Characteristics of Ca2+-stimulated ATPase in rat heart sarcolemma in the presence of dithiothreitol and alamethicin

We have studied the activities of Ca²⁺-stimulated ATPase in rat heart sarcolemma upon modulating the redox state of membrane thiol groups with dithiothreitol (DTT). The suitability of alamethicin to unmask the latent activity of this enzyme was also investigated. The Ca²⁺-stimulated ATPase in sarcolemma exhibited two activation sites — one with low affinity (Km = 0.70 ± 0.2 mM; Vmax = 10.0 ± 2.2 mol Pi/mg/h) and the other with high affinity (Km = 0.16 ± 0.7 mM; Vmax = 4.6 ± 0.8 mol Pi/mg/h) for Mg²⁺ATP. Alamethicin at a ratio of 1:1 with the sarcolemmal protein caused a 3-fold activation of Ca²⁺-stimulated ATPase without affecting its sensitivity to Ca²⁺ or Mg²⁺ATP. Treatment of sarcolemma with deoxycholate or sodium dodecyl sulfate resulted in a total loss of the enzyme activity; high concentrations of alamethicin also showed a detergent-like action on the sarcolemmal vesicles. DTT at 5–10 mM concentrations caused a 4–5 fold activation of Ca²⁺-stimulated ATPase in sarcolemma and this effect was observed to be dependent on the concentration of Mg²⁺ATP. DTT increased the affinity of the enzyme to Mg²⁺ATP at the high affinity site and enhanced the Vmax at the low affinity site in addition to increasing the sensitivity of Ca²⁺-stimulated ATPase to Ca²⁺. DTT protected the Ca²⁺-stimulated ATPase against deterioration by detergents and restored the enzyme activity after treatment with N-ethylmaleimide. The mechanism of action of DTT on Ca²⁺-stimulated ATPase may involve the reduction of essential thiols at the active site of the enzyme or its interaction with specific DTT-dependent inhibitor protein. No changes in the sensitivity of sarcolemmal Ca²⁺-stimulated ATPase to orthovanadate was evident in the absence or presence of DTT and alamethicin. The results suggest the use of both DTT and alamethicin for the determination of Ca²⁺-stimulated ATPase activity in sarcolemmal preparations.     

Evidence for high molecular weight Na−Ca exchange in cardiac sarcolemmal vesicles

Summary Cardiac sarcolemma (SL) vesicles were subjected to irradiation inactivation-target sizing analyses and gel permeation high performance liquid chromatography (HPLC) to ascertain the weight range of native Na–Ca exchange. Frozen SL vesicle preparations were irradiated by electron bombardment and assayed for Na–Ca exchange activity. When applied to classical target sizing theory, the results yielded a minimum molecular weight (M _r) of approximately 226,000±20,000sd (n=6). SL vesicle proteins were solubilized in 6% sodium cholate in the presence of exogenous phospholipid and fractionated by size on a TSK 30XL HPLC column. Eluted proteins were mixed 11 with mobile phase buffer containing 50mg/ml soybean phospholipid and reconstituted by detergent dilution. The resulting proteoliposomes were assayed for Na–Ca exchange activity. Na–Ca exchange activity eluted in early fractions containing larger proteins as revealed by SDS-PAGE. Recovery of total protein and Na–Ca exchange activity were 91±7 and 68±11%, respectively. In the peak fraction, Na–Ca exchange specific activity increased two-to threefold compared to reconstituted controls. Compared to the elution profile of protein standards under identical column conditions, sodium cholate solubilized exchange activity had a minimumM _r of 224,000 Da. Specific⁴⁵Ca²⁺-binding SL proteins withM _r of 234,000, 112,000, and 90,000 Da were detected by autoradiography of proteins transferred electrophoretically to nitrocellulose.These data suggest that native cardiac Na–Ca exchange is approximately 225,000 Da or larger. The exact identification and purification of cardiac Na–Ca exchange protein(s) remains incomplete.     

