The Rockefeller University, New York, NY 10021, U.S.A.
(Received 3 November 1980; accepted 1 March 1981)
Axenically grown trophozoites of Entarnoeba histolytica (NIH-200 strain) contain an active ATPase (170 nmol PO4/min per mg protein) with maximal activity at pH 8.8, a high affinity for ATP (Km ~ 40/zM) and an absolute and specific requirement for Ca 2 ÷. The activation by Ca 2 ÷ shows positive cooperativity (n H = 2.48) at calcium concentrations below 8 /zM and no cooperativity be- tween 8 and 25 WVI. The latter concentration fully saturates the enzyme. The observed activity is insensitive to oligomyein, ouabain and ruthenium red and is unaffected by a range of inhibitors of electron transport and uncouplers of oxidative phosphorylation. The enzyme exhibits structure bound latency and is tightly bound to cellular membranes. It is sedimentable ( > 80%) by high speed centrifugation of cell homogenates which are either protected osmotically or in which subcellular structures are damaged by sonication or treatment with Triton X-100. Arrhenius plots of V in the temperature range of 0 -38°C are linear without breaks, similar to other pyrophosphatases of E. histolytica. The calculated activation energy is 14.8 kcal/mol. This f'mding as well as the failure of phospholipase treatment to affect activity indicate that interactions with membrane lipids play no role in the catalytic function of this tightly membrane-bound ATPase.
[4] provided indications of Mg2+-stimulated ATPase activities of low specific activity. Ihe present investigation, however, has revealed E. histolytica to possess an active ATP-
ase with no Mg 2÷ requirement, but having an unusually strict dependence upon low concentrations of Ca z÷. The activity is tightly bound to cellular membranes and was subsequently reported to be partly associated with the surface membrane of the cell [5 ]. Results of this work have been presented at the 32nd Annual Meeting of the Society of Protozoologists (Stillwater, OK, August 1979) [6].
EXPERIMENTAL PROCEDURES
Culture, collection and homogenization o f cells. The NIH 200 strain of E. histolytica was maintained in vitro as previously described [7]. After 72 h growth the cultures were chilled on ice and the amoebae collected by centrfugation (800 × g for 10 min)
and washed twice with 250 mM sucrose. Cells resuspended in ice-cold 250 mM sucrose, or in some cases 250 mM sucrose, 1.0 mM EGTA, 10 mM Hepes (pH 7.2), were dis- rupted by 8 - 1 0 gentle strokes of a Dounce tissue homogenizer with a large clearance
pestle (Kontes Glass Company, Vinland, NJ). The homogenate was centrifuged in a Sorvall SS-34 rotor, equipped with SorvaU 425 adaptors, at 2500 rpm for 3 min to remove intact cells and nuclei. The supernatant fraction (cytoplasmic extract) was then centrifuged for 1.0 h at 19 000 rpm in the same rotor and adaptors as above. The result-
ing high speed pellet (HSP) was resuspended in 250 mM sucrose and used as a source of
enzyme.
Enzyme assays. Calcium-dependent ATPase (Ca-ATPase) activity was assayed routinely as follows. To 500 #1 60 mM glycylglycine-NaOH (pH 8.8) containing 0.15% Triton X-100 were added 250 ~tl 2.0 mM ATP disodium salt (pH 8.0) and 250/11 2.0 mM calcium acetate. The reaction started with the addition of enzyme (about 60 #g HSP protein).
After 10 rnin at 30°C, 0.25 ml 6.0% sulfosalicylic acid was added, the tubes placed on ice and the released Pi determined [8]. Assay conditions were always such that no more than 3.5% of the available ATP was hydrolyzed. EGTA-Ca buffers used in certain assays
were prepared as described [9]. Since the ATPase activity was highly latent, Triton X-100 was included in all routine
assays, as given above. When the presence of a detergent was to be avoided, samples
kept on ice were sonicated for 4 min with a Branson Sonifier (W-350) equipped with a microprobe (pulse setting at 50% duty cycle and output set at 3).
Assays for nucleoside diphosphatase, acid inorganic pyrophosphatase, acid phosphatase and protein have been described [7,10].
