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Mg2+ ATPase
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 263, No. 2, June, pp. 281-292,1988
Characterization
of Rat Heart Plasma Membrane DAYUAN
ZHAO
AND NARANJAN
Ca2+/Mg2+ ATPase’
S. DHALLA’
Division of Cardiovascular Sciences, St. B&face General Hospital Research Centre and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada RZH 2A6 Received
November
9,1987,
and in revised
form
February
81988
The Ca2’/Mg2’ ATPase of rat heart plasma membrane was activated by millimolar concentrations of Ca2+or Mg2+* other divalent cations also activated the enzyme but to a lesser extent. Sodium azide at high concentrations inhibited the enzyme by about 20%; oligomycin at high concentrations also inhibited the enzyme slightly. Trifluoperazine at high concentrations was found inhibitory whereas trypsin treatment had no significant influence on the enzyme. The rate of ATP hydrolysis by the Ca2+/Mg2+ ATPase decayed exponentially; the first-order rate constants were 0.14-0.18 min-’ for Ca2+ ATPase activity and 0.15-0.30 min-’ for Mg ‘+ ATPase at 37°C. The inactivation of the enzyme depended upon the presence of ATP or other high energy nucleotides but was not due to the accumulation of products of ATP hydrolysis. Furthermore, the inactivation of the enzyme was independent of temperature below 37°C. Con A when added into the incubation medium before ATP blocked the ATP-dependent inactivation; this effect was prevented by a-methylmannoside. In the presence of low concentrations of detergent, the rate of ATP hydrolysis was reduced while the ATP-dependent inactivation was accelerated markedly. Both Con A and glutaraldehyde decreased the susceptibility of Ca2+/Mg2+ ATPase to the detergent. These results suggest that the Ca2+/Mg2+ ATPase is an intrinsic membrane protein which may be regulated by ATP. Q 19% Academic press. I,,~. It is now well established that the Ca’+/ Mg2+ ATPase in various types of mammalian cell membranes (l-7) is different from the Na+-K+ ATPase (Na+ pumping ATPase) or Cazf-stimulated ATPase (Ca2+ pumping ATPase). The Ca2+/Mg2+ ATPase has been suggested to be involved in cell surface processes such as active transport, ameboid motion, and regulation of cell shape (8, 9). The heart sarcolemmal Ca2+/Mg2+ ATPase has been suggested to be involved in the gating mechanism for the translocation of divalent cations across the cell membrane (1, 10-13). Although the exact role of the enzyme in the heart cell function is not clear, the signifir The work reported in this study was supported by a grant from the Medical Research Council of Canada. Dayuan Zhao was a predoctoral fellow of the Canadian Heart Foundation. s To whom correspondence should be addressed. 281
cance of the enzyme activity can be appreciated from the fact that the Ca2+/Mg2+ ATPase activity was depressed in different types of diseased hearts (14,15) as well as by various cardiodepressant agents such as propranolol (16), antiarrhythmic drugs (17), and divalent cations including Co’+ Ni2+, and Mn2+ (18). In order to elucidate the enzyme function, previous work from our laboratory has described some properties of the Ca2+/Mg2+ ATPase in heart sarcolemma prepared by the hypotonic shock-LiBr treatment method (1, 2, 11, 19). This heavy sarcolemmal fraction, which is isolated by low-speed centrifugation, has been shown to differ from the light membrane fraction, which sediments only at high centrifugal force (20). Furthermore, the heavy sarcolemmal fraction from the myocardium, unlike the light membrane fraction, was demonstrated to contain the basement membrane and 0003-9861188 $3.90 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form resewed.
282
ZHAO AND DHALLA
other cell surface material (21). It should also be pointed out that the heavy sarcolemma1 fraction was found to orient itself into predominantly right-side-out vesicles (about 90%) whereas the light membrane fraction from heart showed the predominant presence of inside-out vesicles (80 to 90%) (21-23). The specific activities of various marker enzymes of the plasma membrane were much lower in the heavy sarcolemmal fraction in comparison to those in the light membrane preparation (20,21). In view of these differences in the heavy sarcolemmal and light membrane fractions, it is thought worthwhile to characterize the Ca’+/Mg’+ ATPase activity in the light plasma membrane preparation from the heart. For this purpose, we have employed plasma membranes isolated from the rat heart by the sucrose density gradient technique (22), which are reported to exhibit about 10 times higher activity of Ca2+/Mg2+ ATPase in comparison to that of the heavy sarcolemmal preparation (20). MATERIALS
AND METHODS
Materials. ATP, GTP, ITP, UTP, CTP, ADP, pNPP,’ sodium azide, 2,4-dinitrophenol, trypsin inhibitor, PCMB, trifluoperazine, Ca*+ ionophore A2318’7, valinomycin, concanavalin A, Triton X-100, diadenosine pentaphosphate (AP&, and lysophosphatidylcholine (lysolecithin) were obtained from Sigma Chemical Co. (St. Louis, MO). Oligomycin and FCCP were from Aldrich Chemical Co. (Milwaukee, WI). All other chemicals were of analytical grade. Preparation of rat heart plasma membranes and ATPase measurements. The plasma membranes were isolated from the rat heart by the sucrose density gradient method according to Pitts (22). On the basis of extensive marker enzyme studies (20, 23), this membrane fraction was found to contain minimal (1 to 3%) cross contamination by other subcellular organelles. The Ca2+ ATPase or Mg2+ ATPase activity was assayed at 3’7°C in 1 ml assay medium containing 50 mrd Tris-HCl, pH 7.5, 4 mM Tris-ATP, and 4 mM CaClz or MgClz. The final protein concentration was
3Abbreviations used: Con A, concanavalin A; FCCP, carbonyl cyanide ptrifluoromethoxyphenylhydrazone; SDS, sodium dodecyl sulfate; PCMB, pchloromercuribenzoic acid; APJ, diadenosine pentaphosphate; pNPP, pnitrophenyl phosphate.
0.02 to 0.03 mg/ml. The reaction was started by addition of ATP and stopped 1 min after by the addition of 0.5 ml 10% SDS. Experimental conditions that differed from these described here are given in the text. The Ca2+ ATPase or Mg2+ ATPase activity was calculated as difference between the amount of Pi formed in the presence of 4 mM Ca2+ or Mg” and that in the absence of any divalent cation. The estimation of Pi was carried out by the method of Taussky and Shorr (24) whereas protein concentration was determined by the method of Lowry et al. (25). Unless otherwise indicated at least three different preparations were employed for each observation. RESULTS
The plasma membrane preparation was found to hydrolyze ATP in the presence of Ca2+ as well as Mg2+. The time course of the enzyme reaction (Table I) revealed that the ATPase activities started decreasing 2 min after starting the incubation with ATP. However, the reaction was linear with respect to the protein concentration in the range lo-50 pg/ml. Accordingly, all experiments reported in the study were carried out for 1 min by using 20-30 pg membrane protein in the incubation medium. In one set of experiments, the ATPase activities were measured by varying the concentrations of either Ca2+ or Mg2+ in the presence of 4 mM ATP whereas in the other set, the concentration of ATP was varied in the presence of 4 TABLE
I
Ca’+/M%+ ATPase ACTIVITIESOF HEART SARCOLEMMAATDIFFERENTTIMESOFINCUBATION Time of incubation (min) 1 2 4 8 16
Ca*+ ATPase activity (pm01 Pi/mg/min) 5.9 5.7 3.5 2.2 1.3
f 2 + + +
0.9 0.8 0.4 0.1 0.3
Mgz+ ATPase activity (pm01 Pi/mg/min) 7.6 + 6.7 + 3.5 * 2.1 f 0.9 +
1.4 1.4 0.5 0.3 0.1
Note. The Ca2’/Mgz’ ATPase activities were determined in aliquots taken at different intervals after the addition of 4 mM ATP in a medium containing 4 mM Ca2+ or 4 mM Mgz+. The results represent the rates of ATP hydrolysis during the time periods indicated. Each value is a mean & SE of six experiments.
RAT TABLE KINETIC
PARAMETERS MEMBRANE
HEART
PLASMA
MEMBRANE
TABLE
OF THE RAT HEART Ca2+/Mg2+ ATPase
PLASMA
EFFECT
0.23 * 0.03 7.03 -+ 0.66 0.69 + 0.40 6.73 f 0.80
0.33 * 0.04 9.32 * 0.64 0.71 f 0.40 8.82 + 0.53
Note. ATPase activities were determined by varying the concentration of ATP and keeping the concentrations of Ca*+ or Mg2+ at 4 mbl as well as by varying the concentration of Ca’+ or M$+ and keeping the concentrations of ATP at 4 mbx. The Lineweaver--Burk plots of the data revealed K,, K,,, and V,, values. Each value is a mean f SE of four experiments.
mM Ca2’ or Mg2+. Both Ca2+ ATPase and Mg2+ ATPase were fully activated in the presence of 4 mM ATP and 4 KIM Ca2+ or 4 IllM Mg2+, respectively. The LineweaverBurk plot analysis of the data revealed K, and Km values (Table II) similar to those reported by employing the heavy sarcolemma1 preparations (1, 11,19). However, the V,,, values for both Ca2+ ATPase and Mg2+ ATPase were 10 to 15 times higher than those reported earlier (1, 11,19). Other divalent cations also activated the ATPase activity but were much lesser effective than Mg2+ or Ca2+ (Table III).
III
CATION SPECIFICITY FOR THE ATP BY RAT HEART PLASMA MEMBRANE
Cations
Relative ATP hydrolysis
Cations
Relative ATP hydrolysis
Mg2 Ca2+ co2+ Mn2+ Sr2+
100 75 36 22 14
Cds’ Ni2+ Zn2+ Ba2’ cu2+
11 8 5 0 0
Note. Membranes were incubated in the presence of 4 mM ATP and 4 mM of different cations. The ATP hydrolysis due to the presence of each cation was expressed as a percentage of that in the presence of Mg-.
