Specific stimulation of heart sarcolemmal Ca2+Mg2+ ATPase by concanavalin A

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 268, No. 1, January, pp. 40-48,1989

Specific Stimulation of Heart Sarcolemmal ATPase by Concanavalin A DAYUAN

ZHAO, NAOKI

MAKINO,

AND

NARANJAN

Ca2+/Mg2+

S. DHALLA’

Division of Cardiovascular Department

Sciences, St. Bowface General Hospital Research Centre, 351 Tache Avenue, and of Physiology, Faculty of Medicine, University qf Manitoba, Winnipeg, Canada R2H 2A6 Received June 6,1988, and in revised form September 2,1988

The effects of concanavalin A (Con A) on membrane Ca’+/Me ATPase activities as well as the characteristics of Con A binding were examined by employing rat heart sarcolemmal preparations. Con A stimulated the Ca2+ ATPase and Me ATPase activities in sarcolemma; maximal stimulation in these parameters was seen at a concentration of 10 pg/ml. The observed effects of Con A were blocked by ol-methylmannoside. Sarcolemma1 Na+-K+ ATPase and Ca2+-stimulated ATPase were not affected by Con A. Likewise, Con A did not alter the mitochondrial, sarcoplasmic reticular, and myofibrillar ATPase activities. Con A was found to bind to sarcolemma; cu-methylmannoside prevented this binding. The Scatchard plot analysis of the data on specific Con A binding showed a straight line with a Kd of about 530 nM and a B,,, of 235 pmol/mg protein, thus indicating that there was only one kind of binding site for Con A in sarcolemma. These results suggest that Con A is a specific activator of the low affinity Ca’+/Mc ATPase system in the heart sarcolemmal membrane. o 1989 Academic press, I~~.

tor for ATP (7). In this regard it should be pointed out that the presence of Ca2+/M$+ ATPase in cardiac sarcolemmal preparations has been demonstrated (8-11). Furthermore, the activity of this enzyme system has been shown to alter during the development of heart disease in a wide variety of experimental models (2, 12-14). Since alterations in membrane enzyme activities under pathological conditions are believed to be associated with changes in membrane fluidity (2, 12, 15-18) and since concanavalin A (Con A)’ is commonly used as a probe to alter the fluidity of plasma membrane for studying the organization and behavior of the cell membrane (19-21), it was the purpose of this study to examine the influence of this mitogenic lectin on the

Cardiac muscle depends upon the presence of external Ca2+ for the maintenance of its contractile activity and it is now well known that cardiac cell is equipped with different systems for the translocation of Ca2+ across the sarcolemmal membrane in both directions (l-4). Depolarization of cardiac cell is considered to open Ca2+ channels in sarcolemma for the entry of Ca2+ where Ca2+/Mg+ ATPase has been suggested to serve as a gating mechanism (2, 3). Extracellular ATP has been shown to activate a calcium-permeable channel in arterial smooth muscle cells (5), and to induce Ca2+ transients in cardiac myocytes which are potentiated by norepinephrine (6). These ATP effects can be seen to be mediated through ATP receptors in the cell membrane where the Ca2+/M$+ ATPase may participate in the degradation of the extracellular ATP or itself may be a recep-

1 To whom correspondence 0003-9861189 Copyright All rights

* Abbreviations used: Con A, concanavalin A; Mops, 4-morpholinepropanesulfonic acid; EGTA, ethylene glycol his@-aminoethyl ether) N,N’-tetraacetic acid; a-MM, a-methylmannoside; SDS, sodium dodecyl sulfate.

should be addressed.

$3.00

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

40

RAT HEART

SARCOLEMMAL

sarcolemmal Ca’+/M$+ ATPase activities. The specificity of Con A effects was investigated by studying the interaction of Con A with other types of ATPase in the sarcolemmal, mitochondrial, myofibrillar, and sarcoplasmic reticular preparations. It should be pointed out that Con A has been reported to alter many cell membrane functions (15,22-29). MATERIALS