Attempts to solubilize Ca-A TPase. Various treatments were used in an affort to dissociate the Ca-ATPase from the HSP fraction. These included brief exposure to high pH (0.1 M NH4 OH), low pH (0.2 M acetic acid), high salt concentration (0.4 M KC1) and chelat- ing agents (12.5 mM EDTA or EGTA). A number of different types of detergents were
371
used: Triton X-100 ((3.1, 0.2 and 0.5%), saponin (0.15%); sodium dodecyl sulfate (0.9 mg/mg protein), Lubrol W (0.1,0.2 and 0.5%), acetylpyridinium chloride (0.1, 0.2 and 0.5%), and sodium deoxycholate (0.1, 0.2 and 0.5%). Extraction with organic solvents was also used: 90% 2-chloroethanol; 60% aq. butanol; 90% aq. n-pentanol and chloro- form. Finally, various chaotropic agents were tried: 2 M KSCN; 2 M KI and 6 M urea. After exposure to chelating agents, organic solvents or chaotropic agents, the super- natant fraction or the aqueous phase was dialyzed against 20 mM Tris-acetate, pH 7.2, containing 0.075% Triton X-100.
Phospholipase treatment. Washed HSP was resuspended in 15 mM Tris-maleate, 2 mM calcium acetate, 225 mM sucrose, pH 7.0. To 600/11 of this suspension (about 3.0 mg protein) was added 25 /zl phospholipase C (400 U/ml) or 25/zl phospholipase A2 (600 U/ml) plus 50/A 10% bovine serum albumin and incubation commenced at 25°C for 40 min. At 10-rain intervals, 100./al aliquots were removed to 10/A 37 mM EGTA to stop the reaction, and stored on ice until assayed for ATPase activity. A control incubation prepared without added phospholipase was also sampled.
The structure-bound latency of the Ca-ATPase after phospholipase treatment, as well as of certain other hydrolases was determined as previously [7]. To determine the sedi- mentability of Ca-ATPase, the above volumes were increased and after a 30 min incuba- tion, the stopped reaction mixture centrifuged for 2.0 h at 100 000 X g (using Beckman 2 ml adaptors). The supernatant fraction and resuspended pellet were then assayed for Ca-ATPase.
Materials. The following items were supplied by Sigma Chemical Co. (St. Louis, MO): ADP (sodium salt), 5'-AMP (free acid), ATP (disodium and Tris salts), bovine serum albumin (fatty acid free), calcium acetate, carbonyl cyanide m-chlorophenylhydrazone (CCCP), N,N'-dicyclohexylcarbodiimide (DCCD), ethacrynic acid, ethylene-bis-(fl-amino- ethyl ether)-N,N'-tetraacetic acid (EGTA), glycylglycine (free base), N-2-hydroxyethyl- piperazine-N'-ethanesulfonic acid (Hepes, free acid), p-hydroxymercuribenzoate (sodium salt), iodoacetamide, p-nitrophenylphosphate, oligomycin, ouabain, sodium pyrophos- phate, and quercetin. Thiamine pyrophosphate (HC1) was obtained from Calbiochem (La Jolla, CA), sulfosalicylic acid from Merck (Rahway, NJ), 2,4-dinitrophenol from Eastman (Rochester, NY) and ruthenium red from K & K Laboratories (Plainsview, NY). Phos- pholipase A2 (from porcine pancreas, 600 U/rag) and phospholipase C (from Bacillus cereus 400 U/mg) were both obtained from Boehringer Mannheim (Indianapolis, IN).
RESULTS
The specific activity of the Ca-ATPase in whole homogenates ofE. histolytica assayed at standard conditions was 65 -+ 9 mU/mg protein (mean value -+ S.D. for determinations in 5 experiments). Due to the tight membrane association of this enzyme 92% of this activity is recovered in the HSP, which has a specific activity of 170 + 15 mU/mg protein
100
(12 experiments), resulting in about a 3-fold purification of the activity. All experiments, unless stated otherwise, were performed on such HSP fractions.
Effect ofpH. ATPase activity as a function of pH is shown in Fig. 1 from which a value of pH 8.8 appears to be optimum. A less pronounced and broader peak of activity was
seen between pH 5.0 and 6.0. In contrast to the pH 8.8 activity, activity at low pH
was inhibited 80% by 500/aM NaF. The susceptibility of various E. histolytica acid phos- phohydrolases to fluoride inhibition was noted previously [7].