None W BaP+ 512+ Mn’+ cop+ Nis+ Cd2+ CU+ Zn2+ Las’ Na+ K+ HCO, Vanadate
283 IV
OF DIFFERENT IONS ON THE RAT HEART PLASMA MEMBRANE Ca2+/M8 ATPase ACTIVITIES
Mg2 ATPase Ions added
A. Varying ATP concentrations K,,, for ATP (mu) V,, (pm01 Pi/mg/min) B. Varying cation concentrations K. for Cae+ or Mp (mM) V,, (pm01 PJmg/min)
TABLE
ATPase
II
Ct?' ATPase
DIVALENT HYDROLYSIS
Ca”/Mg’*
Concentration (mW
Relative Gas+ ATPase activity
4 4 4 4 4 4 4 4 4 4 100 100 50 1
Note. Membranes were incubated ATP and 4 mM of Cap+ or Me, and tions of different ions. The ATPase as percentages of Ca*+ ATPase or sence of any other ions.
100 133 109 94 41 38 21 17 13 10 100 97 93 102 96
Me
Relative ATPase activity 100 102 102 88 30 30 12 9 11 1 99 90 90 89 81
in the presence of 4 mM the indicated concentraactivities are expressed Mgs+ ATPase in the ab-
However, in the presence of Ca2+ or Mg2+, most of divalent cations were inhibitory; Zn2’, Cd2+, and Cu2+ were most inhibitory, while Ba2+ was least inhibitory (Table IV). The Ca2+/Mg2+ ATPase activities were not altered by 4 mrvx La3+; 100 mM NaCl, 100 mM KCl, 50 mM NaHC03, and 1 mM vanadate slightly inhibited the Mg2+ ATPase activity. Table V shows the effects of the different inhibitors on the ATPase activity. Trifluoperazine at a low concentration (0.01 mM), which is known to inhibit calmodulin, showed negligible effect whereas in high concentrations (0.02 to 0.05 mM) it produced inhibition. The I& for trifluoperazine varied between 20 and 50 PM. PCMB at high concentrations depressed the Ca2+/Mg2+ ATPase activities. Both oligomyocin and sodium azide at high concentrations also inhibited the Ca2+/ Mg2+ ATPase activities (Table V). Kinetic study of the sodium azide effect showed that this inhibition was apparently competitive with a Ki = 10 mM for both Mg2+ ATPase activity and Ca2+ ATPase activity
284
ZHAO TABLE
AND
V
EFFECTOFINHIBITORSONTHERATHEARTPLASMA MEMBRANE Ca’+/Mp ATPase Ca2+ ATPase Inhibitors
(% inhibition hydrolysis)
Mg2f
ATPase
of ATP
Trifluoperazine 0.01 mM
9
0.02mM 0.05mM
24 63
4 17 60
46 49
36 50
0 13
10 11
0 0
3 6 16 26
PCMB 0.25 PM 250 PM Oiigomycin 3 pgg/ml 30 pg/ml Sodium azide 0.1 mM
1mM 3mM 10 mM
3 22
Note Membranes were preincubated with the inhibitor at 37’C for 3 min. In the case of oligomycin and PCMB, where -0.5-5% ethanol was used in assay medium, control samples were treated with corresponding concentrations of ethanol in the assay medium.
(Fig. 1). Several other agents such as Ca2+ inophore A23187, FCCP, 2,4,-dinitrophenol, gramicidine D, and valinomycin, were found to have no significant effect on the heart membrane Ca2+/Mg2+ ATPase activity (data not shown). Earlier studies (12,13) have shown that treatment of the heavy sarcolemmal preparation with trypsin resulted in the release of Ca2+-dependent ATPase in the supernatant and increase in the Ca2+/Mg2+ ATPase activities in the membrane due to removal of cell surface material. In this study, we examined the effect of trypsin (20 to 300 pg/mg membrane protein) for 5 to 60 min under the experimental conditions described previously (12). Our results indicated that about 80% of protein remained in membrane fraction after trypsin treatment whereas the specific activity of Ca2+/Mg2+ ATPase was increased by about 20% (data not shown). The total activity of the enzyme in the plasma membrane was not changed by trypsin
DHALLA
and no detectable Ca2+-dependent ATPase was found in the supernatant. In one series of experiments, the time course of ATP hydrolysis due to Ca2+/ Mg2+ ATPase was investigated (Fig. 2). We found that the rate of ATP hydrolysis by the Ca2+/Mg2+ ATPase decayed exponentially. The first-order rate constant of inactivation was 0.15 to 0.30 min-’ for Mg2+-ATPase activity and 0.14 to 0.18 min-’ for Ca2+ ATPase activity at 37°C. The Ca2+/Mg2+ ATPase activities were not further inactivated after they were reduced to 20 to 30% of initial ATP hydrolyzing rate. In the absence of ATP, the inactivation rate was about 150 times lower than that in the presence of ATP (Figs. 2 and 3). Addition of 1 InM KH2POI, ADP, and AMP in the assay medium did not show significant change in the initial rates of ATPase activities; similar results were obtained when the reaction was studied in the presence of AP5A, a well-known
i F ?v
0.6 0.4
0
2
4
6
8
l/[ATP]
FIG. 1. Effect of 5 mM NaNa membrane Caa’/Mga+ ATPase. was measured in the presence and varying concentrations of presence (0) or absence (0) of Lineweaver-Burk plots.
on the heart plasma The ATPase activity of 4 mM Ca2+ or Mga+ ATP. The data in the NaNa are presented as
RAT HEART
2 4 6 6 10 12 14 16 0 2 4 Time hid
PLASMA
6
MEMBRANE
8 10 12 14 16 Time (mid
FIG. 2. Rates of ATP hydrolysis by the heart plasma membrane Ca’+/Mg’+ ATPase. (A) Time course of ATP hydrolysis by Ca2+ ATPase in the absence (0) and presence of 20 rg/ml Con A added before (0) or 2 min (A) and 7 min (0) after the addition of ATP; (B) rate of ATP hydrolysis by Ca2* ATPase calculated from the data in A, (C) time course of ATPase hydrolysis by Mg2+ ATPase; (D) rate of ATP hydrolysis by Mg ‘+ ATPase in the absence (0) and presence of Con A added before (0) or 2 min (A) and 7 min (0) after the addition of ATP. The data for ATPase activity are plotted on a semilogarithmic graph.
inhibitor of adenylate kinase (Table VI). This indicated that the inactivation was not due to the product inhibition but was likely an ATP-dependent inactivation. Other high energy nucleotides like CTP, UTP, GTP, ITP, which were hydrolyzed by the ATPase at a rate similar to that for ATP, also induced similar inactivation of the enzyme (Table VII). ADP, on the other hand, was hydrolyzed at a lower rate in comparison to that for ATP but inactivated the enzyme at a higher rate (Table VII). It should also be noted from Table VI that ADP increased the inactivation rate of the enzyme due to ATP. pNPP was not hydrolyzed under same conditions, indicating that the ATPase activity was not due to a nonspecific phosphatase. After pretreating the plasma membrane with ATP for 30 min, when the ATPase activity
Ca’+/Mg*+
ATPase
285
was reduced to 20-30s of original ATP hydrolyzing rate, removal of ATP partially restored the enzyme activity; however, the inactivation rate was not influenced by ATP pretreatment (Table VIII). The pH profiles of the Ca2+/Mg2+ ATPase revealed that the maximal ATPase activity was at pH 7.5, while the inactivation rate of Mg2+ ATPase, unlike Ca2+ ATPase, was lower at the alkaline side (Fig. 4). The ATP-dependent inactivation rate of the enzyme seems to be independent of temperature below 37°C (Fig. 5). The energy of activation of ATP hydrolysis determined from an Arrhenius plot of the data was 11.5 kcal/mol for Ca2+ ATPase and 11.4 kcal/mol for Mg2+ ATPase. Con A, a lectin, was found to prevent ATP-dependent inactivation partially (Fig. 2). Pretreating the membrane with Con A did
Ca*+ATPacle
37%
1 10
20
30 Time
40
50
60
70
(hr)
FIG. 3. Rate of inactivation of the heart plasma membrane Ca*+/Mg’+ ATPase in the absence of ATP. The membranes (0.3 mg/ml) were incubated for 24 to ‘72 h at the indicated temperatures. Aliquots were removed at various times and the initial ATPase activities were measured at 3’7°C. The data are plotted on a semilogarithmic graph.
286
ZHAO
AND
DHALLA
TABLE
VI
EFFECTSOF ADP, AMP,AND Pi ONTHEHEARTPLASMAMEMBRANE ~~~~~~~~~~~~~~~~~~~~~~~ PRESENCEOFDIADENOSINEPENTAPHOSPHATE(AP&) Without Initial (pm01 Control Ca*’ ATPase Mga+ ATPase ADP (1 mM) Ca*’ ATPase M%+ ATPase AMP (1 mM) Cal+ ATPase Mga+ ATPase Pi (1 mM) Ca2+ ATPase Mga+ ATPase ADP (1 mM) + Ca” ATPase Me ATPase
With
APSA
ATPase rate Pi/mg/min)
Inactivation rate constant (min-‘)
Initial (pm01
AP&
ATPase rate Pi/mg/min)
Inactivation rate constant (min-‘)
6.7 8.9
0.15 0.15
5.2 6.3
0.15 0.14
7.8 9.2
0.26 0.31
6.8 9.0
0.17 0.20
6.9 8.7
0.13 0.17
6.5 8.3
0.14 0.14
7.5 8.3
0.14 0.15
4.8 6.7
0.15 0.18
7.2 8.4
0.26 0.26
5.2 6.5
0.25 0.31
Pi (1 mM)
Note. ATPase activities were assayed used as Pi. The concentration of APr,A
in the absence was 10 PM.
not alter the initial hydrolysis rate Ca2+/Mg2+ ATPase, but reduced the dependent inactivation rate constant 0.21 min-’ for Mg2+ ATPase and 0.14
or presence
of the ATPfrom min-’
of various
agents
as indicated.