AND

METHODS

of heart membranes. Isolation and characterization Sarcolemmal membranes were prepared from rat ventricles by the method of Pitts (30). The membrane fraction was characterized with respect to marker enzyme activities by methods used previously (8,17,31). The activity of Na+-K+ ATPase, a well-known marker for plasma membrane, was 22.8 +_ 1.9 pmol Pi/mg/h; this indicated 14- to 16-fold purification with respect to the heart homogenate enzyme activity. Cytochrome c oxidase and rotenone-insensitive NADPHcytochrome c reductase activities in the sarcolemmal fraction indicated minimal (3-5%) contamination with mitochondrial and sarcoplasmic reticular fragments, respectively. Purified myofibrillar, mitochondrial, and sarcoplasmic reticular fractions were also isolated by the methods described elsewhere (31,32). In some experiments, the sarcolemmal membranes were treated with phospholipases A, C, and D and deoxycholate as described elsewhere (10, 33). Treatment of sarcolemma with neuraminidase was conducted by incubating 1 mg membrane fraction in 1 ml medium containing 0.31 unit/ml neuraminidase, 50 mM Tris-HCI, pH 7.4, and 20 mM KC1 at 37°C for 20 min. Treatment of sarcolemma with digitonin was carried out by incubating 1 mg membrane proteins in 1 ml medium containing 0.1% digitonin and 50 mM Tris-HCI, pH 7.5, at 37°C for 10 min. The membranes after different treatments were centrifuged, thoroughly washed with 50 mM Tris-HCl buffer, and then employed for the determination of the enzyme activities. Measurement of ATPase activities. The Ca*+ ATPase and Mgz2’ ATPase activities of the sarcolemma1 fraction in the absence or presence of Con A were measured according to the methods used earlier (10). Membrane proteins (30-50 fig) were incubated in 1 ml of 50 mM Tris-HCI, pH 7.5, with and without 4 mM CaCl, or MgClz (unless otherwise indicated) at 37°C and the reaction was started by adding 4 mM TrisATP (unless otherwise indicated). The reaction was terminated with 1 ml of 12% cold trichloroacetic acid and the ATP hydrolyzed due to the presence of either Ca” or Mga+ was measured to represent Ca2+ or M$ ATPase activities. Na+-K+ ATPase was assayed in a medium containing 100 mM NaCl, 20 mM KCl, 4 mM

Ca2+/M2’

ATPase

41

MgC&, 4 mM Tris-ATP, 5 mM NaN,, 1 mM EDTA, and 50 mM Tris-HCI, pH 7.5; basal ATPase activity (in the absence of K+) was subtracted from the total activity to obtain the NaS-K+ ATPase activity (10). Sarcolemma1 Ca’+-stimulated ATPase was assayed in a medium containing 1 PM free Ca”, 2 mM MgClc, 2 mM Tris-ATP, 160 mM KCl, 5 mM NaN,, and 20 mM MopsTris, pH 7.4; basal ATPase activity (in the absence of Caa’ but in the presence of 1 mM EGTA) was subtracted from the total activity to obtain the Ca’+stimulated ATPase activity (34). For the purpose of comparison, the Ca2+ ATPase and M$+ ATPase of mitochondria and sarcoplasmic reticulum as well as both basal ATPase (Mgz+-ATPase) and Ca*+-stimulated ATPase activities of the sarcoplasmic reticular and myofibrillar fractions were measured in the absence or presence of Con A according to the procedure described elsewhere (31,32,34). Measurement of Con A binding. Con A binding studies were conducted according to a method adapted from that of Brewster et al. (35). Total binding was determined by incubating 75 pg membrane proteins in a medium containing 0.05% bovine serum albumin, 1 pg/ml “‘I-concanavalin A (sp act 6 Ci/mmol), 50 mM Tris-HCI, pH 7.5, in a total volume of 0.2 ml at room temperature for 10 min. The reaction was stopped by adding 1 ml cold Tris buffer; the tubes were centrifuged for 10 min and washed twice with cold buffer. The radioactivity was counted on a Beckman gamma counter. Nonspecific binding was measured by incubating the membrane proteins under the same conditions but in the presence of 0.2 M a-methylmannoside (a-MM) added before the addition of iZ51-Con A. Specific Con A binding was calculated by subtracting the nonspecific binding from the total binding. Statistical anaZ&. Results were expressed as the mean f SE. Statistical analysis was carried out by the Student’s t test, and a P level less than 0.05 was taken to reflect a significant difference between control and experimental values, RESULTS