Cation requirements. The ATPase activity was found to have an absolute and specific re- quirement for Ca 2÷, no detectable activity being apparent in HSP prepared with or without prior exposure to EGTA. Other divalent cations tested could not substitute for calcium; addition of 0 .5 -2 mM Mg 2+, Zn 2÷ or Mn 2÷ gave at most 5% of the maximum activity obtained with Ca 2÷. In contrast to Mg-ATPases, maximum ATPase activities were observed at less than equimolar ratio of metal to ATP; in the presence of 100,250 and 500/aM ATP maximum activity was apparent at 25-50 /aM Ca 2÷. Further increases in Ca 2÷ concentration up to 2 mM had no effect upon ATPase activity. In addition, a com- parison of double-reciprocal plots of activities determiried either at a given fixed ATP level and varying Ca 2÷ or at a fixed level of Ca 2÷ and varying ATP (all within the same
concentration range), revealed a complete lack of identity (Fig. 2), showing that there
is no absolute requirement for Ca-ATP as substrate [11]. Whilst at 100/aM ATP double- reciprocal plots were linear up to 25 -50 /aM Ca 2÷ (Figs. 2 and 3), giving an apparent K a of 2.6-3.2/aM for Ca 2÷, higher ATP levels produced non-linear plots. At 250 and 500
20
E ~= 50
E
o
C o 2 ÷
e ,
f
o" • I0 ,r.i--'--.. ,' /
~ M NoF) I I I I I 0 I 5 6 7 8 9 U
pH
372
I ~ I 0 1 2 0.05 0.1 015 I I c
Fig. 1. Effect of pH on the Ca2÷-ATPase activity ofE. histolytica. 40 mM Tris-acetate buffers, • . . . . . •
in the absence of NaF, ~ in the presence of 500 ~ NaF.
Fig. 2. Double reciprocal plots of E. histolytica ATPase activity using the same fixed levels of either
ATP or Ca 2 ÷ with varying concentrations of Ca 2÷ or ATP (I /c) respectively. ~ 100 ~tM ATP,
5 - 1 0 0 I~M Ca 2 ÷; ~ 100 ~aM Ca 2 ÷, 10-100 I~M ATP. S tandard ATPase assay.
373
/aM ATP an upward concavity was noticeable, diagnostic of positive cooperativity. Hill
plot presentations of these data (Fig. 4) demonstrate a high degree of cooperativity
at both ATP levels up to 6 - 8 /aM Ca 2+, the values for n H indicating at least two Ca 2+ binding sites. At Ca 2÷ concentrations above 6 - 8 /aM, the slope of the plots changes markedly, indicating a much reduced interaction between binding sites.
The effect of magnesium was for the most part inhibitory (Fig. 5). In the presence
of less than 10/aM Ca 2+ low levels of Mg ~÷ ( < 250/aM) had a slight stimulatory effect; however, at or above 10/aM Ca 2+ increases in Mg 2+ concentration resulted in decreasing
ATPase activity. With 50/aM Ca 2÷ and 500/aM ATP the presence of 2 mM Mg 2+ caused a 50% reduction in activity.
No Na ÷- and/or K+-stimulated Mg2+-dependent ATPase could be detected. The addi-
tion of Na ÷ and/or K ÷ ( 2 - 2 0 mM) in the presence of Mg 2+ alone failed to stimulate further activity, nor did these monovalent cations increase Ca-ATPase activity.
Substrate specificity. At the pH optimum for the ATPase (pH 8.8) no discernible hydro- lysis of either ADP or 5'-AMP in the presence of Ca 2÷ or Mg 2+ could be detected. Hydro- lysis of ATP was found to be linear with regard to time for at least 30 min, indicating a lack of the pronounced product (ADP) inhibition characteristic of various other ATP- ases [12, 13].
Double reciprocal plots of velocity as a function of Ca2+-ATP concentration are
shown in Fig. 6. With both 1:1 and 2.5:1 ratios of Ca/ATP linear plots were obtained, displaying almost identical V, but two times higher K m when using Ca/ATP of 1:1. When data shown in Fig. 6 were replotted using direct linear plots [14] the following K m values were obtained: 225/aM for Ca/ATP = 1:1 and 56/aM for Ca/ATP = 2.5:1.