KHaPOl
was
for Ca2+ ATPase to 0.07 and 0.05 min-‘, respectively. The enzyme activities were also stabilized at a much higher level than that in the absence of Con A. However,
TABLE
VII
SUBSTRATESPECIFICITYANDINACTIVATIONRATECONSTANTSFORTHEHEARTPLASMA MEMBRANE Ca*+/Mp ATPase In the presence
Substrate
Hydrolysis rate (pmol/mg/min)
ATP CTP ITP UTP GTP ADP Note. Membranes nucleotides.
of 4
mM Ca2+ Inactivation rate constant (min-‘)
9.3 10.8 8.2 8.9 7.9 1.9 were
incubated
In the presence
Hydrolysis rate (pmol/mg/min)
0.18 0.18 0.18 0.18 0.21 0.32 in the assay
medium
12.3 8.2 10.9 9.9 8.8 1.9 containing
50
mM Tris-HCl,
of 4
mM
Mga+
Inactivation rate constant (min-‘) 0.30 0.32 0.16 0.21 0.25 0.41 pH 7.5, and 4 mM of
RAT
HEART
PLASMA
MEMBRANE TABLE
EFFEC~OFWASHINGONTHEHEARTPLASMA
Caa+/Mg’+
VIII
MEMBRANE Ca2+/M%f
AFTER~RETREATMENTWITH
Initial rate of ATPase (pmol/mg/min) Preincubation medium
Ca2+ ATPase
Control (MgCla) M&la + ATP Control (CaC12) CaCla + ATP
Inactivation rate constant ATPase (mini)
Mga+ ATPase
6.3 5.2 6.8 3.8
287
ATPase
Ca2+ ATPase
7.5 5.5 6.7 5.2
Me
0.21 0.23 0.23 0.21
ATP of
ATPase 0.21 0.21 0.16 0.23
for 30 min at 22°C in a medium containing 50 IIIM Tris-HCl, pH 7.5,4 mM of these membranes were markedly depressed (20 to 30% of the control). Control samples were incubated in medium without the ATP. The samples were then centrifuged at 100,OOOg for 30 min and the pellet was thoroughly washed and resuspended in 50 mM Tris-HCl, pH 7.5, buffer and used for ATPase activity assay. Note.
Membranes
were
incubated
ATP, 4 mM CaC12 or MgC12. The activities
Con A was found to interact with heart membrane Ca2+/Mg2+ ATPase slowly because adding Con A 2 or ‘7 min after the addition of ATP failed to prevent the inactivation immediately (Fig. 2). The effects of Con A on the inactivation of the enzyme were blocked completely by the presence of 50 MM cr-methylmannoside; this indicates that Con A protects the en-
15r
A-._
zyme by binding to oligosaccharide residues, which may form part of the enzyme structure. In the presence of detergents
, 0.15
FIG. 4. pH profiles of the heart plasma membrane Ca2+/Mg2+ ATPase. ATPase activities were measured as described under Materials and Methods except that 50 mM Bistris buffer was used in all pH ranges instead of Tris buffer. (0) The rate of Caa+ ATPase activity; (A) the rate of Mg2+ ATPase activity; (0) and (A) inactivation rates of Ca2’ ATPase and Mga+ ATPase, respectively.
1'
2
4
6
Tii
810
(mid
FIG. 5. Rate of inactivation of the heart plasma membrane Ca2+/Mg a+ ATPase in the presence of ATP. ATPases were assayed at 22°C (O), 30°C (+), 3’7°C (A), and 43°C (0).
288
ZHAO AND DHALLA
such as Triton X-100 and lysophosophatidylcholine, the ATP hydrolyzing rate of the ATPase was reduced, while the rate of ATP-dependent inactivation was accelerated (Fig. 6). Con A blocked the effects of the detergents on the ATP-dependent inactivation rate at all concentrations of the detergents tested, and in the case of lysophosphatidylcholine, partially restored the initial ATP hydrolyzing rate of the enzyme (Table IX). Glutaraldehyde, decreasing the ATP hydrolyzing rate and increasing the ATP-dependent inactivation rate by itself, was able to prevent the effects of the detergents in both aspects (Table X).
DISCUSSION
In this study we have shown that the plasma membranes obtained from rat hearts exhibited a highly active ATPase system, which is capable of hydrolyzing high energy nucleotides in the presence of either Ca2+ or Mg 2+. Although the affinities of the enzyme in this light plasma membrane for Ca2+ or Mg2+ (K, = 0.69 to 0.71 mM) as well as for ATP (K, = 0.23 to 0.33 mM) were similar to those reported for the Ca2+/Mg2+ ATPase in heavy sarcolemma1 fraction (1, 11, 19); the maximal velocities (V,,, = 7.0 pm01 Pi/mg/min for Ca2+ ATPase and 9.3 pmol Pi/mg/min for Mg2+ ATPase) for the light fraction were 10 to 15 times higher than those for the heavy membrane fraction. Such a difference in two types of the heart membrane fraction is primarily due to the presence of cell surface material in the heavy sarcolemma1 fraction. This view is supported by the fact that trypsin treatment, unlike the heavy sarcolemmal fraction (12), was not capable of increasing the Ca2+/Mg2+ ATPase activity in the plasma membrane. However, the patterns of cation activation, substrate specificity, and inhibition exerted by several divalent cations observed in this study as well as those reported earlier (1, 11, 19) suggest that Ca2+/Mg2+ ATPase present in the plasma membranes (light fraction) is similar to
10.0 8.0
Ca*+ATPase
4.0
l t
1.0 0.8 -2 $ P t 5 E
2
l .
2.0 .
0.4 0.2 s’
A-
10.0 8.0
Mg*+ATPass
A
.= .L
40.
2 b3
2.0
A . . .
1.0 0.8
a 0.4
.
0.2
D ‘i
012345 Time (mid
12345 Time (mid
FIG. 6. Effect of Triton X-100 (A, B) and lysophosphatidylcholine (C, D) on the heart plasma membrane Ca*+/Mg*+ ATPase activity. ATPase activities were assayed in the presence of 1 pg/ml (+), 10 pg/ml (A), and 50 pg/ml (0) Triton X-100 or 10 fig/ml (*), 100 fig/ml (A), and 1 mg/ml (0) lysophosphatidylcholine.
that present in the sarcolemmal membranes (heavy fraction). Nonetheless, it should be noted that unlike the heavy sarcolemmal membranes (12, 13), the light plasma membranes did not contain the Ca2+-dependent ATPase, which is not activated by Mg 2+. The observed depression in the activity of Ca2+/Mg2+ ATPase upon treating the membrane with detergents as well as the insensitivity of this enzyme to trypsin digestion suggests that the Ca2+/ Mg2+ ATPase is an intrinsic membrane protein. Millimolar concentrations of Ca2+ or Mg2+ have also been reported to activate the mitochondrial ATPase (26, 27) and in fact azide-sensitive Mg2+-ATPase is usually used as an indication of the mitochondrial contamination in subcellular
RAT
HEART
PLASMA
MEMBRANE TABLE
Ca*‘/Mg*’
289
ATPase
IX
EFFECT OF CON A ON THE HEART PLASMA MEMBRANE Ca2’/Mga+ INTHEPRESENCEOFDETERGENTS
ATPase
INCUBATED
Control Initial
ATPase rate (pmol/mg/min)
Detergents No detergent Ca*+ ATPase Mga+ ATPase Triton X-100, 1 pg/ml Ca*+ ATPase Mga+ ATPase Triton X-100, 10 pg/ml Ca*+ ATPase Mgs+ ATPase Triton X-100, 50 rg/ml Ca*+ ATPase Mga+ ATPase Lysolecithin, 10 fig/ml Ca*+ ATPase Mga+ ATPase Lysolecithin, 100 /*g/ml Ca2+ ATPase Mg2f ATPase Lysolecithin, 1 mg/ml Ca*+ ATPase MgZf ATPase
Note. ATPase
activities
were
Con A Inactivation rate constant (mini)
Initial
ATPase rate (fimol/mg/min)
Inactivation rate constant (min-‘)
6.6 9.1
0.14 0.21
5.8 9.4
0.05 0.07
6.1 8.0
0.28 0.39
5.6 7.6
0.16 0.12
5.8 8.5
0.30 0.55
6.0 8.0
0.16 0.14
6.7 5.9
1.15 2.65
4.5 5.9
0.35 0.35
5.5 6.6
0.28 0.28
7.4 8.5
0.16 0.16
3.9 4.5
1.29 0.92
6.3 8.3
0.32 0.25
3.6 3.0
1.57 1.40
6.1 6.6
0.28 0.48
measured
in the absence
or presence
TABLE
of concanavalin
A (50 fig/ml).
X
EFFECTOFGLUTARALDEHYDEONTHEHEARTPLASMAMEMBRANEC~~+/M~~+ATP~~~ Initial ATPase (~mol/mg/min) Ca*’ Untreated Treated Treated + Triton X-100, 50 rg/ml Treated + lysolecithin, 1 mg/ml
ATPase
rate
Me
Inactivation
ATPase
Ca2+ ATPase
rate (mini)
constant
M$+
ATPase
6.3 5.3
7.5 5.2
0.21 0.37
0.21 0.37
5.5
5.8
0.39
0.51
5.3
5.7
0.55
0.55
Note. Membranes (0.3 mg/ml) was incubated at 1°C in 50 mM Tris-HCl, pH 7.5, with or without 0.25% glutaraldehyde for 30 min. Aliquots (100 11) were diluted with 1 ml of ATPase assay medium to measure ATPase activity.
