATPase system, but activated by an energy-rich in-
termediate in equilibrium with the latter systems.
Jung and Hanson  reported an oligomycin-insensitive
ATPase from cauliflower mitochondria which could be stimulat-
ed by DNP. Cauliflower mitochondrial membrane was apparently
quite leaky, and therefore the organelle could not maintain
an adequate amount of needed ions and cofactors in the matrix
during senescence or starvation. Indirect comparisons of
corn and rat-liver mitochondria did not seem quite as leaky.
It appeared that DNP caused an increase in the ATPase activi-
ty in the mitochondria isolated from Jerusalem artichokes
. This was perhaps due to the collapse of the oxidative
phosphorylation, so that the ATPase had to work faster to
maintain the potential across the membrane.
Inhibitors of ATPase
There are various inhibitors of ATPase reported in the
literature. The state of the science is, however, far from
clear. The same inhibitor which has a large inhibitory ef-
fect in one system may not affect another, even though both
are characterized as ATPases.
Partially purified spinach ATPase was found to be 80%
inhibited by 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole, and
completely reversed by dithiothreitol. This enzyme was also
inhibited by quercetin .
Abrams et al.  reported the effect of DCCD on ATP-
ase derived from Streptococcus faecalis. They looked at
wild-type and mutant forms of the bacteria in respect to sen-
sitivity of the plasma-membrane ATPase to DCCD. They found
that the sensitivity to DCCD required that the enzyme be at-
tached to the membrane. The mutants were 100 times less sen-
sitive to DCCD than the wild types, and the energized uptake
of K4 and cycloleucine was not affected in the mutant Dy DCCD.
The link between sensitivity to inhibitors and its as-
sociation with the membrane is further supported by the work
of Knowles, Guillory and Racker . They found that sen-
sitivity of the ATPase to oligomycin required the presence of
certain p-lipids. When dissociated from the membrane lipids,
the enzyme was not inhibited by oligomycin or DCCD.
Simoni and Shandell  reported a mutant E. coli
which was unable to couple energy frem ATP hydrolysis to the
active transport of proline. They found that the ATPase was
poorly attached to the membrane, and the analysis of the mem-
brane showed the absence of a single polypeptide with a MW of
about 54,000 and the appearance of a new polypeptide with a
NW of about 25,000. The oligomycin-sensitive protein in mi-
tochondria was presumed to be in the stalk. Sensitivity to
DCCD resided elsewhere. This inhibitor was effective only
when the BF1 was attached to the membrane. The ATPase of the
mutant was insensitive to DCCD from 0 to 20 pM. In wild
types, inhibition was maximal at about 5 pM. The conclusion
drawn from these data was that either the strain was defi-
cient in the DCCD-reactive protein, or the BFI was poorly at-
tached to the membrane and thus was insensitive to DCCD.
Similar results were reported with another E. coli mu-
tant with altered sensitivity to DCCD . The wild type
is highly sensitive to 50 pM of DCCD. In the mutant, as much
as 200 pM of DCCD were needed to obtain inhibition. A highly
water-soluble carbodiimide (EDCD), however, was equally ef-
fective on both the wild type and mutant in inhibiting the
ATPase activity. This seemed to indicate that the DCCD pref-
erentially entered the hydrophobic membrane phase of the mu-
tant, thereby effectively decreasing the concentration of
DCCD available for inhibition.
The coupling factor (P1) proteins from beef heart, rat
liver, and yeast mitochondria were similar and had a MW of
about 360,000 . The coupling factors from chloroplast
(CF1) were cold-labile and insensitive to DCCD when isolated
from the membrane. Structurally, CF1 is similar to the coup-
ling factors from various mitochondria.
In mitochondria, DCCD was thought to react specifically
with one polypeptide in the membrane portion of the complex
Plasma-membrane ATPase from corn root  was inhibited
by Ca2+ and La3+ in the presence of Mg2+, but not in its ab-
sence. Oligomycin was not an effective inhibitor; however,
DCCD at concentrations from 50 to 100 pM inhibited the enzyme
by 50%. Complete inhibition occurred with 200 pM of DCCD.
An interesting observation in corn root mitochondrial
ATPase was that DCCD was a good inhibitor of this enzyme when
only low levels were present (less than 50 pM) . Concen-
tration higher than 50 pM did not have an inhibitory effect
on the enzyme.
Lin et al.  isolated vacuoles from petals of Hippe-
astrum and Tulipaand found an ATPase on the tonoplast but not
in the vacuolar contents. This ATPase was not inhibited by
DCCD, but was inhibited by the water-soluble EDCD. The tono-
plast membrane exhibited twice the activity at pH 6 than at 9
and was dependent on Mg2+ with stimulation by KC1. It was
insensitive to oligomycin. They found no acid phosphatase
activity with the tonoplast membrane when PNP was used as a
substrate. They felt that the ATPa3e on the tonoplant might
be on the outer surface of the vacuolar membrane, and impli-
cated it in the transport of ions in and out of the vacuole.
The Mg2+--dependent ATPase from the envelope of spinach
chloroplast was also insensitive to DCCD .
Sapir and Pederson  reported that ATPase from rat
liver mitochondria was inhibited by 95% by 2.5 pM of oligomy-
cin and 60% with 1-8 pM of DCSD. The isolated ATPase was
less sensitive to oligomycin, venturicidin, and DCCD than was
membrane-bound ATPase. They concluded that this may indicate
the need for a conformational specificity. They also report-
ed an oligomycin-insensitive ATPase in the same system. When
oligomycin-sensitive ATPase was purified from rat liver mito-
chondria, detergent treatment decreased the enzyme's sensi-
tivity to oligomycin.
Riebeling and Jungermann  reported an 80% inhibition
of clostridial membrane-bound ATPase by DCCD, but oligomycin,
ouabain and NaN3 had no effect at concentrations ranging from
10-8 to 10-3 M.
Triphenyltin chloride (less than 1 pM) inhibited ATP
formation and coupled electron transport in isolated spinach
chloroplasts . When the ATPase was purified, the Ca2+-
Mg2+ ATPase CF1 protein was found to be insensitive to TPT.