60
50
4O I V 3O
20
10
500~M /"
!
I I •
~" 250~M / / /
/1" ~"
g t.~" ,ff/ IO0,uM
p;o 2;0 3;0 4;0 5;o [Co2"1" ]-r ~M
ATP
,q-
0.5 :=,.
0.1
0.05
A /H=22 ,95 I I I I I I I I 2 5 I0 20 50 2 5 I0 20 50
Ca2+]juM
Fig. 3. Double reciprocal plots of E. histolytica ATPase as a function of Ca 2+ concentration at fLxed ATP levels. 0......43, 100 WM;&---A, 250 #M; o-----e, 500 tAf ATP. Standard ATPase assay.
Fig. 4. Hill plots of data from Fig. 3 showing biphasic cooperative kinetic behavior orE. histolytica ATPase with respect to Ca 2 + concentration. (A) 250 #M ATP; (B) 500 #M ATP.
374
150
.E
o a_ IO0
5O
5O
~0 [C02+] pM
250 750 1500 2500
[Wl,,,,
I V
16 214 32 410 [ATP]-' mM
Fig. 5. Effect of Mg 2÷ on E. histolytica ATPase in the presence of various concentrations of Ca 2÷ established with Ca-EGTA buffers. Free Ca 2÷ concentration calculated according to reference [7].
Fig. 6. Double reciprocal plots of E. histolytica ATPase reaction velocity as dependent on the ratio of Ca ~÷ to ATP. ~-~i , : 1 to I; ~----o: 2.5 to I.
Inhibitors and modulators. The following uncouplers and inhibitors of mitochondrial
metabolism were without effect on Ca-ATPase (final concentrations in parentheses):
DCCD (0.05; 0.4 mM); quercetin (10/ag/ml), NaN3 (0.2, 2.0 mM) and KCN (0.2, 2.0 mM). The Ca-ATPase was also insensitive to sulfhydryl blocking reagents (iodoacetamide,
chloromercuribenzoate and ethacrynic acid), ouabain (0.2, 0.4 taM) and ruthenium red
(0.2 mM) an inhibitor specific for most Ca-ATPases [15, 16]. The lack of effect of
ouabain, a specific inhibitor for Na-K-Mg-ATPase, is in agreement with the lack of sti-
mulation in the presence of Na ÷ and K ÷. Only NaF, at high levels, produced any notice-
able inhibition (Fig. 7).
All compounds were tested with untreated, Triton X-100-treated, and sonicated HSP
fractions.
Membrane association and sedimentability. The ATPase activity in HSP fractions was found to be highly (80-85%) latent, and its full expression required prior sonication or
addition of the detergent Triton X-100. The enzyme, similar to two other pyrophos- phatases of 17. histolytica [7], has so far resisted all attempts at solubilization with the treatments listed under Methods. After sonication, low and high pH treatment or salt
extraction o f the HSP fraction, more than 90% of the original activity remained sediment- able with the centrifugation conditions used to prepare the HSP fraction. In contrast to certain other ATPases [1, 17, 18; McLaughlin, J., unpublished results], no inhibition was noted in the presence of Triton X-100 (up to 1.5 mg/mg protein) and up to 80% of the total activity remained sedimentable. All other detergents caused extensive inactivation at the highe~ concentrations tested. The organic solvents inactivated the ATPase from
375
50% (n-butanol) to 95% (2-chloroethanol). The remaining activity was always in the residual pellet and none in the supernatant fraction.
Arrhenius plots based on V values [19] obtained through the use of a range of sub- strate concentrations at each temperature for the ATPase as well as for three other phosphohydrolases of E. histolytica were linear, without breaks in the temperature range 0-38°C (Fig. 8) in contrast to various membrane-bound ATPases [20-22] . Whilst sonication might modify certain enzyme-lipid interactions thus giving rise to linear Arrhenius plots, previous work on various mitochondrial enzymes [23] found no changes in temperature breaks after such treatment.