290
ZHAO
AND
preparations (20). In this study we have shown that low concentrations of sodium azide, which markedly inhibited mitochondrial ATPase, had little effect on the plasma membrane Ca2+/Mg2+ ATPase; sodium azide in high concentrations, however, inhibited the plasma membrane Ca2+/Mg2+ ATPase by about 20%. Kinetic study revealed that the Ki of sodium azide for the plasma membrane enzyme is 10 IIIM, which is 2 orders higher than that for the mitochondrial ATPase (26, 27). Oligomycin, another mitochondrial ATPase inhibitor also depressed the plasma membrane enzyme at high concentrations. Furthermore, ADP, which is a potent competitive inhibitor of mitochondrial ATPase (26), and HC03, which is an activator of the mitochondrial ATPase (26), were found to exert no such effects on the plasma membrane ATPase. Therefore, it appears that the inhibition of plasma membrane Ca2+/Mg2+ ATPase by high concentrations of sodium azide or oligomycin may not be due to mitochondrial contamination but may represent the property of this enzyme system. It should be pointed out that oligomycin has been shown to inhibit Na+, K+ ATPase whereas sodium azide was reported to inhibit the smooth muscle membrane ATPase (28, 29).
The plasma membrane ATPase was inhibited by trifluoperazine, a well-known inhibitor of calmodulin (30). This would suggest that this enzyme is sensitive to calmodulin; however, it should be noted that the inhibitory effect of trifluoperazine was evident only at high concentrations. Furthermore, this agent inhibited the ATPase activity in the presence of either Ca2+ or Mg2+. Since trifluoperazine is a hydrophobic compound and has been shown to exert a fluidizing effect on the cell membrane (31), it is possible that this drug may affect the plasma membrane Ca2+/Mg2+ ATPase by altering the microenvironment of the enzyme in some nonspecific manner. The depressant effect of PCMB on the Ca2+/Mg2+ ATPase may indicate the importance of sulfhydryl groups in the functional activity of this enzyme.
DHALLA
An ecto-ATPase with properties similar to those reported here for the rat heart plasma membrane Ca2+/Mg2+ ATPase has been shown to be located on the outer surface of different types of cell membranes (32). The fact that Con A interacts with the sarcolemmal Ca2+/Mg2+ ATPase supports the view that the sarcolemmal Ca2+/Mg2+ ATPase is an ecto-enzyme because Con A is considered to exert its effects by binding to saccharide residues, which are normally present at the cell surface. However, it is not clear at present why a high activity of Ca2+/Mg2+ ATPase is found and why Con A is effective in a plasma membrane preparation with predominately inside-out orientation. One of the explanations is that some of the inside-out vesicles are not sealed and thus allow ATP and Con A to act from the inside of the vesicles. The possibility that the ATP and Con A binding sites of the enzyme may change their orientation during the isolation procedure cannot be ruled out. Furthermore, it should be pointed out that the predominant inside-out orientation of the preparation employed here was essentially based upon the degree of inhibitory effect of ouabain on Na+-Kf ATPase with and without treatment of the membrane vesicles with detergents (23) and it is possible that more rigorous criteria of vesicular orientation have to be used for a meaningful conclusion. It can also be argued that the measured Ca2+/Mg2+ ATPase activity may only represent the portion of the enzyme activity in the right-side-out vesicles present in our preparation. However, our attempts to unmask the latent activity with detergents were unsuccessful because these agents have been shown to decrease the sarcolemmal Ca2+/Mg2+ ATPase (33). We have observed that the rat heart Ca2+/Mg2+ ATPase was inactivated in the presence of ATP or other high energy nucleotides. This is consistent with the observations of Beeler et al. (3) who have reported inactivation of Mg2+ ATPase in skeletal muscle microsomes by ATP. The Ca2+-pump ATPase in the red blood cell membrane, on the other hand, was shown to be inactivated by Ca’+; an effect which
RAT
HEART
PLASMA
MEMBRANE
is prevented by the presence of ATP in the medium (34). Such differences in the behavior in the inactivation properties of Ca2+/Mg2+ ATPase and Ca’+-pump ATPase may be due to differences in the basic properties of two enzymes or origin of membrane preparations in which these enzymes are embedded. In this regard, it should be noted that detergents such as Triton X-100 and lysolecithin, which are known to disturb the membrane structure, were found to accelerate the inactivation of Ca’+/Mg”+ ATPase. Furthermore, Con A and gluteraldehyde, which have been shown to decrease the disturbance of protein-lipid and protein-protein interaction in the membrane bilayer (3,7,35,36), were observed to prevent the inactivation of the enzyme in the absence or presence of detergents. It is therefore likely that the process of inactivation of the Ca2+/Mg2+ ATPase may depend upon the functional integrity of the plasma membrane. Although the exact mechanism for the ATP-induced inactivation of the plasma membrane Ca2+/Mg2+ ATPase is not clear at present, it should be noted that this process was found to be reversible. It is therefore possible that ATP may play a regulatory role where the enzyme can be seen to respond to the local concentrations of ATP in the form of activation and inactivation. Since the process of ATP-dependent inactivation of Ca2+/Mg2+ ATPase was modified by Con A, it is conceivable that a lectin-like protein similar to Con A is present in the heart cell surface material and this may modulate the regulatory effect of ATP on this enzyme. In this regard it should be noted that Beyer et al. (37) have recently isolated endogenous lectins from skeletal muscle and Moulton et al. (38) have also proposed the modulatory action of these lectin-like substances on the transverse tubular membrane Mg2+ ATPase. Extensive further work in cardiac muscle is required to substantiate these viewpoints. REFERENCES 1. ANAND-SRIVASTAVA, M. DHALLA, N. S. (1982) 359-371.
B., PANAGIA, V., AND Adv. Myocardiol. 3,
Ca2+/MgzC
291
ATPase
2. ANAND, M. B., CHAUHAM, M. S., AND DHALLA, N. S. (1977) J. Biochem (Tokyo) 82,1731-1’739. 3. BEELER, T. J., GABLE, K. S., AND KEFFER, J. M. (1983) Biochim. Biophys. Acta 734,221-234. 4. HIDALGO, C., GONZALEZ, M. E., AND LAGOS, R. (1983) J. Biol. Chem. 258,13937-13945. 5. OERTLE, M., VAN DYKE, R., AND SCHARSCHMIDT, B. F. (1984) Arch. Znt. PhysioL B&hem. 92, 107-11s. 6. MODOLELL, J. B., AND MOORE, R. 0. (1967) Biochim. Biophys. A& 135,319-332. 7. CARRAWAY, C. H. C., CORRADO IV, F. J., FOGLE, D. D., AND CARRAWAY, K. L. (1980) B&hem. .I 191,45-51. 8. WALLACH, D. F. H., AND ULLREG, D. (1962) Canm! Res. 22,228-234. 9. CHAMBERS, D. A., SALZMAN, E. W., AND NERI, L. L. (1967) Arch. Biochem. Biophys. 119, 173-178. 10. ZIEGELHOFFER, A., AND DHALLA, N. S. (1987) Mol. CelL B&hem. 77,135-141. 11. MCNAMARA, D. B., SINGH, J. N., AND DHALLA, N. S. (1974) J. Biochem (Tokyo) 76,603-609. 12. DHALLA, N. S., ANAND-SRIVASTAVA, M. B., TUANA, B. S., AND KHANDELWAL, R. L. (1981) J. Mel Cell. Cardiol. 13,413-423. 13. TUANA, B. S., AND DHALLA, N. S. (1982) J. Biol. Chem. 257,14440-14445. 14. DHALLA, N. S., SINGH, J. N., BAJUSZ, E., AND JASMIN, G. (1976) Clin. Sci. Mol. Med 51,233-242. 15. TOMLINSON, C. W., LEE, S. L., AND DHALLA, N. S. (1976) Circ. Res. 39,82-92. 16. DHALLA, N. S., LEE, S. L., ANAND, M. B., AND CHAUHAN, M. S. (1977) B&hem. Pharmacol. 26,2050-2060. 17. DHALLA, N. S., HARROW, J. A. C., AND ANAND, M. B. (1978) Biochem. Pharmacol. 27, 1281-1283. 18. HARROW, J. A. C., DAS, P. K., AND DHALLA, N. S. (1978) B&hem Pharmucol. 27,2605-2609. 19. PANAGIA, V., LAMER& J. M. J., SINGAL, P. K., AND DHALLA, N. S. (1982) Znt. J. Biochem. 14, 387-397. 20. DHALLA, N. S., AND PIERCE, G. N. (1984) in Methods in Studying Cardiac Membranes (Dhalla, N. S., Ed.), Vol. 1, pp. 3-18, CRC Press, Boca Raton, FL. 21. MOFFAT, M. P., SINGAL, P. K., AND DHALLA, N. S. (1983) Basic Res. Cardiol 78,451-461. 22. PITTS, B. J. R. (1979) J. Biol Chem 254,6232-6234. 23. MAKINO, N., DHALLA, K. S., ELIMBAN, V., AND DHALLA, N. S. (1987) Amer. J Physiol. 253, E202-E207. 24. TAUSSKY, H. H., AND SHORR, E. (1953) J. Biol. Chem. 202,675-685. 25. LOWRY,
0. H., ROSEBROUGH,
N. J., FARR,
A. L.,
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AND RANDALL,R.J. (1951)J. Biol C’~~WZ.193, 265-2'75. 26. PULLMAN,M. E., PENEFSKY, H. S., DATTA,A., ANDRACKER,E. (1960)J. Biol. Chem. 235, 3322-3329.
27. PENEFSKY, H. S., PULLMAN,M. E., DATTA,A., ANDRACKER,E. (1960)J. Biol. Chem. 235, 3330-3336. 28. MATSUI,H., ANDSCHWARTZ, A. (1966)Biochim Biophys. Acta 128,380-390. 29. KWAN,C. Y., ANDKOSTKA,P. (1984)Biochim. Biuphys. Acta 776,209-216. 30. LEVIN, R. M., ANDWEISS,B. (1978) B&him. Biophys.Acta 660.197-206. 31. SEEMAN,P. (1977)Biochem. Pharmacol. 26, 1741-1748.