Role of sulfhydryl groups in phospholipid methylation reactions of cardiac sarcolemma

The effect of reagents that modify sulfur-containing amino acid residues in the phosphatidylethanolamine N-methyltransferase was studied in the isolated rat cardiac sarcolemma by employing S-adenosyl-L-[methyl-³H]methionine as a methyl donor. Dithiothreitol protected the sulfhydryl groups in the membrane and caused a concentration- and time-dependent increase of phospholipid N-methylation at three different catalytic sites. This stimulation was highest (9-fold) in the presence of 1 MM MgCl₂ and 0.1 µM S-adenosyl-L-[methyl-³H]methionine at pH 8.0 (catalytic site 1), and was associated with an enhancement of V_max without changes in K_m for the methyl donor. Thiol glutathione was less stimulatory than dithiothreitol; glutathione disulfide inhibited the phosphatidylethanolamine N-methylation by 50%. The alkylating reagents, N-ethylmaleimide and methylmethanethiosulfonate, inhibited the N-methylation with IC_5O of 6.9 and 14.1 µM, respectively; this inhibition was prevented by 1 mM dithiothreitol. These results indicate a critical role of sulfhydryl groups for the activity of the cardiac sarcolemmal phosphatidylethanolamine N-methyltransferase and suggest that this enzyme system in cardiac sarcolemma may be controlled by the glutathione/glutathione disulfide redox state in the cell.Abbreviations AdoMet S-Adenosyl-L-methionine - AdoHey S-adenosyl-L-homocysteine - DTNB 5,5dithiobis (2-nitrobenzoate) - NEM N-ethylmaleimide - MMTS methylmethanethiosulfonate - DTT dithiothreitol - EDTA Ethylenediaminetetraacetic acid - GSH glutathione - GSSG glutathione disulfide - PE phosphatidylethanolamine - PMME phosphatidyl-N-monomethylethamolamine - PDME phosphatidyl-N-dimethylethanolamine - PC phosphatidylcholine - NPL nonpolar lipids - SL sarcolemma     

Effect of platelet-activating factor (PAF) on sodium calcium exchange in cardiac sarcolemmal vesicles

Summary The effects of platelet-activating factor (PAF) on Na⁺-dependent calcium uptake in myocardial sarcolemmal vesicles were examined in order to clarify its mechanism of inotropic action on the heart. PAF (40 and 20 µM) significantly inhibited Na⁺-Ca²⁺ exchange by 61% and 37%, respectively. Both initial rate of exchange and maximal exchange were inhibited. The Km for the reaction was not altered but Vmax was lowered 55% by PAF. Lyso-PAF inhibited Na⁺-Ca²⁺ exchange to a similar degree as PAF. CV-3988, a specific PAF receptor antagonist, failed to diminish the inhibitory effect of PAF on Na⁺-Ca²⁺ exchange, suggesting that the effect of PAF on Na⁺-Ca ²⁺ exchange is not via a receptor mechanism. The passive permeability of sarcolemmal vesicles to Ca²⁺ was markedly elevated after PAF treatment. However, this effect could not account for the decrease in Na⁺-Ca²⁺ exchange. Interestingly, passive Ca²⁺ binding to cardiac sarcolemma was increased by 40 µM PAF. This study indicates that a depression of Na⁺-Ca²⁺ exchange probably does not play a role in the negative inotropic effect of PAF on the myocardium under physiological conditions. Its mechanism of action on Na⁺-Ca²⁺ exchange is discussed.     