Values calculated for the activation energy (Ea) are all below 20 kcal (Fig. 8). Values in excess of this are typical for most integral membrane-bound enzymes [24], especially at temperatures below 30°C. At such temperatures it is postulated that the more rigid
ordering of surrounding phospholipid fatty acid chains induce conformational restrictions affecting enzyme function [25].
Although sedimentability data suggest that the Ca-ATPase is tightly membrane associ- ated, lipid interactions seem to have no direct effect upon the catalytic properties of this enzyme. Further support for this conclusion comes from the lack of effect of prior treatment of HSP fractions with either phospholipase A2 or C upon the latency, sedi- mentability or specific activity of the ATPase as well as both pyrophosphatases. Thus both latency and sedimentability remained unchanged at 82-87% and 90-96%, re- spectively. The acid pyrophosphatase lost 60-70% of its activity after exposure to
f :~ 5O
o
i i ,.o ~.o 3'0 ,'0 5'.0
[NoF] mM
:#
I000
500
200
LO0
50
I000
500
200
I00
3.15
TEMPERATURE 30* 20* 10" 0 °
72 ' " C" D
EQ=t28
30* 20* iO ° O*
' ' ' B'
335 355 315 335 355
(K) -I xlO 3
Fig. 7. Effect of NaF on E. histolytica ATPase.
Fig. 8. Arrhenius plots for ATPase and other hydrolases of E. histolytica in the temperature range 0-38°C. (A) Ca-ATPase; (B) Ca-NDPase (thiamine pyrophosphatase); (C) acid inorganic pyrophos- phatase; (D) acid phosphatase (p-nitrophenylphosphate). Ordinate shows V values obtained for each substrate using sonicated high speed pellets as enzyme without addition of Triton X-100.
376
phospholipase A2 if serum albumin was omitted from the assay mixture, possibly due to an inhibitory effect of released long chain fatty acids.
DISCUSSION
The enzyme activity described is somewhat unique in not requiring Mg 2+, in contrast
to most ATPases [12, 26, 27]. ATPase activities that can utilize Ca 2+ and Mg 2+ equally well [28, 29] or are Mg2*-dependent but stimulated by Ca 2÷ [30] have been previously described. Among the few examples of an absolute and specific requirement for Ca 2+ are the high affinity Ca-ATPase from vascular smooth muscle [31], a macrophage plasma
membrane Ca-ATPase [18], Acanthamoeba myosin [32], and a Trypanosoma cruzi Ca-ATPase [33]. In agreement with present findings of E. histolytica enzyme, Mg 2÷ was inhibitory in two instances [31,32], and free ATP rather than the metal complex
was suggested to be the active substrate in the smooth muscle ATPase [31 ]. No informa- tion is presented as to how strict the Ca 2+ requirement is for the T. cruzi ATPase or the influence of Mg 2÷, probably reflecting the difficulty of separating this activity from the
much more active mitochondrial ATPase. However, the data [33] show a K a for Ca 2÷ 25 times greater than in E. histolyHca and present no evidence for cooperativity with respect to Ca 2÷. Although previous reports [2 -4 ] have described Mg-ATPase activities
in various Entamoeba species, it should be noted that the reported specific activities were
less than 5 mU/mg protein (total cell homogenate), about 8% of the Ca-ATPase. This is within the 10% activity measurable in the present study and is probably due to residual low levels of Ca 2+ in the cell homogenate or in the components of the assay mixture.
A notable feature of the E. histolytica ATPase is the tight control exerted on its kinetic properties by very low levels of Ca 2÷. This is well illustrated by the sudden loss of cooperativity at Ca 2÷ concentrations above 8 laM, and suggests the enzyme to be involved in some regulatory process. Biphasic kinetic plots, with respect to Ca 2+ concentration, exhibiting varying degrees of cooperativity have been reported for the Ca2+-stimulated
Mg-ATPase of erythrocytes [34]. The biphasic nature of such plots was attributed to the presence of a membrane-bound protein activator of the ATPase [35], synonymous with calmodulin [36, 37], a widely distributed calcium-binding protein.