DHALLA
32. BANERJEE, R. K. (1981)Mol CeU Biochem 37, 91-99.
33. PANAGIA,V.,LAMERS,J.M.J.,SINGAL,P.K.,AND DHALLA,N. S. (1982)Int. J. Biochem. 14, 387-397. 34. PEDEMONTE, C. H., ANDBALEGNO, H. F. (1982) Mol. Cell B&hem
47.31-34.
35. LYLIS,D. S., ANDLANDSBERGER, F. R. (1976) Proc. Nat1 Acad Sci. USA 73,3497-3501.
36. MYRDAL,~.E., ANDDEHAAN,R.L.(~~~~)J.CeU Physiol. 117,319-325. 37. BEYER,E.C.,ZWEIG,S.E.,ANDBARONDES,S.H. (1980)J. Biol. Chem 255,4236-4239. 38. MouLToN,M.P.,SABBADINI,R.A.,NORTON,K.C., ANDDAHMS,A. S. (1986).I Biol Chem 261, 12244-12251.
Characterization
of Rat Heart Plasma Membrane DAYUAN
ZHAO
AND NARANJAN
Ca2+/Mg2+ ATPase’
S. DHALLA’
Division of Cardiovascular Sciences, St. B&face General Hospital Research Centre and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada RZH 2A6 Received
November
9,1987,
and in revised
form
February
81988
The Ca2’/Mg2’ ATPase of rat heart plasma membrane was activated by millimolar concentrations of Ca2+or Mg2+* other divalent cations also activated the enzyme but to a lesser extent. Sodium azide at high concentrations inhibited the enzyme by about 20%; oligomycin at high concentrations also inhibited the enzyme slightly. Trifluoperazine at high concentrations was found inhibitory whereas trypsin treatment had no significant influence on the enzyme. The rate of ATP hydrolysis by the Ca2+/Mg2+ ATPase decayed exponentially; the first-order rate constants were 0.14-0.18 min-’ for Ca2+ ATPase activity and 0.15-0.30 min-’ for Mg ‘+ ATPase at 37°C. The inactivation of the enzyme depended upon the presence of ATP or other high energy nucleotides but was not due to the accumulation of products of ATP hydrolysis. Furthermore, the inactivation of the enzyme was independent of temperature below 37°C. Con A when added into the incubation medium before ATP blocked the ATP-dependent inactivation; this effect was prevented by a-methylmannoside. In the presence of low concentrations of detergent, the rate of ATP hydrolysis was reduced while the ATP-dependent inactivation was accelerated markedly. Both Con A and glutaraldehyde decreased the susceptibility of Ca2+/Mg2+ ATPase to the detergent. These results suggest that the Ca2+/Mg2+ ATPase is an intrinsic membrane protein which may be regulated by ATP. Q 19% Academic press. I,,~. It is now well established that the Ca’+/ Mg2+ ATPase in various types of mammalian cell membranes (l-7) is different from the Na+-K+ ATPase (Na+ pumping ATPase) or Cazf-stimulated ATPase (Ca2+ pumping ATPase). The Ca2+/Mg2+ ATPase has been suggested to be involved in cell surface processes such as active transport, ameboid motion, and regulation of cell shape (8, 9). The heart sarcolemmal Ca2+/Mg2+ ATPase has been suggested to be involved in the gating mechanism for the translocation of divalent cations across the cell membrane (1, 10-13). Although the exact role of the enzyme in the heart cell function is not clear, the signifir The work reported in this study was supported by a grant from the Medical Research Council of Canada. Dayuan Zhao was a predoctoral fellow of the Canadian Heart Foundation. s To whom correspondence should be addressed. 281
cance of the enzyme activity can be appreciated from the fact that the Ca2+/Mg2+ ATPase activity was depressed in different types of diseased hearts (14,15) as well as by various cardiodepressant agents such as propranolol (16), antiarrhythmic drugs (17), and divalent cations including Co’+ Ni2+, and Mn2+ (18). In order to elucidate the enzyme function, previous work from our laboratory has described some properties of the Ca2+/Mg2+ ATPase in heart sarcolemma prepared by the hypotonic shock-LiBr treatment method (1, 2, 11, 19). This heavy sarcolemmal fraction, which is isolated by low-speed centrifugation, has been shown to differ from the light membrane fraction, which sediments only at high centrifugal force (20). Furthermore, the heavy sarcolemmal fraction from the myocardium, unlike the light membrane fraction, was demonstrated to contain the basement membrane and 0003-9861188 $3.90 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form resewed.
282
ZHAO AND DHALLA
other cell surface material (21). It should also be pointed out that the heavy sarcolemma1 fraction was found to orient itself into predominantly right-side-out vesicles (about 90%) whereas the light membrane fraction from heart showed the predominant presence of inside-out vesicles (80 to 90%) (21-23). The specific activities of various marker enzymes of the plasma membrane were much lower in the heavy sarcolemmal fraction in comparison to those in the light membrane preparation (20,21). In view of these differences in the heavy sarcolemmal and light membrane fractions, it is thought worthwhile to characterize the Ca’+/Mg’+ ATPase activity in the light plasma membrane preparation from the heart. For this purpose, we have employed plasma membranes isolated from the rat heart by the sucrose density gradient technique (22), which are reported to exhibit about 10 times higher activity of Ca2+/Mg2+ ATPase in comparison to that of the heavy sarcolemmal preparation (20). MATERIALS
AND METHODS
Materials. ATP, GTP, ITP, UTP, CTP, ADP, pNPP,’ sodium azide, 2,4-dinitrophenol, trypsin inhibitor, PCMB, trifluoperazine, Ca*+ ionophore A2318’7, valinomycin, concanavalin A, Triton X-100, diadenosine pentaphosphate (AP&, and lysophosphatidylcholine (lysolecithin) were obtained from Sigma Chemical Co. (St. Louis, MO). Oligomycin and FCCP were from Aldrich Chemical Co. (Milwaukee, WI). All other chemicals were of analytical grade. Preparation of rat heart plasma membranes and ATPase measurements. The plasma membranes were isolated from the rat heart by the sucrose density gradient method according to Pitts (22). On the basis of extensive marker enzyme studies (20, 23), this membrane fraction was found to contain minimal (1 to 3%) cross contamination by other subcellular organelles. The Ca2+ ATPase or Mg2+ ATPase activity was assayed at 3’7°C in 1 ml assay medium containing 50 mrd Tris-HCl, pH 7.5, 4 mM Tris-ATP, and 4 mM CaClz or MgClz. The final protein concentration was
3Abbreviations used: Con A, concanavalin A; FCCP, carbonyl cyanide ptrifluoromethoxyphenylhydrazone; SDS, sodium dodecyl sulfate; PCMB, pchloromercuribenzoic acid; APJ, diadenosine pentaphosphate; pNPP, pnitrophenyl phosphate.
0.02 to 0.03 mg/ml. The reaction was started by addition of ATP and stopped 1 min after by the addition of 0.5 ml 10% SDS. Experimental conditions that differed from these described here are given in the text. The Ca2+ ATPase or Mg2+ ATPase activity was calculated as difference between the amount of Pi formed in the presence of 4 mM Ca2+ or Mg” and that in the absence of any divalent cation. The estimation of Pi was carried out by the method of Taussky and Shorr (24) whereas protein concentration was determined by the method of Lowry et al. (25). Unless otherwise indicated at least three different preparations were employed for each observation. RESULTS
The plasma membrane preparation was found to hydrolyze ATP in the presence of Ca2+ as well as Mg2+. The time course of the enzyme reaction (Table I) revealed that the ATPase activities started decreasing 2 min after starting the incubation with ATP. However, the reaction was linear with respect to the protein concentration in the range lo-50 pg/ml. Accordingly, all experiments reported in the study were carried out for 1 min by using 20-30 pg membrane protein in the incubation medium. In one set of experiments, the ATPase activities were measured by varying the concentrations of either Ca2+ or Mg2+ in the presence of 4 mM ATP whereas in the other set, the concentration of ATP was varied in the presence of 4 TABLE
I
Ca’+/M%+ ATPase ACTIVITIESOF HEART SARCOLEMMAATDIFFERENTTIMESOFINCUBATION Time of incubation (min) 1 2 4 8 16
Ca*+ ATPase activity (pm01 Pi/mg/min) 5.9 5.7 3.5 2.2 1.3
f 2 + + +
0.9 0.8 0.4 0.1 0.3
Mgz+ ATPase activity (pm01 Pi/mg/min) 7.6 + 6.7 + 3.5 * 2.1 f 0.9 +
1.4 1.4 0.5 0.3 0.1
Note. The Ca2’/Mgz’ ATPase activities were determined in aliquots taken at different intervals after the addition of 4 mM ATP in a medium containing 4 mM Ca2+ or 4 mM Mgz+. The results represent the rates of ATP hydrolysis during the time periods indicated. Each value is a mean & SE of six experiments.
RAT TABLE KINETIC
PARAMETERS MEMBRANE
HEART
PLASMA
MEMBRANE
TABLE
OF THE RAT HEART Ca2+/Mg2+ ATPase
PLASMA
EFFECT
0.23 * 0.03 7.03 -+ 0.66 0.69 + 0.40 6.73 f 0.80
0.33 * 0.04 9.32 * 0.64 0.71 f 0.40 8.82 + 0.53
Note. ATPase activities were determined by varying the concentration of ATP and keeping the concentrations of Ca*+ or Mg2+ at 4 mbl as well as by varying the concentration of Ca’+ or M$+ and keeping the concentrations of ATP at 4 mbx. The Lineweaver--Burk plots of the data revealed K,, K,,, and V,, values. Each value is a mean f SE of four experiments.
mM Ca2’ or Mg2+. Both Ca2+ ATPase and Mg2+ ATPase were fully activated in the presence of 4 mM ATP and 4 KIM Ca2+ or 4 IllM Mg2+, respectively. The LineweaverBurk plot analysis of the data revealed K, and Km values (Table II) similar to those reported by employing the heavy sarcolemma1 preparations (1, 11,19). However, the V,,, values for both Ca2+ ATPase and Mg2+ ATPase were 10 to 15 times higher than those reported earlier (1, 11,19). Other divalent cations also activated the ATPase activity but were much lesser effective than Mg2+ or Ca2+ (Table III).