This compound is thought to inhibit the phosphorylation ex-
change and membrane-bound ATPase in chloroplasts by specifi-
cally blocking the transport of protons through the membrane-
bound channel located in a hydrophobic region of the membrane
at or near the functional binding site for the coupling fac-
The ATPase activity of the isolated coupling factor was
insensitive to DCCD . if the ATPase was attached to the
membrane, the DCCD did inhibit the enzyme. It was thought
that DCCD did not inhibit ATP formation by interfering di-
rectly with the coupling factor protein, but rather by react-
ing with another tightly bound component within the membrane
itself. It also seemed to act as an electron transport in-
It appeared that TPT behaved similarly to DCCD  but
at much lower concentrations. It too inhibited electron
transport as well as ATP formation.
Evans concluded  that the Mg2+-Ca2+ ATPase of E.
coli is tightly integrated in the cell membrane, and that the
electrical phenomenon or conformational changes, or both,
play important roles in the functioning and regulation of
this enzyme in intact membranes.
Phlorizin, phloretin, and DES  are inhibitors of
Na -dependent sugar transport, and they inhibit Na+-K+ ATPase
activity but do not seem to affect the Hg2+ ATPase. In ani-
mal cells, the uphill sugar transport was dependent either
directly or indirectly on the activities of the Na+ pump.
This pump could be inhibited by inhibiting the Na -K AT'ase
activity or by inhibiting the metabolic generation of ATP
itself. Other common inhibitors tried were ouabain, oligo-
mycin and NEM.
The intracellular r.et abclic function was not permanently
altered by PCMBS, and there vas good evidence that the site
of inhibition was the nlasm a membrane . Another sulfhy-
dryl group inhibitor, i'EM, is known to act on the intracellu-
lar processes as well.
Phlorizin and NEM, like TPT, did not act on the coupling
factor directly, but on a component either within or closely
associated with the membrane .
Cell-surface Phosphatase Activity of Fresh Scutellum Slices
Scutellum slices were incubated in distilled water for
one hour, and then placed in a buffered ATP solution. Phos-
phatase activity of these slices was determined by measuring
Pi in the bathing solution. The effect of pH on phosphatase
activity is shown in Fig. 1. The addition of Mg2+ stimulat-
ed the activity at all pH's tested. The peak activity with-
out Mg2+ was at pH 6.5, and with Mg2+ it was at pH 5.7.
Humphreys [117,118] found that maltase activity on
fresh maize scutellum slices could be nearly eliminated by
washing the slices for 30 to 60 min in 0.0111 HCI. Such
treatment did not interfere with sucrose synthesis, cellular
respiration, or the transport of maltose or sucrose across
the plasmalemma. When fresh scutellum slices were treated
with 0.01N HC1 prior to testing for phosphatase activity,
there was a drop in the activity over the entire pH range
from 4 to 7 (Fig. 1). When Mg2+ was added, however, the HCI-
washed and H20.-washed slices showed nearly equal phosphatase
activities at oH 6.5. Acid treatment decreased phosphatase
activity at the lower pII's (Fig. 1), but appeared to have no
effect on the phosphatase having peak activity at pH 6.5 in
the presence of added Mg2+. The addition of Mg2+ to acid-
treated scutellum slices greatly stimulated the phosphatase
Figure 1. Phosphatase activity of fresh scutellum slices.
Fresh maize scutellum slices (0.5 g) were treated with
H20 or 0.01N HC1 for 1 hr with a change of bathing solu-
tion after 30 min. Magnesium chloride (20 mM) was added
for the + Mg2+ studies. Each vessel contained 50 mM MES
(pH 3.5-6.5) or 50 mM MOPS (pH 6.5-7.5) and 3 im ATP.
The experiments were run at 300 for 60 min. Each point
is an average of 5 values.
4 5 6 7
activity especially between pH 6 and 5.5 (Fig. 1).
These results indicate that the phosphataese are at the
surfaces of the scutellum cells. The activity was sensitive
to bathing-solution pH, the substrate was added to the bath-
ing solution and the product appeared in the bathing solu-
tion, the addition of Mg2+ to the bathing solution stimulated
activity and HC1 treatment destroyed part of the activity.
Phosphatase Activity of Frozen Scutellum Slices
Frozen scutellum slices (which allowed substrate access
to both sides of the plasmalemma) were thawed quickly by the
addition of either H20 or HC1 (0.01N), and then incubated at
30 for 30 or 60 min before measuring phosphatase activity.
Frozen slices that were tested without pretreatment with
either H20 or HC1 showed the highest phosphatase activity
(Table 1). Phosphatase activity was decreased 25% after the
slices were H20-treated for 30 min, and 65% after 1 hr.
Frozen HC1-treated slices showed greater decreases in enzyme
activity. After 30 min in HC1, the activity decreased from
153 pmol/g hr to 67 umol/g hr. After 1 hr in HC1, the phos-
phatase activity was only 11 umol/g hr. When ig2+ was added
to the reaction mixture, the phosphatase activity of the
frozen, HCI-treated slices rose from 11 pmol/g hr to
110 ,mol/g hr. The latterrate is nearly equal to that ob-
tained with fresh slices at the same pH (Fig. 1).
The effect of pH on phosphatase activity of frozen,
H20-treated slices is shown in Fig. 2. The activity in the
pH 4 to 6 range remained relatively high, then decreased
above pH 6. The addition of Mg2+ increased the activity
Table 1. Effect of H20 or HCI treatment on phosphatase
activity of frozen-thawed scutellum slices
Pi, ramol/g hr
30 min in H20
60 min in H2O
30 min in HC1
60 min in HCI
60 min in HCl; + Mg2+
60 min in HC1; no ATP
Note: The substrate (3 mM ATP) was added to reaction mixture
after the various pretreatments. Added Mg2+ was absent in
the reaction mixture in all but #6. In #6, 20 mM of MgC12
was added to the reaction mixture.
The reaction was allowed to proceed for 60 min at 300 in
50 mM MIES pH 6.5.