A further striking property of the E. histolytica ATPase is its tight binding to mem- branes similar to the acid pyrophosphatase and nucleoside diph'osphatase of this species
[7]. The surface membrane ATPase of Neurospora crassa [38] has also been found to resist attempts at solubilization with procedures similar to those used in the present investigation. By contrast, most bacterial surface membrane ATPases can be displaced
by simple changes in pH or salt concentration [12] and the various extraction proce- dures tested in this study can be successfully employed in preparing mitochondrial, chloroplast and sarcoplasmic reticulum ATPases [27, 39]. In addition, Ca-ATPase and the pyrophosphatases give linear Arrhenius plots, possess low energies of activation and are unaffected by phospholipase treatment. This might indicate that either these enzymes (despite having quite different requirements for optimum activity) occupy physically
377
related membrane sites, or they occupy distinct sites in E. histolytica membranes that
exhibit little differentiation in lipid-protein interactions. Lipid might exert little in- fluence over the catalytic properties of these enzymes, as is apparently the case with the
membrane associated glycerol-3-phosphate dehydrogenase of Escherichia coli [40]. In addition, the high cholesterol content ofE. histolytica membranes [4] would be ex- pected to abolish phase transitions in the lipid bilayer [41]. It has in turn been demon- strated that incorporation of cholesterol eliminates the temperature breaks observed for several enzymes associated with cholesterol depleted membranes, including at least one
ATPase [42]. The subcellular localization of the Ca-ATPase remains unknown at present. Latency
is easily eliminated with sonication and detergents, suggesting that activity is within membrane limited structures. Other hydrolases of E. histolytica (acid phosphatase,
acid pyrophosphatase and nucleoside diphosphatase [7], plus/~-N-acetylglucosaminidase and neutral thiol proteinase (McLaughlin, J. and Lindmark, D.G., unpublished) also show latency and are in sedimentable structures. Isopycnic centrifugation in a sucrose gradient showed identical distribution patterns with a median equilibrium density of 1.15 g/ml for all these activities (Lindmark, D.G. and McLaughlin, J., unpublished).
These results might reflect their localization in the same particles or the cosedimentation of various particles including vesiculated fragments of the surface membrane. The latter possibility is supported by the results of fractionation ofE. histolytica after cell surface
labeling with ~2s I which suggested that Ca-ATPase is partly a component of the surface membrane whereas acid phosphatase is not [5]. The resolution of this question is ham- pered by the absence from Entamoeba species of suitable marker enzymes for the sur- face membrane, e.g. alkaline 5'-mononucleotidase (McLaughlin, J., unpublished) or Na-K-Mg-ATPase.
The functional role of the E. histolytica Ca-ATPase remains unknown, but it is tempt- ing to suggest a specific regulatory function, possibly in maintaining intracellular Ca 2÷ levels. In view of the comparatively high levels of PPi found in E. histolytica and its importance as an energy source [43], an ability to closely maintain low levels of Ca 2÷
would seem a distinct advantage.
ACKNOWLEDGEMENTS
We thank Dr. Louis S. Diamond (Bethesda, MD) for the E. histolytica strain and Mr. Donald Fishel for technical assistance. This research was supported by grant PCM 76 - 16657 from the National Science Foundation.
REFERENCES
1 Lloyd, D., Lindmark, D.G. and Miiller, M. (1979) Adenosine triphosphatase activity of Tri- trichomonas foetus. J. Gen. Microbiol. 115, 301- 307.
2 McLaughlin, J. and Meerovitch, E. (1976) The surface membrane and cytoplasmic membranes of Entamoeba invadens (Rodhain 1934). Comp. Biochem. Physiol. 52B, 477-486.
378
3 Van Vliet, M.M.D.M., Spies, F., Linnemans, W.A.M., Klepke, A., Op den Kamp, J.A.F. and van Deenen, L.L.M. (1976) Isolation and characterization of subcellular membranes of Entamoeba invadens. J. Cell Biol. 71 ,357-369.
4 Serrano, R., Deas, J.E. and Warren, L.G. (1976) Entamoeba histolytica: Membrane fractions. Exp. Parasitol. 41 ,370-384 .
5 Aley, S.B., Scott, W.A. and Cohn, Z.A. (1980) Plasma membrane ofEntarnoeba histolytica. J. Exp. Med. 152,391 400.
6 McLaughlin, J. and Miiller, M. (1979) Calcium dependent ATPase in Entamoeba histotytica. J. Protozool. 26, 10A.
7 McLaughlin, J., Lindmark, D.G. and Mfiller, M. (1978) Inorganic pyrophosphatase and nucleo- side diphosphatase in the parasitic protozoon, Entamoeba histolytica. Biochem. Biophys. Res. Commun. 82 ,913-920.