III
CATION SPECIFICITY FOR THE ATP BY RAT HEART PLASMA MEMBRANE
Cations
Relative ATP hydrolysis
Cations
Relative ATP hydrolysis
Mg2 Ca2+ co2+ Mn2+ Sr2+
100 75 36 22 14
Cds’ Ni2+ Zn2+ Ba2’ cu2+
11 8 5 0 0
Note. Membranes were incubated in the presence of 4 mM ATP and 4 mM of different cations. The ATP hydrolysis due to the presence of each cation was expressed as a percentage of that in the presence of Mg-.
None W BaP+ 512+ Mn’+ cop+ Nis+ Cd2+ CU+ Zn2+ Las’ Na+ K+ HCO, Vanadate
283 IV
OF DIFFERENT IONS ON THE RAT HEART PLASMA MEMBRANE Ca2+/M8 ATPase ACTIVITIES
Mg2 ATPase Ions added
A. Varying ATP concentrations K,,, for ATP (mu) V,, (pm01 Pi/mg/min) B. Varying cation concentrations K. for Cae+ or Mp (mM) V,, (pm01 PJmg/min)
TABLE
ATPase
II
Ct?' ATPase
DIVALENT HYDROLYSIS
Ca”/Mg’*
Concentration (mW
Relative Gas+ ATPase activity
4 4 4 4 4 4 4 4 4 4 100 100 50 1
Note. Membranes were incubated ATP and 4 mM of Cap+ or Me, and tions of different ions. The ATPase as percentages of Ca*+ ATPase or sence of any other ions.
100 133 109 94 41 38 21 17 13 10 100 97 93 102 96
Me
Relative ATPase activity 100 102 102 88 30 30 12 9 11 1 99 90 90 89 81
in the presence of 4 mM the indicated concentraactivities are expressed Mgs+ ATPase in the ab-
However, in the presence of Ca2+ or Mg2+, most of divalent cations were inhibitory; Zn2’, Cd2+, and Cu2+ were most inhibitory, while Ba2+ was least inhibitory (Table IV). The Ca2+/Mg2+ ATPase activities were not altered by 4 mrvx La3+; 100 mM NaCl, 100 mM KCl, 50 mM NaHC03, and 1 mM vanadate slightly inhibited the Mg2+ ATPase activity. Table V shows the effects of the different inhibitors on the ATPase activity. Trifluoperazine at a low concentration (0.01 mM), which is known to inhibit calmodulin, showed negligible effect whereas in high concentrations (0.02 to 0.05 mM) it produced inhibition. The I& for trifluoperazine varied between 20 and 50 PM. PCMB at high concentrations depressed the Ca2+/Mg2+ ATPase activities. Both oligomyocin and sodium azide at high concentrations also inhibited the Ca2+/ Mg2+ ATPase activities (Table V). Kinetic study of the sodium azide effect showed that this inhibition was apparently competitive with a Ki = 10 mM for both Mg2+ ATPase activity and Ca2+ ATPase activity
284
ZHAO TABLE
AND
V
EFFECTOFINHIBITORSONTHERATHEARTPLASMA MEMBRANE Ca’+/Mp ATPase Ca2+ ATPase Inhibitors
(% inhibition hydrolysis)
Mg2f
ATPase
of ATP
Trifluoperazine 0.01 mM
9
0.02mM 0.05mM
24 63
4 17 60
46 49
36 50
0 13
10 11
0 0
3 6 16 26
PCMB 0.25 PM 250 PM Oiigomycin 3 pgg/ml 30 pg/ml Sodium azide 0.1 mM
1mM 3mM 10 mM
3 22
Note Membranes were preincubated with the inhibitor at 37’C for 3 min. In the case of oligomycin and PCMB, where -0.5-5% ethanol was used in assay medium, control samples were treated with corresponding concentrations of ethanol in the assay medium.
(Fig. 1). Several other agents such as Ca2+ inophore A23187, FCCP, 2,4,-dinitrophenol, gramicidine D, and valinomycin, were found to have no significant effect on the heart membrane Ca2+/Mg2+ ATPase activity (data not shown). Earlier studies (12,13) have shown that treatment of the heavy sarcolemmal preparation with trypsin resulted in the release of Ca2+-dependent ATPase in the supernatant and increase in the Ca2+/Mg2+ ATPase activities in the membrane due to removal of cell surface material. In this study, we examined the effect of trypsin (20 to 300 pg/mg membrane protein) for 5 to 60 min under the experimental conditions described previously (12). Our results indicated that about 80% of protein remained in membrane fraction after trypsin treatment whereas the specific activity of Ca2+/Mg2+ ATPase was increased by about 20% (data not shown). The total activity of the enzyme in the plasma membrane was not changed by trypsin
DHALLA
and no detectable Ca2+-dependent ATPase was found in the supernatant. In one series of experiments, the time course of ATP hydrolysis due to Ca2+/ Mg2+ ATPase was investigated (Fig. 2). We found that the rate of ATP hydrolysis by the Ca2+/Mg2+ ATPase decayed exponentially. The first-order rate constant of inactivation was 0.15 to 0.30 min-’ for Mg2+-ATPase activity and 0.14 to 0.18 min-’ for Ca2+ ATPase activity at 37°C. The Ca2+/Mg2+ ATPase activities were not further inactivated after they were reduced to 20 to 30% of initial ATP hydrolyzing rate. In the absence of ATP, the inactivation rate was about 150 times lower than that in the presence of ATP (Figs. 2 and 3). Addition of 1 InM KH2POI, ADP, and AMP in the assay medium did not show significant change in the initial rates of ATPase activities; similar results were obtained when the reaction was studied in the presence of AP5A, a well-known
i F ?v
0.6 0.4
0
2
4
6
8
l/[ATP]
FIG. 1. Effect of 5 mM NaNa membrane Caa’/Mga+ ATPase. was measured in the presence and varying concentrations of presence (0) or absence (0) of Lineweaver-Burk plots.
on the heart plasma The ATPase activity of 4 mM Ca2+ or Mga+ ATP. The data in the NaNa are presented as
RAT HEART
2 4 6 6 10 12 14 16 0 2 4 Time hid
PLASMA
6
MEMBRANE
8 10 12 14 16 Time (mid
FIG. 2. Rates of ATP hydrolysis by the heart plasma membrane Ca’+/Mg’+ ATPase. (A) Time course of ATP hydrolysis by Ca2+ ATPase in the absence (0) and presence of 20 rg/ml Con A added before (0) or 2 min (A) and 7 min (0) after the addition of ATP; (B) rate of ATP hydrolysis by Ca2* ATPase calculated from the data in A, (C) time course of ATPase hydrolysis by Mg2+ ATPase; (D) rate of ATP hydrolysis by Mg ‘+ ATPase in the absence (0) and presence of Con A added before (0) or 2 min (A) and 7 min (0) after the addition of ATP. The data for ATPase activity are plotted on a semilogarithmic graph.
inhibitor of adenylate kinase (Table VI). This indicated that the inactivation was not due to the product inhibition but was likely an ATP-dependent inactivation. Other high energy nucleotides like CTP, UTP, GTP, ITP, which were hydrolyzed by the ATPase at a rate similar to that for ATP, also induced similar inactivation of the enzyme (Table VII). ADP, on the other hand, was hydrolyzed at a lower rate in comparison to that for ATP but inactivated the enzyme at a higher rate (Table VII). It should also be noted from Table VI that ADP increased the inactivation rate of the enzyme due to ATP. pNPP was not hydrolyzed under same conditions, indicating that the ATPase activity was not due to a nonspecific phosphatase. After pretreating the plasma membrane with ATP for 30 min, when the ATPase activity
Ca’+/Mg*+
ATPase
285
was reduced to 20-30s of original ATP hydrolyzing rate, removal of ATP partially restored the enzyme activity; however, the inactivation rate was not influenced by ATP pretreatment (Table VIII). The pH profiles of the Ca2+/Mg2+ ATPase revealed that the maximal ATPase activity was at pH 7.5, while the inactivation rate of Mg2+ ATPase, unlike Ca2+ ATPase, was lower at the alkaline side (Fig. 4). The ATP-dependent inactivation rate of the enzyme seems to be independent of temperature below 37°C (Fig. 5). The energy of activation of ATP hydrolysis determined from an Arrhenius plot of the data was 11.5 kcal/mol for Ca2+ ATPase and 11.4 kcal/mol for Mg2+ ATPase. Con A, a lectin, was found to prevent ATP-dependent inactivation partially (Fig. 2). Pretreating the membrane with Con A did
Ca*+ATPacle
37%
1 10
20
30 Time
40
50
60
70
(hr)
FIG. 3. Rate of inactivation of the heart plasma membrane Ca*+/Mg’+ ATPase in the absence of ATP. The membranes (0.3 mg/ml) were incubated for 24 to ‘72 h at the indicated temperatures. Aliquots were removed at various times and the initial ATPase activities were measured at 3’7°C. The data are plotted on a semilogarithmic graph.