In the 60 min H20 or HC1 pretreatments, a change in bath-
ing solution was made after 30 min.
Figure 2. Phosphatase activity of frozen scutellum slices.
Frozen tissue (0.5 g) was thawed with 0.01N HC1 or H20
and treated for 1 hr with a change after 30 min. Mag-
nesium chloride (20 mM) was added for the + Mg2+ stud-
ies. Each reaction vessel contained 50 mM buffer (MES
pH 3.0-6.5 or MOPS pH 6.5-7.0) and 3 mi ATP, and was run
at 300 for 60 min. Each point is an average of 5 exper-
4 5 6 7
less than 15% in the pH range of 4 to 6. The addition of
Mg2+ shifted the peak phonphatase activity from pH 6.5 to
pH 5.9. The addition of Mg2+ to fresh H20-treated sices
also shifted the pH optimum to a lower value (Fig. 1).
The phosphatase activity of the frozen, HCl-treated
slices in the absence of added Mg2+ was low at all pH's
tested. In the presence of E g2, however, there was a sharp
rise in activity near pH 5 and a peak activity at pH 6.5.
Since Pi was measured as the indicator of phosphatase
activity, the amount of Pi leakage and uptake by fresh acid-
treated slices was measured. The results in Table 2 show
only 1 to 2 pmol of Pi change in the bathing solution after
1 hr of incubation. When frozen acid-treated slices were
tested for Pi leakage, only 1 pmol had leaked into the bath-
ing solution after 1 hr. The results were nearly identical
over the pH range of 4.5 to 8.0 (Table 2).
All the results in this study are presented in terms of
fresh weight. Totals for protein, lipid and phosphorus were
determined for 1 gm of fresh and frozen tissue to give an in-
dication of the composition of the tissues. The results are
given in Table 3. Small differences (13%) in total protein
were observed between the fresh and frozen acid-treated tis-
sues. The frozen tissue had a greater amount of extractable
lipid (38%) than the fresh tissue, and the fresh tissue had
23% more total phosphorus than the frozen.
From Table 3 it is clear that after acid treatment there
still remains a great deal of phosphorus in both fresh and
Table 2. Pi leakage and uptake
Initial Pi in bath-
ing solution, Ymol
APi in bathing
pH solution, pmol
Note: Each reaction vessel contained 0.5 g of HCl-treated
scutellum slices, 50 mM buffer (MES pH 4.5-6.5, MOPS pH 8.0),
added Pi, and 20 mM MgC12. The experiments were run for
60 min at 300.
Table 3. Total phosphorus, nitrogen, and lipid
Ethyl Ether Ex-
Tissue Total Total tractable Lipid,
issue hosphorus/g Nitrogen/g mg/g
Fresh 150.5 pmol 9.0 mg 89.2 mg
Frozen .26.0 pmol 8.0 mg 123.5 rg
Note: HCl-treated scutellum slices were analyzed for total
phosphorus, nitrogen and lipid per g of fresh weight. The
total lipid is the ethyl ether extractable lipid in the scu-
tellum slices. More lipid was extracted from the frozen
tissue, perhaps due to the disruption of the membranes dur-
frozen scutellum slices. This phosphorus may be bound in
various organic compounds such as phytic acid . These
organically bound sources of phosphorus do not seem to be
used as substrates by the phosphatase enzyme, nor do they
leak from the tissue.
Electron micrographs of the frozen, HC1-treated slices
showed few distinct organelles. There were no distinguish-
able mitochondria, ER, dictyosomes or nuclei. There were
some lipid globules, and patches of cytoplasm. When these
cells were specifically stained for plasmalemma, with the
PTA-CrOr method , many vesiculated and nonvesiculated
plasmalemma fragments were observed (Fig. 3).
From the above results, it is concluded that there is an
acid-stable, tightly bound phosphatase located at the cell
surfaces of the maize scutellum. This phosphatase is acti-
vated by Mg2+ and has a peak activity at pH 6.5. The nearly
identical results obtained with acid-treated preparations of
both fresh and frozen slices strongly suggest that the same
phosphatase was being observed in both cases.
The following sections are concerned with the properties
and location of this phosphatase.
Both fresh and frozen acid-treated preparations were
used to test various substrates (Table 4). Nucleoside mono-
phosphates were poor substrates, as were sugar phosphates,
PNPP, and P-Pi. The ADP and LCP were hydrolyzed at rates
Figure 3. Phosphotungstic acid stain for plant plasmalemma.
Sections are of frozen, HCl-treated scutellum tissue
stained with phosphotungstic-chromic acid, and showing
p]asmalemma (pl), cytoplasm (cy), and cell wall (CW).
Table 4. Substrate reference
% of ATP Activity
Note: Both fresh and frozen tissues were pretreated for 1 hr
in 0.01N HC1. Each reaction vessel contained 3 mM substrate,
20 nM MgC12, 50 nm MES pH 6.5 and 0.5 g tissue, and was run
for 1 hr at 300. Each value is an average of 4-5 experiments.
between 67% and 86% of that of ATP. The UDP, however, gave
only 5% to 7% of the ATP activity. The CTP was hydrolized
at rates equal to that of ATP. The other nucleoside triphos-
phates were hydrolyzed at rates 53% to 86% of that of ATP.
The greatest difference in phosphatase activity between
fresh and frozen tissue preparations was obtained with P-Pi.
In the frozen preparation, P-Pi gave 34% of the ATP activity,
but only 10% with the fresh preparation.
Stoichiometry of the Phosphatase Reaction with ATP as the
End point stoichiometric studies were run in triplicate
with fresh HCl-treated slices. At the end of 60 min, the
AATP was 24.2 pmol/g, APi was 44.0 vmol/g, AADP was 6.3 umol/g
and AAMP was 13.8 Pmol/g. (Each value is an average of 3
replicates.) Thus, 83% of the ATP lost from the bathing so-
lution was recovered as ADP and AMP.
Time course stoichiometric studies were done with both
fresh and frozen acid-treated slices. In the fresh prepara-
tion, as the level of ATP in the bathing solution decreased
with time, the Pi and the ADP levels increased (Fig. 4).