8 McLaughlin, J. and Meerovitch, E. (1976) A simple and sensitive modification of the Chen procedure for orthophosphate determination in the presence of Triton X-100. Anal. Biochem. 70 ,643-644.
9 Ogawa, Y., Marigaya, S., Ebashi, S. and Lee K.S. (1971) Sarcoplasmic reticulum: Calcium uptake and release systems in muscle. In: Methods in Pharmacology, (Schwartz, A., ed.), Vol. 1, pp. 327- 346, Appleton-Century-Crofts, New York, NY.
10 Leighton, F., Poole, B.. Beaufay, H., Baudhuin, P., Coffey, J.W., Fowler, S. and de Duve, C.
(1968) The large-scale separation of peroxisomes, mitochondria, and lysosomes from the livers of rats injected with Triton WR-1339. J. Cell Biol. 37 ,483-513.
11 Dixon, M. and Webb, E.C. (1964) Enzymes, P. 440, 2nd edition, Academic Press, New York. 12 Abrams, A. and Smith, J.B. (1974) Bacterial membrane ATPases. In: The Enzymes, (Boyer, P.
D., ed.), Vol. 10, pp. 395-429, Academic Press, New York. 13 Bowman, B.J. and Slayman, C.W. (1977) Characterization of plasma membrane adenosine
triphosphatase ofNeurospora crassa. J. Biol. Chem. 252, 3357- 3363. 14 Eisenthal, R. and Cornish-Bowden, A. (1974) The direct linear plot. A new graphical procedure
for estimating enzyme kinetic parameters. Biochem. J. 139,715-720. 15 Scherr, F. and Gunther, T. (1978) Inhibition ofMg, Ca-ATPase fromEscherichia coli by ruthenium
red. Z. Naturforsch. 33C, 61-64 . 16 Watson, E., Vincenzi, F.F. and Davis, P.W. (1971) Ca2÷-~ictivated membrane ATPase: Selective
inhibition by ruthenium red. Biochim. Biophys. Acta 249,606-610. 17 Cooper, P.H. and Stanworth, D.R. (1976) Characterization of a calcium ion activated adenosine
triphosphatase in the plasma membrane of rat mast cells. Biochem. J. 156,691-700. 18 Gennaro, R., Mottola, C., Schneider, C. and Romeo, D. (1979) Ca 2 *-dependent ATPase activity
of alveolar macrophage plasma membrane. Biochim. Biophys. Acta 567, 238-246. 19 Silvius, J.R., Read, B.D. and McElhaneny, R.N. (1978) Enzymes: Artifacts in Arrhenius plots
due to temperature dependence of substrate-binding affinity. Science 199,902-904. 20 Lenaz, G., Sechi, A.M., Parenti-Castelli, G., Landi, L. and Bertoli, E. (1972) Activation energies
of different mitochondrial enzymes: Breaks in Arrhenius plots of membrane bound enzymes occur at different temperatures. Biochem. Biophys. Res. Commun. 49, 536 542.
21 Watson, K., Bertoli, E. and Griffiths, D.E. (1973) Phase transitions in yeast mitochondrial mem- brane. The transition temperatures of succinate dehydrogenase and F~-ATPase in mitochondria of aerobic and anaerobic cells. FEBS Lett. 30, 120-124.
22. Saeki, H., Nozawa, Y., Shimonaka, H., Kawai, K., lto, M. and Yamamoto, M. (1979) Effects of anesthetics, dibucaine and methoxyflurane on the ATPase activity and physical state of Tetra- hymena surface membranes. Biochem. Pharmacol. 28, 1095-1098.
23 Raison, J.K., Lyons, J.M. and Thomson, W.W. (1971) The influence of membranes on the tem- perature-induced changes in the kinetics of some respiratory enzymes of mitochondria. Arch. Biochem. Biophys. 142, 83-90.