286
ZHAO
AND
DHALLA
TABLE
VI
EFFECTSOF ADP, AMP,AND Pi ONTHEHEARTPLASMAMEMBRANE ~~~~~~~~~~~~~~~~~~~~~~~ PRESENCEOFDIADENOSINEPENTAPHOSPHATE(AP&) Without Initial (pm01 Control Ca*’ ATPase Mga+ ATPase ADP (1 mM) Ca*’ ATPase M%+ ATPase AMP (1 mM) Cal+ ATPase Mga+ ATPase Pi (1 mM) Ca2+ ATPase Mga+ ATPase ADP (1 mM) + Ca” ATPase Me ATPase
With
APSA
ATPase rate Pi/mg/min)
Inactivation rate constant (min-‘)
Initial (pm01
AP&
ATPase rate Pi/mg/min)
Inactivation rate constant (min-‘)
6.7 8.9
0.15 0.15
5.2 6.3
0.15 0.14
7.8 9.2
0.26 0.31
6.8 9.0
0.17 0.20
6.9 8.7
0.13 0.17
6.5 8.3
0.14 0.14
7.5 8.3
0.14 0.15
4.8 6.7
0.15 0.18
7.2 8.4
0.26 0.26
5.2 6.5
0.25 0.31
Pi (1 mM)
Note. ATPase activities were assayed used as Pi. The concentration of APr,A
in the absence was 10 PM.
not alter the initial hydrolysis rate Ca2+/Mg2+ ATPase, but reduced the dependent inactivation rate constant 0.21 min-’ for Mg2+ ATPase and 0.14
or presence
of the ATPfrom min-’
of various
agents
as indicated.
KHaPOl
was
for Ca2+ ATPase to 0.07 and 0.05 min-‘, respectively. The enzyme activities were also stabilized at a much higher level than that in the absence of Con A. However,
TABLE
VII
SUBSTRATESPECIFICITYANDINACTIVATIONRATECONSTANTSFORTHEHEARTPLASMA MEMBRANE Ca*+/Mp ATPase In the presence
Substrate
Hydrolysis rate (pmol/mg/min)
ATP CTP ITP UTP GTP ADP Note. Membranes nucleotides.
of 4
mM Ca2+ Inactivation rate constant (min-‘)
9.3 10.8 8.2 8.9 7.9 1.9 were
incubated
In the presence
Hydrolysis rate (pmol/mg/min)
0.18 0.18 0.18 0.18 0.21 0.32 in the assay
medium
12.3 8.2 10.9 9.9 8.8 1.9 containing
50
mM Tris-HCl,
of 4
mM
Mga+
Inactivation rate constant (min-‘) 0.30 0.32 0.16 0.21 0.25 0.41 pH 7.5, and 4 mM of
RAT
HEART
PLASMA
MEMBRANE TABLE
EFFEC~OFWASHINGONTHEHEARTPLASMA
Caa+/Mg’+
VIII
MEMBRANE Ca2+/M%f
AFTER~RETREATMENTWITH
Initial rate of ATPase (pmol/mg/min) Preincubation medium
Ca2+ ATPase
Control (MgCla) M&la + ATP Control (CaC12) CaCla + ATP
Inactivation rate constant ATPase (mini)
Mga+ ATPase
6.3 5.2 6.8 3.8
287
ATPase
Ca2+ ATPase
7.5 5.5 6.7 5.2
Me
0.21 0.23 0.23 0.21
ATP of
ATPase 0.21 0.21 0.16 0.23
for 30 min at 22°C in a medium containing 50 IIIM Tris-HCl, pH 7.5,4 mM of these membranes were markedly depressed (20 to 30% of the control). Control samples were incubated in medium without the ATP. The samples were then centrifuged at 100,OOOg for 30 min and the pellet was thoroughly washed and resuspended in 50 mM Tris-HCl, pH 7.5, buffer and used for ATPase activity assay. Note.
Membranes
were
incubated
ATP, 4 mM CaC12 or MgC12. The activities
Con A was found to interact with heart membrane Ca2+/Mg2+ ATPase slowly because adding Con A 2 or ‘7 min after the addition of ATP failed to prevent the inactivation immediately (Fig. 2). The effects of Con A on the inactivation of the enzyme were blocked completely by the presence of 50 MM cr-methylmannoside; this indicates that Con A protects the en-
15r
A-._
zyme by binding to oligosaccharide residues, which may form part of the enzyme structure. In the presence of detergents
, 0.15
FIG. 4. pH profiles of the heart plasma membrane Ca2+/Mg2+ ATPase. ATPase activities were measured as described under Materials and Methods except that 50 mM Bistris buffer was used in all pH ranges instead of Tris buffer. (0) The rate of Caa+ ATPase activity; (A) the rate of Mg2+ ATPase activity; (0) and (A) inactivation rates of Ca2’ ATPase and Mga+ ATPase, respectively.
1'
2
4
6
Tii
810
(mid
FIG. 5. Rate of inactivation of the heart plasma membrane Ca2+/Mg a+ ATPase in the presence of ATP. ATPases were assayed at 22°C (O), 30°C (+), 3’7°C (A), and 43°C (0).
288
ZHAO AND DHALLA
such as Triton X-100 and lysophosophatidylcholine, the ATP hydrolyzing rate of the ATPase was reduced, while the rate of ATP-dependent inactivation was accelerated (Fig. 6). Con A blocked the effects of the detergents on the ATP-dependent inactivation rate at all concentrations of the detergents tested, and in the case of lysophosphatidylcholine, partially restored the initial ATP hydrolyzing rate of the enzyme (Table IX). Glutaraldehyde, decreasing the ATP hydrolyzing rate and increasing the ATP-dependent inactivation rate by itself, was able to prevent the effects of the detergents in both aspects (Table X).
DISCUSSION
In this study we have shown that the plasma membranes obtained from rat hearts exhibited a highly active ATPase system, which is capable of hydrolyzing high energy nucleotides in the presence of either Ca2+ or Mg 2+. Although the affinities of the enzyme in this light plasma membrane for Ca2+ or Mg2+ (K, = 0.69 to 0.71 mM) as well as for ATP (K, = 0.23 to 0.33 mM) were similar to those reported for the Ca2+/Mg2+ ATPase in heavy sarcolemma1 fraction (1, 11, 19); the maximal velocities (V,,, = 7.0 pm01 Pi/mg/min for Ca2+ ATPase and 9.3 pmol Pi/mg/min for Mg2+ ATPase) for the light fraction were 10 to 15 times higher than those for the heavy membrane fraction. Such a difference in two types of the heart membrane fraction is primarily due to the presence of cell surface material in the heavy sarcolemma1 fraction. This view is supported by the fact that trypsin treatment, unlike the heavy sarcolemmal fraction (12), was not capable of increasing the Ca2+/Mg2+ ATPase activity in the plasma membrane. However, the patterns of cation activation, substrate specificity, and inhibition exerted by several divalent cations observed in this study as well as those reported earlier (1, 11, 19) suggest that Ca2+/Mg2+ ATPase present in the plasma membranes (light fraction) is similar to
10.0 8.0
Ca*+ATPase
4.0
l t
1.0 0.8 -2 $ P t 5 E
2
l .
2.0 .
0.4 0.2 s’
A-
10.0 8.0
Mg*+ATPass
A
.= .L
40.
2 b3
2.0
A . . .
1.0 0.8
a 0.4
.
0.2
D ‘i
012345 Time (mid
12345 Time (mid
FIG. 6. Effect of Triton X-100 (A, B) and lysophosphatidylcholine (C, D) on the heart plasma membrane Ca*+/Mg*+ ATPase activity. ATPase activities were assayed in the presence of 1 pg/ml (+), 10 pg/ml (A), and 50 pg/ml (0) Triton X-100 or 10 fig/ml (*), 100 fig/ml (A), and 1 mg/ml (0) lysophosphatidylcholine.
that present in the sarcolemmal membranes (heavy fraction). Nonetheless, it should be noted that unlike the heavy sarcolemmal membranes (12, 13), the light plasma membranes did not contain the Ca2+-dependent ATPase, which is not activated by Mg 2+. The observed depression in the activity of Ca2+/Mg2+ ATPase upon treating the membrane with detergents as well as the insensitivity of this enzyme to trypsin digestion suggests that the Ca2+/ Mg2+ ATPase is an intrinsic membrane protein. Millimolar concentrations of Ca2+ or Mg2+ have also been reported to activate the mitochondrial ATPase (26, 27) and in fact azide-sensitive Mg2+-ATPase is usually used as an indication of the mitochondrial contamination in subcellular
RAT
HEART
PLASMA
MEMBRANE TABLE
Ca*‘/Mg*’
289
ATPase
IX
EFFECT OF CON A ON THE HEART PLASMA MEMBRANE Ca2’/Mga+ INTHEPRESENCEOFDETERGENTS
ATPase
INCUBATED
Control Initial
ATPase rate (pmol/mg/min)
Detergents No detergent Ca*+ ATPase Mga+ ATPase Triton X-100, 1 pg/ml Ca*+ ATPase Mga+ ATPase Triton X-100, 10 pg/ml Ca*+ ATPase Mgs+ ATPase Triton X-100, 50 rg/ml Ca*+ ATPase Mga+ ATPase Lysolecithin, 10 fig/ml Ca*+ ATPase Mga+ ATPase Lysolecithin, 100 /*g/ml Ca2+ ATPase Mg2f ATPase Lysolecithin, 1 mg/ml Ca*+ ATPase MgZf ATPase
Note. ATPase
activities
were
Con A Inactivation rate constant (mini)
Initial
ATPase rate (fimol/mg/min)
Inactivation rate constant (min-‘)
6.6 9.1
0.14 0.21
5.8 9.4
0.05 0.07
6.1 8.0
0.28 0.39
5.6 7.6
0.16 0.12
5.8 8.5
0.30 0.55
6.0 8.0
0.16 0.14
6.7 5.9
1.15 2.65
4.5 5.9
0.35 0.35
5.5 6.6
0.28 0.28
7.4 8.5
0.16 0.16
3.9 4.5
1.29 0.92
6.3 8.3
0.32 0.25
3.6 3.0
1.57 1.40
6.1 6.6
0.28 0.48
measured
in the absence
or presence
TABLE
of concanavalin
A (50 fig/ml).