The ADP level reached a plateau afterabout 10 min, and re-
mained nearly constant over the remainder of the 60-min per-
iod. The Pi level, however, continued to increase. After
about 20 min, the rate of Pi production rose and then re-
mained linear. After 60 min, 46 imol of Pi were found in
the bathing solution, along with 11 pmol of ADP and 25 pmol
of ATP. There was little difference in Pi production between
fresh and frozen preparations. Initially 51 limol of ATP were
present (Table 5). These values are close to the theoretical
values. The total Pi as a result of ATP hydrolysis and the
Figure 4. Tine-course stoichiometry with fresh and frozen
HCl-treated tissue. Fresh scutellum slices 0.5 gw-ere
HCl-treated for 1 hr with a change after 30 min. The
reaction mixtures contained 20 mM MgC12, 50 mM MES
pH 6.5, and 2.5 mM ATP, and were run at 300 for 60 min.
All points are an average of 3 experiments.
20 40 60
Table 5. Stoichiometry with fresh and frozen HCl-treated
24 26 11 11
Note: HCl-treated scutellum slices (0.5 g) were placed in a
reaction mixture containing 2.5 mM ATP, 50 mM MES pH 6.5, and
20 mM MgC12, and was run at 300. Each value is an average of
hydrolysis of ADP subsequently formed should total 43 umol.
the amount measured was 16 pmol.
The results with acid-treated, frozen slices were simi-
lar to those of the fresh preparations (Table 5 and FiE. 5).
The kinetic studies for ATP were run for 10 min with
fresh and frozen acid-treated preparations. The substrate
concentration curve is shown in Fig. 6.
The Lineweaver-Burke plot of the combined data from
fresh and frozen tissue preparations is shown in Fig. 7. The
Km was calculated to be 0.65 mM.
The kinetic studies with ADP in fresh, ICl-treated
slices resulted in a Km of 5.0 and a 1/Vmax identical to that
of ATP (Fig. 7).
Effect of Divalent Cations on the Phosphatase Activity
Most ATPases require Mg2+ for activity. Magnesium ions
have been shown to combine with ATP to form MgATP, which then
acts as the true substrate for the enzyme . The phos-
phatase activity of both the fresh and the frozen HCl-treated
preparations was stimulated by the addition of Mg2+ (Figs. 1
and 2). The effect of Mg2+ concentration on phosphatase ac-
tivities of these preparations is shown in Fig. 8. The rate
of phosphatase reaction increased with increasing M.g2+ con-
centration at least up to 20 mM. This is far in excess of
the ATP concentration of 3 mM. Storer and Cornish-Bowden
 have shown that the true MgATP concentration cannot be
Figure 5. Pi formation in fresh and frozen scutellum slices
Fresh and frozen HCl-treated scutellum slices O .5 g
were placed in a reaction vessel containing 20 mM MgCI2,
50 rmM MES pH 6.5, and 2.5 mJi ATP and run for 60 min at
300. Each point is an average of 3 experiments.
Figure 6. Rate of phosphatase activity with increasing ATP
concentration. Fresh and frozen HCl-treated scutellum
slices were placed in a reaction vessel containing var.-
ous concentrations of ATP, 20 mM IMgCl1 and 50 mM MES
pll 6.5 and run at 30 for 10 min. Each point is an av-
erage of 2 experiments.
0 1 2 3
II I Cr F5S -q
2 0. I C
L- g 1 -
CC" 0 G *
Iso CD a
0 3 c+
o* >o pfi
C C (tD' CB
03 0 0 I a
S P0D cD
1> O P.
OC 'o 00S H
UJ tB(D Fl
0 C H- -St
O P SD
Ot c (
-c- (f (D
F 0 CD
0 "5 (H 0C'
CO r'4 P
0 C O C0
Figure 8. Effect of Ng2+ on phosphatase activity. Fresh and
frozen HCl-treated scutellum slices were placed in a re-
action vessel containing 3 mM ATP, varying concentra-
tions of MgC12, and 50 mM MES ph 6.5, and run at 300 for
60 min. Each point is an average of 3 experiments.
O I I.
0 10 20
derived by merely adding equimolar concentrations of the ion
and ATP. They suggested that in cases where the enzyme being
studied was not inhibited by high metal ion concentrations,
5 mM or more excess of i1g2+ over ATP was likely to give a
better stimulatory effect. It is assumed in calculating the
Km's for ATP and ADP (Fig. 7) that in 20 nmM MgC12 all of the
nucleoside was present in solution as the Mg2+ complex.
Phosphatase activity in both fresh and frozen slice
preparations was also stimulated by :n2+, Ca2+ and Co2+
(Fig. 9). The relative effectiveness of the cations was
Mn2+ > Mg2+ > Ca2+ > Co2+. At high I.In2+ concentrations, the
stimulatory effect was sharply decreased; at 20 mM Mn2+,
phosphatase activity was only one-half of that obtained with
5 mM Mn2+ (Fig. 9). Calcium ions gave maximum stimulation at
10 mM, but at higher concentrations of Ca2+ the stimulatory
effect decreased. Cobalt ions were much less effective than
the other divalent ions tested, and maximum stimulation was
obtained with 5-10 mM Co2+
Effect of Na' and K+ on the Phosphatase Activity
The stimulatory effect of Na+ and K+ was tested in the
fresh and frozen preparations with and without Mg2+. When
various ratios of Na+ and K+ were added in the presence of
1ig2+, the additional stimulation was very small (Table 6) in
fresh tissue preparations. With a Na+/K+ ratio of 0.25 the
additional stimulation was only 10% more than with Mg2+ alone.
The other ratios gave smaller amounts of stimulation. In the
frozen tissue preparation, the addition of ?'a+ and K+ gave
Figure 9. Effect of divalent cations on phosphatase activity.