Membrane ATPase activities. In one series of experiments, the effects of different concentrations of Con A were studied on the sarcolemmal ATPase activities. The data shown in Table I indicate that the sarcolemmal Ca2+ ATPase activity when measured at 1.25 or 4.0 mM Ca” was increased by 2 to 50 pg/ml Con A significantly. Likewise, the activity of sarcolemmal M$+ ATPase was increased by 2 to 50 pg/ml Con A (Table II). Maximal stimulation of the sarcolemmal Ca2+ or M$+ ATPase activities was seen at 10 pug/ml Con A. In order to test the specificity of Con A effect on the

ZHAO,

MAKINO, TABLE

AND

DHALLA

I

EFFECTOFC~~AONRATHEARTSARCOLEMMAL,MITOCHONDRIAL,ANDSARCOPLASMICRETICULAR (MICROSOMES) Caa+ATPase ACTIVITIESINTHE PRESENCEOF~.~~OR~~M Ca2+ ATPase activity

(pm01 Pi/mg protein/h)

Sarcolemma

Mitochondria

Microsomes

Concentration of Con A

1.25 mM

4.0mM

1.25mM

4.0mM

1.25 mM

4.0mM

(dml)

Ca2+

Caa+

Ca2+

Caa+

Ca2+

Caa+

Control 2 5

117+- 8.1 166k 9.6* 179 i 11.6* 204 AZ13.2* 166k 10.0* 153 + 8.9*

11.8 10.6 14.4 9.6 11.2 10.0

36.2 30.4 34.6 33.4 32.8 32.2

76.4 75.9 80.0 81.5 83.6 84.1

87.1 93.6 101.6 104.3

10 20 50

161 f 244 + 280 f 284 + 272 f 252 f

9.6 8.1* 10.0 * ll.O* 8.9* 12.0 *

100.5 102.2

Note. Each value for sarcolemma is a mean I SE of six experiments whereas all other values are the average of three experiments. These activities were corrected for ATP hydrolysis in the absence of Ca*+. This ATP hydrolysis generally accounted for less than 20% of the total activities measured. * Significantly (P < 0.05) different from the control value.

sarcolemmal Ca2+/Mga+ ATPase, the ATPase activities of other subcellular fractions were measured in the absence or presence of different concentrations of Con A. No appreciable effect of Con A was seen on mitochondrial or microsomal (sarcoplasmic reticular) ATP hydrolysis in the presence of Mga+ or Ca2+(Tables I and II). The sarcolemmal Na+-K+ ATPase activity

TABLE

EFFECTOFC~~AONRATHEARTMEMBRANE Me

was also not influenced by Con A. Although sarcolemmal Ca’+-stimulated ATPase activity (ATP hydrolysis due to micromolar concentrations of Ca2+ in the presence of Mp) was not affected by Con A, the basal ATPase activity, which mainly represents Me ATPase activity, in sarcolemma was markedly increased by 2 to 50 pg/ml Con A (Table III). The basal ATPase and Ca2+-

II

ATPase AND~ARCOLEMMALNa+-K+ATPase

ATPase activity Sarcolemma Concentration of Con A (hdnl) Control 2 5 10 20 50

147 f 10

191a11' 307 _+18* 317f21’ 236 f 16* 230 + 14*

protein/h)

Mitochondria Mg2f ATPase

Mg2f ATPase

(4.0mM)

(pm01 Pi/mg

Na+-K+ ATPase 21 f 21 f 23 + 26 f 24 f 20 +

3.1 2.0 1.8 1.6 2.1 2.4

ACTIVITIES

Microsomes MgZf ATPase

(4.0mM)

(4.0mM)

48.3 47.4 44.7 45.7 45.3 47.1

98.6 97.4 98.5 103.2 105.6 104.3

Note. Each value for sarcolemma is a mean f SE of six experiments whereas all other values are the average of three experiments. The Mga+ ATPase activities were corrected for ATP hydrolysis in the absence of Me. * Significantly (P i 0.05) different from control.