X
EFFECTOFGLUTARALDEHYDEONTHEHEARTPLASMAMEMBRANEC~~+/M~~+ATP~~~ Initial ATPase (~mol/mg/min) Ca*’ Untreated Treated Treated + Triton X-100, 50 rg/ml Treated + lysolecithin, 1 mg/ml
ATPase
rate
Me
Inactivation
ATPase
Ca2+ ATPase
rate (mini)
constant
M$+
ATPase
6.3 5.3
7.5 5.2
0.21 0.37
0.21 0.37
5.5
5.8
0.39
0.51
5.3
5.7
0.55
0.55
Note. Membranes (0.3 mg/ml) was incubated at 1°C in 50 mM Tris-HCl, pH 7.5, with or without 0.25% glutaraldehyde for 30 min. Aliquots (100 11) were diluted with 1 ml of ATPase assay medium to measure ATPase activity.
290
ZHAO
AND
preparations (20). In this study we have shown that low concentrations of sodium azide, which markedly inhibited mitochondrial ATPase, had little effect on the plasma membrane Ca2+/Mg2+ ATPase; sodium azide in high concentrations, however, inhibited the plasma membrane Ca2+/Mg2+ ATPase by about 20%. Kinetic study revealed that the Ki of sodium azide for the plasma membrane enzyme is 10 IIIM, which is 2 orders higher than that for the mitochondrial ATPase (26, 27). Oligomycin, another mitochondrial ATPase inhibitor also depressed the plasma membrane enzyme at high concentrations. Furthermore, ADP, which is a potent competitive inhibitor of mitochondrial ATPase (26), and HC03, which is an activator of the mitochondrial ATPase (26), were found to exert no such effects on the plasma membrane ATPase. Therefore, it appears that the inhibition of plasma membrane Ca2+/Mg2+ ATPase by high concentrations of sodium azide or oligomycin may not be due to mitochondrial contamination but may represent the property of this enzyme system. It should be pointed out that oligomycin has been shown to inhibit Na+, K+ ATPase whereas sodium azide was reported to inhibit the smooth muscle membrane ATPase (28, 29).
The plasma membrane ATPase was inhibited by trifluoperazine, a well-known inhibitor of calmodulin (30). This would suggest that this enzyme is sensitive to calmodulin; however, it should be noted that the inhibitory effect of trifluoperazine was evident only at high concentrations. Furthermore, this agent inhibited the ATPase activity in the presence of either Ca2+ or Mg2+. Since trifluoperazine is a hydrophobic compound and has been shown to exert a fluidizing effect on the cell membrane (31), it is possible that this drug may affect the plasma membrane Ca2+/Mg2+ ATPase by altering the microenvironment of the enzyme in some nonspecific manner. The depressant effect of PCMB on the Ca2+/Mg2+ ATPase may indicate the importance of sulfhydryl groups in the functional activity of this enzyme.
DHALLA
An ecto-ATPase with properties similar to those reported here for the rat heart plasma membrane Ca2+/Mg2+ ATPase has been shown to be located on the outer surface of different types of cell membranes (32). The fact that Con A interacts with the sarcolemmal Ca2+/Mg2+ ATPase supports the view that the sarcolemmal Ca2+/Mg2+ ATPase is an ecto-enzyme because Con A is considered to exert its effects by binding to saccharide residues, which are normally present at the cell surface. However, it is not clear at present why a high activity of Ca2+/Mg2+ ATPase is found and why Con A is effective in a plasma membrane preparation with predominately inside-out orientation. One of the explanations is that some of the inside-out vesicles are not sealed and thus allow ATP and Con A to act from the inside of the vesicles. The possibility that the ATP and Con A binding sites of the enzyme may change their orientation during the isolation procedure cannot be ruled out. Furthermore, it should be pointed out that the predominant inside-out orientation of the preparation employed here was essentially based upon the degree of inhibitory effect of ouabain on Na+-Kf ATPase with and without treatment of the membrane vesicles with detergents (23) and it is possible that more rigorous criteria of vesicular orientation have to be used for a meaningful conclusion. It can also be argued that the measured Ca2+/Mg2+ ATPase activity may only represent the portion of the enzyme activity in the right-side-out vesicles present in our preparation. However, our attempts to unmask the latent activity with detergents were unsuccessful because these agents have been shown to decrease the sarcolemmal Ca2+/Mg2+ ATPase (33). We have observed that the rat heart Ca2+/Mg2+ ATPase was inactivated in the presence of ATP or other high energy nucleotides. This is consistent with the observations of Beeler et al. (3) who have reported inactivation of Mg2+ ATPase in skeletal muscle microsomes by ATP. The Ca2+-pump ATPase in the red blood cell membrane, on the other hand, was shown to be inactivated by Ca’+; an effect which
RAT
HEART
PLASMA
MEMBRANE
is prevented by the presence of ATP in the medium (34). Such differences in the behavior in the inactivation properties of Ca2+/Mg2+ ATPase and Ca’+-pump ATPase may be due to differences in the basic properties of two enzymes or origin of membrane preparations in which these enzymes are embedded. In this regard, it should be noted that detergents such as Triton X-100 and lysolecithin, which are known to disturb the membrane structure, were found to accelerate the inactivation of Ca’+/Mg”+ ATPase. Furthermore, Con A and gluteraldehyde, which have been shown to decrease the disturbance of protein-lipid and protein-protein interaction in the membrane bilayer (3,7,35,36), were observed to prevent the inactivation of the enzyme in the absence or presence of detergents. It is therefore likely that the process of inactivation of the Ca2+/Mg2+ ATPase may depend upon the functional integrity of the plasma membrane. Although the exact mechanism for the ATP-induced inactivation of the plasma membrane Ca2+/Mg2+ ATPase is not clear at present, it should be noted that this process was found to be reversible. It is therefore possible that ATP may play a regulatory role where the enzyme can be seen to respond to the local concentrations of ATP in the form of activation and inactivation. Since the process of ATP-dependent inactivation of Ca2+/Mg2+ ATPase was modified by Con A, it is conceivable that a lectin-like protein similar to Con A is present in the heart cell surface material and this may modulate the regulatory effect of ATP on this enzyme. In this regard it should be noted that Beyer et al. (37) have recently isolated endogenous lectins from skeletal muscle and Moulton et al. (38) have also proposed the modulatory action of these lectin-like substances on the transverse tubular membrane Mg2+ ATPase. Extensive further work in cardiac muscle is required to substantiate these viewpoints. REFERENCES 1. ANAND-SRIVASTAVA, M. DHALLA, N. S. (1982) 359-371.
B., PANAGIA, V., AND Adv. Myocardiol. 3,
Ca2+/MgzC
291
ATPase
2. ANAND, M. B., CHAUHAM, M. S., AND DHALLA, N. S. (1977) J. Biochem (Tokyo) 82,1731-1’739. 3. BEELER, T. J., GABLE, K. S., AND KEFFER, J. M. (1983) Biochim. Biophys. Acta 734,221-234. 4. HIDALGO, C., GONZALEZ, M. E., AND LAGOS, R. (1983) J. Biol. Chem. 258,13937-13945. 5. OERTLE, M., VAN DYKE, R., AND SCHARSCHMIDT, B. F. (1984) Arch. Znt. PhysioL B&hem. 92, 107-11s. 6. MODOLELL, J. B., AND MOORE, R. 0. (1967) Biochim. Biophys. A& 135,319-332. 7. CARRAWAY, C. H. C., CORRADO IV, F. J., FOGLE, D. D., AND CARRAWAY, K. L. (1980) B&hem. .I 191,45-51. 8. WALLACH, D. F. H., AND ULLREG, D. (1962) Canm! Res. 22,228-234. 9. CHAMBERS, D. A., SALZMAN, E. W., AND NERI, L. L. (1967) Arch. Biochem. Biophys. 119, 173-178. 10. ZIEGELHOFFER, A., AND DHALLA, N. S. (1987) Mol. CelL B&hem. 77,135-141. 11. MCNAMARA, D. B., SINGH, J. N., AND DHALLA, N. S. (1974) J. Biochem (Tokyo) 76,603-609. 12. DHALLA, N. S., ANAND-SRIVASTAVA, M. B., TUANA, B. S., AND KHANDELWAL, R. L. (1981) J. Mel Cell. Cardiol. 13,413-423. 13. TUANA, B. S., AND DHALLA, N. S. (1982) J. Biol. Chem. 257,14440-14445. 14. DHALLA, N. S., SINGH, J. N., BAJUSZ, E., AND JASMIN, G. (1976) Clin. Sci. Mol. Med 51,233-242. 15. TOMLINSON, C. W., LEE, S. L., AND DHALLA, N. S. (1976) Circ. Res. 39,82-92. 16. DHALLA, N. S., LEE, S. L., ANAND, M. B., AND CHAUHAN, M. S. (1977) B&hem. Pharmacol. 26,2050-2060. 17. DHALLA, N. S., HARROW, J. A. C., AND ANAND, M. B. (1978) Biochem. Pharmacol. 27, 1281-1283. 18. HARROW, J. A. C., DAS, P. K., AND DHALLA, N. S. (1978) B&hem Pharmucol. 27,2605-2609. 19. PANAGIA, V., LAMER& J. M. J., SINGAL, P. K., AND DHALLA, N. S. (1982) Znt. J. Biochem. 14, 387-397. 20. DHALLA, N. S., AND PIERCE, G. N. (1984) in Methods in Studying Cardiac Membranes (Dhalla, N. S., Ed.), Vol. 1, pp. 3-18, CRC Press, Boca Raton, FL. 21. MOFFAT, M. P., SINGAL, P. K., AND DHALLA, N. S. (1983) Basic Res. Cardiol 78,451-461. 22. PITTS, B. J. R. (1979) J. Biol Chem 254,6232-6234. 23. MAKINO, N., DHALLA, K. S., ELIMBAN, V., AND DHALLA, N. S. (1987) Amer. J Physiol. 253, E202-E207. 24. TAUSSKY, H. H., AND SHORR, E. (1953) J. Biol. Chem. 202,675-685. 25. LOWRY,
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