Fresh and frozen HC1-treated scutellum slices were
placed in a reaction vessel containing 3 mM ATP, varying
ion concentrations and 50 mM MES pH 6.5 and run at 300
for 60 min. Each point is an average of 3 experiments.
f_ V i2+
0 j/ o, A, o Fresh
0, *,t Frozen
I I I ;
0 10 20
Table 6. Effect of Na+ and K' on phosphatase activity
Pi, imol/g hr
Na+/K+ ratio g2, molg hr
No Na+ or K+ + 45 2.2 30 + 2.0
25 + 5.6 6 2.5
Na+ only + 46 + 0 35 + 0.8
35 0.6 9 0.6
4 + 48 0.8 34 + 1.0
1.5 + 48 0.8 36 1.0
36 0.8 9 + 0.6
0.66 + 48 0.6 36 + 0.6
0.25 + 50 0.6 36 0.6
36 1.0 9 0
K+ only + 50 0.6 36 1.0
37 + 0.6
Note: Total monovalent ions equalled 50 mM. Mg2+ concentra-
tion was 10 mM. Only Na+- and K+-free ATP was used in this
study. Each value is an average of 3-1 experiments followed
by the standard deviation.
from 13% to 20% additional stimulation over Mg2 alone.
In the absence of Mg2+, uaa+ alone at 50 mM stimulated
the phosphatase activity by 40%, and K+ alone at the same
concentration by 48%, in the fresh tissue preparations. The
various Na+/K+ ratios gave amounts of stimulation very simi-
lar to Na+ alone or K+ alone. The phosphatase activity in
the frozen tissue preparations was stimulated 50% by the ad-
dition of various ratios of Na+/K+.
Effect of DNP on the Phosphatase Activity
In mitochondria , chloroplasts , bacteria ,
and intact ascites tumor cells , DNP stimulates ATPase
activity. In fresh HC1-treated scutellum slices, DNP in-
creased the phosphatase activity at pH's below 5 (Fig. 10).
At pH 3.8, DNP caused a fourfold increase in phosphatase ac-
tivity. In contrast to the results with fresh tissue, DNP
had no effect on phosphatase activity in frozen tissue. With
fresh H20- or HC1-treated scutellum slices, DNP also causes a
proton influx and sucrose efflux at pH's below 5 [99,103].
Although the pH of the bathing solution affects the amount of
DNP that enters the cells , it has a much greater effect
on DNP-induced proton influx  and sucrose efflux ,
and on DNP stimulation of the phosphatase activity (Fig. 10).
Several enzyme inhibitors, some specific for ATPase, were
tested with fresh and frozen acid-treated preparations (Ta-
ble 7). In most cases, responses of fresh and frozen prep-
arations to the added inhibitors were quite similar.
Figure 10. Effect of DNP on phosphatase activity of fresh
scutellum slices. Fresh and frozen HCl-treated slices
were placed in a reaction vessel with or without
5 X 10- M DNP, and with 3 m1 ATP, 20mM MgC12 and 50 mM
MES (pH 3.5-6.0), and run at 30 for 60 min.
In the absence of substrate, DNP caused a small
amount (7 pmol/g) of Pi efflux, but the data reflect a
corrected value. Each point is an average of 3-4 ex-
Frozen + DNP
A Fresh + DNP
A Fresh /
Table 7. Inhibitors
Oligomycin 1 pg/ml
Na Azide 5 mM
Note: Fresh and frozen HCl-treated slices were placed in
a vessel with 3 mM ATP, 20 mM MgC12, 50 mn MES pH 6.5 and
various concentrations of inhibitors. Since oligomycin
and DCCD have low water solubilities, they were made up
in 95% EtOH. The controls received equal quantities of
95% EtOiI. Since the DCCD, at higher concentrations, pre-
cipitated when added to the bathing solution, the true
concentration of DCCD in these cases was not known. The
dal.a are presented as a % of the controls. The reaction
was run at 300 for 60 min.
Although DCCD is a specific and potent inhibitor of some
plant and animal ATPases, it did not greatly inhibit the phos-
phatases of the scutellum preparations. The maximum inhibi-
tion by DCCD was only 20%. A more water-soluble analogue of
DCCD (EDCD) caused a 30% to 33% inhibition, but only at rela-
tively high concentrations (10 mM). A 5 mM concentration of
PCMBS also gave 32% inhibition. Both PCMB and sodium azide gave
little or no inhibition, and oligomycin did not inhibit the
To establish the localization of the phosphatase reaction
within the cell, a cytological study was done. Fresh HC1-
treated slices were fixed in glutaraldehyde and then placed
in a reaction mixture containing buffered ATP, Mg2+ and lead
A 1% glutaraldehyde pretreatment for 30 min inhibited the
phosphatase activity 56%. When glutaraldehyde treatment was
followed by a 1% lead nitrate treatment for 30 min, the enzyme
activity was inhibited 72%. Thus, there remained sufficient
enzyme activity for a cytological localization study.
Precipitates of lead phosphate were deposited at the site
of the ATPase activity (Fig. 11). The activity was localized
on the plasmalemma. There were a few scattered lead phosphate
deposits in the cytoplasm, but they were not associated with
any particular organelle. The control without ATP showed no
Figure 11. Electron micrograph of ATPase localization in
fresh scutellum slices.
A. Control, without ATP, showing plasmalemma (pl),
cytoplasm (cy), plasmodesma (pd), lipid (L), and cell
B. Enzyme localization, showing lead phosphate on the
plasmalemma (pl) and along the plasmodesma (pd).
Phosphatase localization in the frozen slices was not
attempted, because freezing and thawing releases the vacuolar
contents, and disrupts other organelles (Fig. 3), causing the
appearance of electron-dense granules throughout the cell.
Presumably, these granules are Mg and K salts of phytic acid
; they resemble the lead phosphate deposits. Such vacu-
olar interference would have made localization studies in the
frozen tissue extremely difficult.
In the frozen-thawed slices, a possible source of phos-
phatase activity may be the mitochondrial ATPase.
Mitochondrial preparations from fresh maize scutella
were tested for ATPase activity with the same concentrations
of Mg2- and ATP that were used in whole-tissue experiments.
The ATPase activity at pH 8.5 was 40% greater than at pH 6.5.