RAT HEART

SARCOLEMMAL TABLE

Caa+/Me

43

ATPase

III

INFLUENCEOFC~~AONRATHEARTSARCOLEMMAL,SARCOPLASMICRETICULAR(MICROSOMES),AND MYOFIBRILLARBASALANDCa'+-STIMULATEDATPase ACTIVITIES ATPase activity

Basal ATPase

Control 2 5 10 20 50

14lk 12 198k 9* 286+ 14* 295kll” 248 f 12* 242+ 11*

Ca’+-stimulated ATPase 15.0 t 14.4 f 13.8 k 13.4 f 12.1 f 11.3 +

protein/h)

Microsomes

Sarcolemma Concentration of Con A (bdml)

(pm01 Pi/mg

1.43 0.92 0.98 1.05 0.88 0.93

Basal ATPase 94.3 89.1 89.8 100.4 107.3 105.5

Ca’+-stimulated ATPase 13.2 10.9 10.6 13.0 10.5 10.8

Myofibrils Basal ATPase 4.5 4.0 4.0 3.9 3.9 4.2

Ca’+-stimulated ATPase 17.9 17.4 17.1 17.4 17.1 16.9

Note. Each value for sarcolemma is a mean + SE of six experiments whereas all other values are an average of three experiments. Ca’+-stimulated ATPase activities were measured in the presence of 1 FM Ca2+ whereas basal ATPase represents ATP hydrolysis in the absence of Caa+. * Significantly (P < 0.05) different from control.

stimulated ATPase activities of microsomal fraction and myofibrils were not affected appreciably by Con A (Table III). It should be pointed out that Ca’+-stimulated ATPase cannot be detected in mitochondria, unlike other subcellular organelles. In another set of experiments, the actions of 10 pg/ml Con A were examined on the sarcolemmal Ca’+/M$+ ATPase by keeping the concentration of Ca2+ or M$+ at 4 mM and varying the concentration of ATP in the reaction medium (Figs. 1 and 2). Although the results as shown in these figures do not show apparent stimulatory action of Con A at lower concentrations of ATP, the Lineweaver-Burk plots of the data for the Con A-treated and control sarcolemmal Ca’+/M$+ ATPase were linear (r > 0.99). The K, of sarcolemmal Ca2+ ATPase was increased from 0.64 + 0.08 to 1.53 + 0.16 mM ATP and V,,,,, was increased from 273 f 25 to 494 + 36 pmol PJmg/h by 10 pg/ml Con A. The K, value for the sarcolemmal Me ATPase was increased from 0.62 f 0.07 to 1.24 + 0.11 mM ATP and V,,, was increased from 244 +_ 18 to 473 + 29 pmol Pi/mg/h by 10 pg/ml Con A. The activities of both Ca2+ ATPase and M$ ATPase in heart sarcolemma were also

measured in the absence or presence of 10 pg/ml Con A by using different concentrations of Ca2+ or Mp in the presence of 4 mM ATP (Figs. 1 and 2). The K, for the sarcolemmal Ca2+ ATPase was increased from 0.68 + 0.07 to 2.3 -t 0.36 mM Ca2+ and V,, was increased from 230 + 31 to 580 k 42 pmol Pi/mg/h by 10 pg/ml Con A. For the sarcolemmal M$+ ATPase the K, value was increased from 0.57 + 0.08 to 2.4 + 0.42 mM Mg+ and V,,,,, was increased from 248 t 26 to 590 + 45 pmol Pi/mg/h by 10 pg/ml Con A. The stimulatory effects of 10 pug/ml Con A on the sarcolemmal Ca2+ ATPase or M$+ ATPase activities were blocked by cu-methylmannoside in a dosedependent manner (Table IV). ol-methylmannoside (50 mM), which was found to have no actions of its own, completely blocked the effect of Con A. In order to examine whether any alteration in membrane integrity or membrane components would affect the stimulatory actions of Con A on sarcolemmal Ca’+/ Mg2i ATPase, the sarcolemmal membrane was subjected to various treatments with detergents (digitonin and deoxycholate), phospholipases, and neuraminidase, and then the effects of Con A on Ca’+/M$’ ATPase were tested. Table V shows that

44

ZHAO, Ca*+-dependent

MAKINO,

ATPase

ATP(mM)

FIG. 1. Influence of Con A on Ca2+ ATPase in rat heart sarcolemma at different concentrations of ATP and Ca2+. When the concentration of ATP (top) was varied, the concentration of Ca2+ was kept at 4 t?IM whereas when the concentration of Ca2+ (bottom) was varied, the concentration of ATP was 4 mM. Cazf ATPase activities were measured in the absence (0) or presence (0) of 10 fig/ml Con A. Values are averages of three experiments.