379
24 Fourcans, B. and Jain, M.N. (1976) Role of phospholipids in transport and enzymatic reactions. In: Advances in Lipid Research, (R. Paoletti and D. Kritchevsky, eds.), Vol. 12, pp. 147-226 , Academic Press, New York.
25 Lenaz, G. (1979) The role of lipids in the structure and function of membranes. In: Subcellular Biochemistry, (Roodyn, D.B., ed.), Vol. 6, pp. 233 -243 , Plenum Press, New York.
26 Penefsky, H.S. (1974) Mitochondrial and chloroplast ATPases. In: The Enzymes, (Boyer, P.D., ed.), Vol. 10, pp. 375-394, Academic Press, New York.
27 Tada, M., Yamamoto, T. and Tonomura, Y. (1979) Molecular mechanism of active calcium transport by sarcoplasmic reticulum. Physiol. Rev. 58, 1 - 79.
28 Shami, Y. and Radde, I.C. (1971) Calcium-stimulated ATPase of guinea pig placenta. Biochim. Biophys. Acta 249 ,345 -352 .
29 Mirsky, R. and Barlow, V. (1971) Purification and properties of ATPase from the cytoplasmic membrane of Bacillus megaterium KM. Biochim. Biophys. Acta 2 4 1 , 8 3 5 - 345.
30 Vincenzi, F.F. and Hinds, T.R. (1976) Plasma membrane calcium transport and membrane bound enzymes. In: The Enzymes of Biological Membranes, (Martonosi, A., ed.), Vol. 3, pp. 261 - 281.
31 Thorens, S. (1979) Ca2÷-ATPase and Ca uptake without requirement of Mg 2÷ in membrane frac- tions of vascular smooth muscle. FEBS Lett. 98 ,177-180 .
32 Maruta, M. and Korn, E.D. (1977)Acanthamoeba myosin. J. Biol. Chem. 252, 6501-6509 . 33 Frasch, A.C.C., Segura, E.L., Cazzulo, J.J. and Stoppani, A.O.M. (1978) Adenosine triphos-
phatase activities in Trypanosoma cruzi. Comp. Biochem. Physiol. 60B, 271 - 275. 34 Scharf, O. (1976) Ca 2÷ activation of membrane-bound (Ca2÷)-dependent ATPase from human
erythrocytes prepared in the presence or absence of Ca 2÷. Bioehim. Biophys. Acta 443, 2 0 6 - 218.
35 Scharf, O. and Focler, B. (1978) Reversible shift between two states of Ca2*-ATPase in human erythrocytes mediated by Ca 2÷ and a membrane-bound activator. Biochim. Biophys. Acta 509, 6 7 - 7 7 .
36 Luthra, M.G., Hildebrandt, G.R. and Hannahan, D.J. (1976) Studies on an activator of the (Ca2÷-Mg2÷)-'ATPase of human erythrocyte membranes. Biochim. Biophys. Aeta 419, 164-179.
38 Scarborough, G.A. (1977) Properties of the Neurospora crassa plasma membrane ATPase. Arch. Biochem. Biophys. 180, 384-393 .
39 Hasselbach, W. (1974) Sarcoplasmic membrane ATPase. In: The Enzymes, (Boyer, P.D., ed.), Vol. 10, pp. 431 -467 , Academic Press, New York.
40 Mavis, R.D. and Vagelos, P.R. (1972) The effect of phospholipid fatty acid composition on membranous enzymes in E. coli. J. Biol. Chem. 247 ,652 -659 .
41 Chapman, D. (1973) Some recent studies of lipids, lipid-cholesterol and membrane systems. In: Biological Membranes, (Chapman, D. and WaUaeh, D.F.A., eds.), Vol. 2, pp. 91 -144 , Academic Press, London.
42 Rottem, S., Cirillo, V.P., De Kruyff, B., Shinitzky, M. and Razin, S. (1973) Cholesterol in Myco- plasma membranes: Correlation of enzymic and transport activities with physical state of lipids in membranes of Mycoplasma mycoides varicapri adapted to grow with low cholesterol concen- tration. Biochim. Biophys. Acta 323 ,509-515 .
43 Reeves, R.E. (1976) How useful is the energy in inorganic pyroph0sphate. Trends Biochem. SCI. 1, 5 3 - 5 5 .