This is in agreement with pH optima for mitochondrial ATPases
Equivalent aliquots from the same initial mitochondrial
preparations were frozen for 48 hours at -50, thawed, and
washed with 0.01N HCI for 30 min. The mitochondrial prepara-
tion was then rinsed with buffer and tested for ATPase activ-
ity at pH 6.5 and 8.5. There was no phosphatase activity at
Acid phosphatases (E.C. 188.8.131.52) and alkaline phosphat-
ases (E.C. 184.108.40.206) have wide specificity and are not stimu-
lated by metal ions [13,124]. By contrast, ATPases
(E.C. 220.127.116.11) show a more narrow specificity, primarily for
nucleoside 5'-triphosphates , and are activated by cer-
tain cations, usually Mg2+ and Ca2+. Some ATPases are addi-
tionally stimulated by Na and K .
The results of this study strongly suggest that the
phosphatase activity observed in the HC1-treated preparations
of both fresh and frozen scutellum slices is a plasmalemma-
bound ATPase. Evidence in support of this conclusion is sum-
marized below under four headings:
Fresh scutellum slices
Frozen scutellum slices
Effect of DNP
Fresh Scutellum Slices
1. Fresh HCi-treated slices have a phosphatase activity
with a sharp pH optimum at 6.5 (Fig. 1). HC1 treatment
causes considerable loss of phosphatase activity below this
optimum pH, but very little loss in activity at pH 6.5. This
is similar to the pH optima for ATFases from plasmalem.na
fractiotn- of various plant sources [10,11,39,40,50,51].
2. The addition of Mg2+ stimulated the phosphatase ac-
tivity in both the H20- and HCl-treated slices. With added
!'2+, the HC1-treated preparations showed nearly identical
'*etiv.Lty at pH 6.5 as the H20-treated preparations (Fig. 1).
3. Adding Ca2+ was as effective as adding Mg2+, and the
enzyme could, perhaps, be called a Mg2+-Ca2+ ATPase. Other
divalent cations were also effective stimulators of the en-
zyme (Fig. 9). With Mn2+, the enzyme was maximally stimulated
at 5 mM, then the stimulatory effect decreased sharply. This
is similar to the results reported by Jolliot et al.  on
potato tuber plasmalemma ATPase. They found the greatest
stimulation with Mn2+ occurred at 2 mM, and dropped sharply
as the concentration of Mn2+ increased. With Mg2+ and Ca2+,
peak stimulation was obtained at 4 mM and decreased with in-
creasing concentration, but the drop was more gradual. At
20 mM, Mn2+ gave only 50% of the stimulation of Mg2+ or Ca2+.
4. The monovalent cations Na+ and K+ in the presence of
vg2+ gave a small additional stimulation, but in the absence
of ME2+ both Na+ and K+ gave 40% to 50% stimulation. Leonard
and Hodges  and Balke and Hodges  reported only slight
stimulation of the basal ATPase activity of oat root plasma-
lemr. by N'. or K+ in the absence of Mg2+. However, bean
cotyledor plasmalemma ATPase was reported to be stimulated by
Na+ or K' to a greater amount in the absence of added Mg2+
than in its presence .
5. The scutellum phosphatase showed a clear preference
for ITP arid CTP over the other substrates tested. The
substrate PNPF, which is commonly used to detect acid and
alkaline phosphatases , gave low activity with the scu-
tellum' preparations. Although UTP gave 73% to 83% of the
activity of ATP, the diphosphate, UD?, gave only 57 to 7%.
However, ADP gave 67% to 77% of the activity of ATP (Table 4).
The high activity obtained with ADP is similar to results
obtained with other isolated plant plasmalemma preparations
[7,'4 It is possible that the high activity with ADP may
be a result of a nucleoside diphosphatase. This enzyme has
been reported  to hydrolyze nucleoside diphosphates,
but not tri- or monophosphates. in the maize scutellum
slices, however, very low activity was observed with UDP.
The Vmax (Fig. 7) could indicate that ADP is being acted on
by the same enzyme which is acting on ATP. This relatively
high specificity for ADP has been reported with other plant
plasmalcmma ATPases [7,40,47].
6. The Km for the phosphatase from fresh and frozen
HC.-treated slices was 0.65 with ATP as the substrate and 5.0
with ADP as the substrate (Fig. 7). The Km value for ATP is
similar to other published values for higher plant plasmalem-
ma ATPases [49,51,124].
Frozen Scutellum Slices
The results indicate that the enzyme activity observed
in the frozen-thawed, HC1-treated scutellum slices is the
same as that observed with fresh HCl-treated slices.
1. The pH optimum (6.5) of the frozen HCl-treated prep-
aration was identical to that of the fresh slice preparation
(Figs. 1, 2). In the absence of Mg2+, the frozen HCl-treated
slices gave very low activity over a pH range from 4.4 to 7.0
(Fig. 2). Acid treatment of the frozen slices decreased the
phosphatase activity to 11 ;imol/g hr without added Mg2+ at
pH 6.5. Addition of ig2+ resulted in a 364% rise in phos-
phatase activity (Table 1) at pH 6.5. This level is ccmpara-
ble to that obtained with fresh HCl-treated slices with added
IMg2+ at pH 6.5.
2. The stoichiometry, kinetics, monovalent and divalent
stimulations, effects of inhibitors, and substrate specific-
ity of the phosphatase in the HC1-treated frozen preparations
were similar to or identical with those of the HCl-treated
Effect of DNP
The addition of DNP to fresh HCl-treated preparations
stimulated the rate of ATP hydrolysis. In contrast, DNP had
no effect on the phosphatase activity in frozen tissue
(Fig. 10). It seems that the DNP effect can only be seen if
the plasmalemma is intact either as whole cells, organelles
or sealed vesicles, thus being relatively impermeable to H+
[97,123,126,127]. Adding DNP to reaction vessels containing
intact ascites tumor cells stimulated the ATPase activity by
177% . Mitchell and Moyle  have proposed that in
mitochondria, DNP acts as an uncoupler of ATPase, by increas-
ing the permeability of the membrane to protons . When
the ATPase was purified from rat liver mitochondria, Lambeth
and Hardy  reported that DNP decreased the activity of
thi3 purified enzyme.