AND

DHALLA

About 50% of the total and specific binding occurred during the first minute. Thereafter the Con A binding rose more gradually until 10 min after initiation of the reaction, and then started to decline slightly. The nonspecific Con A binding, as measured in the presence of 200 mM cr-methylmannoside, was always less than 10% of the total binding; 50 mM cu-methylmannoside was not able to prevent the Con A binding completely. The mitrochondrial, myofibrillar, and microsomal fractions did not exhibit any specific Con A binding. Figure 4 shows the Con A binding to the sarcolemma at varying concentrations of Con A; specific binding increased with an increase in the concentration of Con A. Scatchard plot analysis of the data showed a straight line with a Kd of about 530 nM (57.0 pg/ml) and a B,,, of 235 pmol/mg protein; this indicated that Con A had one kind of binding site on the isolated heart sarcolemmal membrane.

‘loo

1

I%**-dependent

300

these treatments by themselves decreased the Ca2+/M$+ ATPase, but the stimulatory effects of Con A on Ca’+/M$+ ATPase were not affected except in the case of neuraminidase treatment, where the percentage stimulation of Ca2+/Me ATPase by Con A was increased from 111% (control) to 229%. The treatments with digitonin, deoxycholate, phospholipase D, and neuraminidase increased the ouabain-inhibitable Na+-K+ ATPase activity (Table V) indicating that the sarcolemmal vesicles were made permeable to ouabain by these treatments. Treatment of heart sarcolemma with phospholipase A depressed the Na+-Kf ATPase activities markedly whereas the activity of Na+-K+ ATPase was significantly increased with phospholipase C treatment without any changes in the ouabain-inhibitable Na+-K+ ATPase (Table V). Sarcolemmal Con A binding. The time course of the binding of Con A to isolated rat heart sarcolemma is shown in Fig. 3.

ATPase

o/O I

/

ATP (mM)

0.5 1

2

4

Mg*+hM)

FIG. 2. Influence of Con A on Mg2+ ATPase in rat heart sarcolemma at different concentrations of ATP and Me. When the concentration of ATP (top) was varied, the concentration of Me was 4 mM whereas when the concentration of Me (bottom) was varied, the concentration of ATP was 4 mM. Mp ATPase activities were measured in the absence (0) or presence (0) of 10 *g/ml Con A. Values are averages of three experiments.

RAT HEART

SARCOLEMMAL TABLE

Caa+/Me IV

EFFECTOFWMETHYLMANNOSIDEONCaa+ATPase AND Me

ATPase OFRATHEARTSARCOLEMMA Me ATPase (pm01 Pi/m&h)

Ca2+ ATPase (rmol Pi/mg/h)

Concentration of a-methylmannoside (mm

Without

0

Con A

158 + 8.4 158 f 7.2 161 f 7.0 160 + 4.5 160 -+ 7.8 158 f 8.9

2 5 10 20 50

45

ATPase

Without

With Con A 271 f 272 + 235 + 190 + 172 + 156,

8.4 11.9 11.7 7.0 6.4 7.6

159 t 159 + 166k 171 2 165 f 165 +

Note. The ATPase activities were assayed in the presence of 4 mM Me wg/ml when present. Each value is a mean rt SE of six experiments.

Con A 7.3 5.5 9.7 6.6 8.7 10.4

With Con A 386 + 344 f 256 i 197 f 170 + 164 f

or Ca’+. Con A concentration

19.9 17.0 14.2 9.4 6.9 9.1

was 10

chondrial, and myofibrillar fractions were not altered significantly by Con A. Other In this study we have demonstrated that investigators have also reported the stimCon A was capable of stimulating the Ca2+ ulation of Ca2+ATPase and M$’ ATPase ATPase and Me ATPase activities in activities by Con A in mammary glands, heart sarcolemma. The action of Con A on liver, and skeletal muscle membranes, althe sarcolemmal Ca’+/M$+ ATPase ap- though the extent of stimulation and the pears to be of specific nature since the sar- concentrations of ATP, M$+, or Ca2+ colemmal Na+-K+ ATPase and Cazt-stimshowing the stimulatory actions of Con A ulated ATPase activities were not affected were different (22,23,26). Such a difference by this lectin. Furthermore, ATPase activ- may be due to the species and tissue reities in the sarcoplasmic reticular, mito- lated differences in the characteristics of DISCUSSION