Since DiP is a lipid-soluble weak acid with a pK of 4.0,
the amount that enters the cells increases as the pH is low-
ered. However, previous studies with the maize scutelluni
h vc shown that below pH 5, DNP induced a proton influx, and
that the bathing solution pH had a much greater effect on
proton influx than it did on DNP content in the tissue .
The DNP was reported to cause an efflux of sucrose from maize
cuLtellum slices and an influx of H across the plasmalemma,
and both fluxes increased as the pH was lowered . The
effect of DNP on sucrose offlux and the stimulation of the
ATPase in the maize scutellum slices may both result from the
proton influx and the resulting loss of membrane potential.
The following studies support the idea that the ATPase
is located in the plasmalemma.
1. The cytological localization (Fig. 11) clearly
showed that the areas of ATPase activity were localized on
the plasmalemma. There are several reports of similar re-
sults obtained with phloem sieve cells  and sieve tube
2. In both the fresh and the frozen tissue prepara-
tJon, HC1 treatment for 1 hr did not destroy the enzyme ac-
tivity. This rigorous treatment should denature most pro-
teins. However, the plasmalemma proteins of the scutellurm
cells apparently are not affected, since after HC1 treatment
rates of sucrose and maltose uptake , sucrose synthesis
i'ron fructose , and respiration  are not affected.
The levels of enzyme activity at pII 6.5 in fresh and frozen
tissue preparations were nearly identical (Figs. 1, 2). How-
ever, HCI treatment decreased the activity in the pH 4-5 range
(Fig. 1), presumably by destroying the acid phosphatases.
3. In studies with Pi leakage in both fresh and frozen
tissue preparations, after 1 hr less than 2 pmol of Pi leaked
into the bathing solution (Table 2). In the fresh tissue,
there was no uptake of Pi from the bathing solution. It is
unlikely, therefore, that the ATP entered the cell, was hydro-
lyzed and the Pi leaked out to the bathing solution. The re-
sults indicate that the reaction occurred in or on the cell
surface (membrane), the ATP was hydrolyzed from the outside
of the cell, and the Pi remained in the bathing solution.
The stoichiometric studies confirm that the substrate
and products remain in the bathing solution (Table 5). This
enzyme seems to be capable of recognizing substrate coming
from outside the cell. Similar results were reported with
intact ascites tumor cells . These tumor cells hydro-
lyzed ATP to produce AMP and Pi, and both products were found
in the bathing solution. Under normal in vivo conditions,
the ATPase would only encounter the substrate from the inside.
Nurminen et al.  reported various enzymes on the
cell wall and plasmalemma of yeast. When the wall was enzy-
matically digested and the released enzymes assayed, saccha-
rases and acid phosphatases were released, but the Mg2+ ATP-
ase was not. This was interpreted to mean that this enzyme
was bound to the plasma membrane.
Most ATPases are considered to be composed of two func-
eiornal parts, each with several globular proteins precisely
arranged in some specific functional quaternary structure.
The port-ion of the enzyme which is associated with the actual
synthesis or hydrolysis of AT? is referred to as the FI por-
tion, and the portion thought to be involved in proton trans-
port as the Fo portion [74,97]. The Fl portion of the enzyme
is attached to the F portion, and the latter is tightly em-
bedded in the lipid bilayer, -hereas the former protrudes
from the membrane. Thus, most membrane-bound ATPases are
thought to be vectorially oriented. Models of ATPases are
often drawn with the substrate recognition portion (F1)
sticking into the cytoplasm, matrix of the mitochondria, or
stroma of the chloroplast [74,97].
Much of the information upon which ATPase models are
based comes from inhibitor studies. The compound DCCD is an
inhibitor of some ATPases from plant and animal sources, act-
ing on the membrane-bound portion (Fo) of the ATPase but not
on the portion (F,) that is known to catalyze the hydrolysis
of ATP [83,109,110,114]. Intact ATPases attached to the mem-
brane are inhibited by DCCD in many instances; however, the
purified enzyme which contains only the Fl portion is not
susceptible to inhibition by DCCD [107,114).
The maize scutellum plasmalemma ATPase was not inhibited
by DCCD, although the membrane was intact. ATPase from maize
root plasmalemma has been reported to be inhibited by 50%
when 50 to 100 pM DCCD was present . This was not as
large an inhibition as was seen with other ATPases where 90,
to 95% inhibition was reported [109,110]. There are reports
of bacterial ATPases which seem to be resistant to DCCD 
and an ATPase on the tonoplast of Hipoeastrum  which was
not inhibited by DCCD.
It is possible that the ATPase on the maize scutellum
slices is placed in the membrane in such a way that it is in-
accessible to the added inhibitors. The water-soluble ana-
logue of DCCD (EDCD) did inhibit the enzyme activity by 33%,
but at a relatively high (10 imM) concentration.
At this time, the vectorial model described is consid-
ered to be the best model for the mitochondrial and chloro-
plast ATPase [74,97]. Not as much is known about the ATPase
in the plasmalemma of higher plants. Post of the information
comes from work with partially purified plant plasma mem-
branes, and the structure of the enzyme is not known [40,51,
The sarcoplasmic reticulum, Na+-K+ ATPase has been shown
to span the membrane, and the Ca2+ ATPase from the same
source is thought to be predominantly membrane-bound with a
small portion sticking out into the cytoplasm . Both are
thought to be functionally vectorial. iost ATPases are rela-
tively large proteins and could easily span the lipid bilayer
The Na+-K+ ATPase from the microsomal fraction of canine
renal medulla  is thought to be composed of two subunits,
the large and small pol!peptides. From studies with cardiac
glycoside binding experiments, it was found that the larger
of the two subunits spans the membrane, and is seen from the
outside as well as the inside of the cell. This large poly-
peptide is capable of binding with the cardiac glycoside from
the outside, and is also responsible for the hydrolysis of
ATP, presumably from the inside.
Bacterial membranes obtained by osmotic lysis  yield
nearly all right-side-out vesicles. Such vesicles showed a
large ATPase activity when the substrate was introduced from
the outside. The high rate of ATP hydrolysis was not expect-
ed, since the model presumed a vectorial orientation with the
substrate recognition portion of the enzyme on the inside.
Thia model is supported by results using spheroplasts of E.
coli. indicating that the ATPase was localized on the inner
part of the cytoplasmic membrane .
Penefsky  reported that an analogue of ATP (AMP-PNP)
was a potent competitive inhibitor of mitochondrial ATPase,
but did not inhibit oxidative phosporylation. He concluded
that the hydrolytic site of the enzyme was not identical to
the synthetic site.
It is possible that plant plasmalemma ATPases are dif-
ferent in orientation to mitochondrial or bacterial ATPases.
The hydrolytic site may respond to substrate levels, as well
as other factors such as ion concentrations and pH, and un-
dergo some conformational changes accordingly. Stein et al.
 presented a model to explain the transport of ATPase-
mediated Ua+ and K+. They suggested a tetrameric model which
depends oi conformational changes to bring about transport.
In the maize scutellum tissue studies, the results with
fresh and frozen tissue preparations are very similar. It
appears, then, that the scutellum plasmalemma ATPase is
tightly bound to the lipid bilayer, and is capable of sub-
strate recognition from either side of the plasmalemma.
There is evidence for the existence of a proton pump in
the maize scutellum plasmalemma . The ATPase on the
plasmalerama may be acting as the pump. The ATPase could set
up a proton gradient which would drive cation and sugar
transport [10,11,12,13,14,99] across the membrane.
Preparation of the Scutellum Slices
Corn grains (Zea mays L. c.v. Mc;Nair 508) were soaked in
running water for 24 hr, then placed on moist paper towels
and grown in the dark at 24o-250 for 72 hr. The scutella
were excised, and cut transversely with a razor blade into
slices of 0.5 mm or less in thickness.
The scutellum slices were washed in deionized water sev-
eral times, until the wash water remained clear. They were
then blotted dry on filter paper, and weighed into groups of
0.5 g each (80-90 slices). Scutellum slices to be frozen
were similarly prepared, weighed, and placed in the freezer
at -o to -50. The frozen slices could be stored at least
27 days witn no loss of phosphatase activity.
The thickness of the scutellum slices, in one sampling,
was ;measured under a dissecting scope fitted with an ocular
micrometer. A single slice, so measured, was then sectioned
and observed under a light microscope, and the number of
cells that made up the thickness was counted. The cells of
the scutellum are nearly uniform, and each cell measured ap-
proxiiatbely 50 un in diameter. The thickness of a random
nur.ber of slices was then measured. The results showed that
the slices were made up of from 3 to 7 cells in thickness.
There were many intercellular spaces.
Unless otherwise noted, all incubations were carried out
with 0.5 g of scutellum slices in 10 ml bathing solution at
30" in a "Gyrotory" water bath (New Brunswick Scientific Co.,
New Brunsw.ick, N. J.).
Hil0- or Ho_20-Treatment
Slices (0.5 g) were incubated in 0.01N HCI at 30 for
1 hv. The bathing solution was replaced with fresh HCl after
30 min. The slices were then rinsed twice with distilled H20.
Groups of slices to be H20-treated were similarly han-
died, with glass-distilled H20 in place of HC1.
Analysis of Pi
Pi was measured as the product of hydrolysis of the var-
ious substrates. The modified Fiske-Sabbarow method of
Bartlett was used .
The method described by Larmprecht and Trautschold was
followed for the ATP determinations .
ADP and AMP Analysis
The method described by Adam [1303 was followed for ADP
and AMP determinations.
Preparation of Na+- and K+-Free ATP
To a certain volume of ATP '( 50 mMN4 was added 4 g of
JDo.ex-50. After stirring for 1 min the solution was filtered
and neutralized with Tris-base. The concentration of ATP was
then determined spectrophotonetrically . The final con-
'entration was determined to be 44.7 mM.
Mitochondria were isolated from fresh, whole scutella
according to the method of Hanson et al. . The purity
of the preparation was checked b; electron microscopy. Many
intact as well as partially broken mitochondria were seen.
Total lipids were determined by a method outlined in the
AOAC Method of Analysis . One g of tissue was used in
Total Nitrogen and Phosphorus
Scutellum slices (0.5 g) were digested in H2S04. The
digest was diluted to 35 ml with H20 and aliquots were taken
for total nitrogen, determined by Nesselerization , and
for total phosphorus, determined by the method of Bartlett
ATP Product Localization
The method of Wachstein and Meisel was modified for the
ATPase localization study . Fresh scutellum slices were
HC1-treated and then incubated in buffered 1% glutaraldehyde
(50 pTM MES pH 6.5) for 30 min. The slices were then rinsed
twice with buffer and treated with:
1. Lead nitrate (Y1%) + Mg2+ (20 rM) + buffer (50 mM MES
pH 6.5) + ATP (3 mi);
2. Controlsconsisted of: Mg2+ alone; lead nitrate
alone; buffer alone; and lead nitrate + Mg2+ +
The reactions were allowed to proceed for 30 min at 300. The
slices were then rinsed with buffer and fixed in 4% buffered
OsC%! for 30 min. They were then dehydrated in a series of
ethanol solutions, and finally in 100% acetone. The slices
were embedded in an Epon-Araldite plastic mixture, sectioned,
and observed under a Hitachi HU-11C electron microscope.
Phosphotungstic-Chromic Oxide Stain for Plasmalemma of Frozen
Frozen slices were prepared for EM as described above,
without the incubation in lead nitrate and ATP. Following
dehydration steps, the frozen slices were embedded as before
and sectioned. The specific plant plasmalemima stain with
phosphotungstic acid and chromic oxide stain was carried out
as described by Roland et al. .
All biochemicals were purchased from Sigma Chemical Co.,
St. Louis, Mo. The adenosine 5'-triphosphate, Sigma grade II
was used throughout this study .
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