TABLE

V

EFFECTSOFVARIOUSTREATMENTSONTHEEFFECTOFC~~AONRATHEARTSARCOLEMMALATP~~~ Ca” ATPase Without Con A

Treatment Control Digitonin Deoxycholate Phospholipase Phospholipase Phospholipase Neuraminidase

A C D

163 i 106 f 85 + 36 f 71 f 130 i 76 f

6.4 7.1 8.6 3.3 2.4 2.2 6.0

With Con A 289 k 9.1 194 +- 9.3 168 k 14.1 54 +- 3.8 115 +- 2.4 214 + 4.9 184 k 2.3

M$+ ATPase Without Con A 153 -+ 5.9 95 + 6.1 76 f 8.0 12 t 0.7 35 f 2.5 109 f 3.2 63 + 1.5

With Con A 324 + 235 + 154 -t 20f 82 f 258 f 207 +

11.8 8.6 13.0 1.0 2.5 8.0 3.9

Na+-K+ ATPase 25.5 +- 0.7 20.3 3~2.1 28.1 f 2.3 1.7 i 0.9 30.2 f 0.6 16.2 f 3.2 24.5 + 1.4

Ouabaininhibitable Na+-K+ ATPase 4.7 + 1.6 12.9 F 1.7 27.9 -I- 3.5 1.5 f 1.0 3.3 f 0.6 13.4 -I- 1.4 15.9 F 2.8

Note. ATPase activities are expressed as Nmol Pi/mg/h. Con A concentration was 10 pg/ml when present. MgZ+ and Cazi ATPases were measured in the presence of 4 mM MgClz or 4 mM CaCl,, respectively. The ouabain-inhibitable Na+-K+ ATPase activity represents the depression of the enzyme in the presence of 2 mM ouabain. Each value is a mean 4 SE of four experiments. The concentration of digitonin was O.l%, deoxylcholate 0.02%, phospholipase A 1.6 unit/ml, phospholipase C 1.0 unit/ml, phospholipase D 1.0 unit/ml, and neuraminidase 0.31 unit/ml.

46

ZHAO,

MAKINO,

4 c l3

0

20

10

30

Tome (mm)

FIG. 3. Time course of Con A binding to rat heart sarcolemma. The Con A binding was measured in the absence (0) and presence of 50 mM (A) and 200 mM (0) a-methylmannoside.

different membrane preparations. This view is supported by the fact that the Na’K+ ATPase activity in rabbit thymocytes and lymph node cells, unlike rat heart sarcolemma, was stimulated by Con A (27). Our inability to detect the stimulatory

Con

AND

DHALLA

effect of Con A on heart sarcolemmal Na+K’ ATPase was not due to any isolation artifact because the sarcolemmal preparation obtained by the hypotonic shock-LiBr treatment method also did not show any effect of Con A on Nat-K+ ATPase (15). Nonetheless, the observed increase in Ca’+/M$+ ATPase activity due to Con A may be a result of an increased ability of sarcolemma to bind Ca2+or Mga+ since earlier studies from this laboratory have revealed a stimulatory action of Con A on the ATP-independent Ca2+ binding with the cardiac cell membrane (15). In order to examine whether changes in any specific phospholipid may mediate the stimulatory effect of Con A on Ca2+/Mg2 ATPase, we pretreated the sarcolemmal membranes with phospholipase A, C, or D. The phospholipases reduced the activity of Ca’+/M$+ ATPase, which is consistant with a previous report (33), but these enzymes did not alter the effect of Con A on Ca’+/Mc ATPase. Therefore it seems unlikely that Con A exerted its effect through its interaction with some specific phospholipids. The treatment of heart sarcolemma with detergents such as digitonin and deoxycholate, which are likely to increase

A ()lg/ml)

FIG. 4. Effect of Con A concentration on the specific binding of Con A to rat heart sarcolemma. inset is the Scatchard plot of the same data.

The

RAT HEART

SARCOLEMMAL

the accessibility of Con A to the active sites in the membrane, also had no effect on the stimulation of Ca2’/Mp ATPase by Con A. The observed decrease in the sarcolemma1 Ca2+/Mp ATPase by detergents is in agreement with our earlier results (10,ll). On the other hand, treatment with neuraminidase, which hydrolyzes the sialic acid residues in the membrane, markedly inhibited the Ca2+/Mp ATPase but increased the effect of Con A on the enzyme. This finding indicates that the sialic acid residues may play some role in the activity of Ca2’/MF ATPase as well as in the action of Con A on this ATPase system. Such a view is supported by the observations that a-methylmannoside, which is known to prevent the binding of Con A to carbohydrate residues in the membrane, was found to inhibit the Con A-induced stimulation in sarcolemmal Ca2+/M$+ ATPase activity. Okamoto et al. (36) and Moulton et al. (25) have found that Con A bound to a protein (M, 102,000 on SDS electrophoresis gel) in the transverse tubule membrane of the skeletal muscle. Accordingly, these investigators proposed that this protein might represent M$+ ATPase in the transverse tubule and its stimulation by Con A occurred through a direct interaction of Con A with the protein rather than through an indirect mechanism (25,36). In this study we found that Con A bound to the isolated heart sarcolemma and this binding appeared to be specific since amethylmannoside, a well-known antagonist of Con A, prevented this binding. Results obtained in this study show that the maximal Con A binding to the sarcolemma1 membrane occurred at about 200 pg/ ml Con A whereas its maximal stimulatory action on the sarcolemmal Ca2+/Mg+ ATPase was evident at 10 pg/ml. These results suggest that the binding of Con A to some specific sites in the heart sarcolemma1 membrane may be intimately associated with the activation of Ca2+/M$+ ATPase. It should be pointed out that Con A has been reported to alter the membrane permeability (19), membrane potential (24), cell surface charge (28), and crosslinking of membrane proteins (22). Since

Caa+/Mp

ATPase

47

Con A is also known to affect membrane fluidity (19-21), it is likely that the augmentation of the sarcolemmal Ca2+/M2+ ATPase by Con A may also be due to its action on the fluidity of heart sarcolemma. It should be noted that Con A-induced changes in membrane fluidity are considered to involve both carbohydrate groups and hydrophobic ligands in the plasma membrane (37-39). However, the action of Con A on phospholipids has also been demonstrated in vesicles containing neither glycoproteins nor glycolipids (26, 40) and there is evidence that the lipid environment of the membrane protein is altered as a result of Con A binding (41). Thus it appears that the binding of Con A to the Ca2+/M$+ ATPase as well as the alterations in the lipid microenvironment of the ATPase in heart sarcolemma by Con A may result in stimulating the Ca2+t/M2+ activities. ACKNOWLEDGMENTS The research work reported in this paper was supported by a grant from the Medical Research Council of Canada. Dayuan Zhao was a predoctoral fellow of the Canadian Heart Foundation. REFERENCES 1. CARAFOLI, E. (1985) J. Mol. Cell. CardioL 17,203212. 2. DHALLA, N. S., PIERCE, G. N., PANAGIA, V., SINGAL, P. K., AND BEAMISH, R. E. (1982) Basic Res. CardioL 77,117139. 3. DHALLA, N. S., ZIEGELHOFFER, A., AND HARROW, J. A. C. (197’7) Canad. J. Physiol. Pharmacol. 55, 1211-1234. 4. LANGER, G. A. (1982) Annu. Rev. Physiol. 44,435449. 5. BENHAM, C. D., AND TSIEN, R. W. (1986) in Cell Calcium and the Control of Membrane Transport (Mandel, L. J., and Eaton, D. C., Eds.), pp. 46-64, Rockefeller Univ. Press, New York. 6. DEYOUNG, M. B., AND SCARPA, A. (1987) FEBS Lett. 223,53-58. 7. PEARSON, J. D., AND GORDON, J. L. (1985) Annu. Rev. Physiol. 47,617-627. 8. ANAND, M. B., CHAUHAN, M. S., AND DHALLA, N. S. (1977) .I Biochem. (Tokyo) 82,1731-1739. 9. MALOUF, N. N., AND MEISSNER, G. (1980) J. Histothem. Cytochem. 28,1286-1294. 10. PANAGIA, V., LAMERS, J. M. J., SINGAL, P. K., AND DHALLA, N. S. (1982) Int. J. Biochem. 14, 387397.

48

ZHAO,

MAKINO,

AND

DHALLA

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