The calmodulin-activated form of the Ca2+-pumping ATPase of the cardiac sarcolemmal membrane produces Ca2+ gradients with a thermodynamic efficiency of 100%

The thermodynamic efficiency of the calmodulin-activated form of the Ca²⁺-pumping ATPase of the bovine cardiac sarcolemma (SL) was evaluated in sealed vesicles under reversible conditions. The free internal Ca²⁺ concentration ([Ca²⁺]_i) established in the SL vesicle lumen by action of the ATPase was determined as a function of the [ATP]/([ADP][P_i]) ratio for the following experimental conditions: 250mM sucrose, 100mM KCI, 0.1mM Mg²⁺, 25mM HEPES, 25mM Tris, pH 7.40, at 37°C, [Ca²⁺]_o=50nM (1mM Ca/EGTA buffer), 0.75mM Mg-ATP, 0.1mM P_i, variable [ADP]. Under these conditions, with the pump working near itsK _m of 64nM, the [Ca²⁺]_i achieved was 18mM, decreasing with increasing [ADP] for [ADP] 0.84mM. A plot of the square of the [Ca²⁺]_i/[Ca²⁺]_o ratio against [ATP]/([ADP][P_i]) gave a straight line with a slope of 1.5×10⁷M. This was in agreement, within the experimental error, with the equilibrium constant for ATP hydrolysis under these conditions (1.09×10⁷M). These results demonstrate (1) tight coupling between Ca²⁺ transport and ATP hydrolysis with a stoichiometry of 2 Ca²⁺ moved per ATP split and (2) a low degree of passive leakage. Analysis at low [ADP] (<0.83mM) showed the unexpected result that ADP increases the rate of theforward reaction of the pump. The maximal effect on the initial rate is a 96±5% increase, with an EC₅₀ of approximately 0.4mM (ADP). Similar but lesser stimulation was observed with CDP. The implications of the above results for the energetics of the pump and for its physiological function in the beating heart are discussed.     

A rapid method for preparation of sarcolemma from frog skeletal muscle

A rapid method for the preparation of sarcolema from frog skeletal muscle has been described. The purified cell segments were transparent and devoid of contractile material. The Na⁺, K⁺ -ATPase and 5'-nucleotidase activities in sarcolemma purified by this method were comparable to those reported for sarcolemmal preparations purified by density gradient centrifugation. The preparation also possessed acid phosphatase, alkaline phosphatase and K⁺ -activated, ouabain-sensitive p-nitrophenyl phosphatase activities. The cholesterol to phospholipid ratio of the sarcolemma was 0.33, indicating its high purity; further, the preparation was free from mitochondria and contractile proteins.     

Localization of (Ca2+ + Mg2+)-ATPase,Ca2+ pump and other ATPase activities in cardiac sarcolemma

N-Ethylmaleimide was employed as a surface label for sarcolemmal proteins after demonstrating that it does not penetrate to the intracellular space at concentrations below 1·10^?4 M. The sarcolemmal markers, ouabain-sensitive (Na⁺ + K⁺)-ATPase and Na⁺/Ca²⁺-exchange activities, were inhibited in N-ethylmaleimide perfused hearts. Intracellular activities such as creatine phosphokinase, glutamate-oxaloacetate transaminase and the internal phosphatase site of the Na⁺ pump (K⁺-p-nitrophosphatase) were not affected. Almost 20% of the (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ pump were inhibited indicating the localization of a portion of this activity in the sarcolemma. Sarcolemma purified by a recent method (Morcos, N.C. and Drummond, G.I. (1980) Biochim. Biophys. Acta 598, 27–39) from N-ethylmaleimide-perfused hearts showed loss of approx. 85% of its (Ca²⁺ + Mg²⁺-ATPase and Ca²⁺ pump compared to control hearts. (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ pump activities showed two classes of sensitivity to vanadate ion inhibition. The high vanadate affinity class ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for inhibition approx. 1.5 μM) may be localized in the sarcolemma and represented approx. 20% of the total inhibitable activity in agreement with estimates from N-ethylmaleimide studies. Sucrose density fractionation indicated that only a small portion of Mg²⁺-ATPase and Ca²⁺-ATPase may be associated with the sarcolemma. The major portion of these activities seems to be associated with high density particles.     

Na+−Ca2+ exchange and Ca2+ channel characteristics in bovine aorta and coronary artery smooth muscle sarcolemmal membranes

Tension generation and Ca²⁺ flux in smooth muscle varies depending upon the diameter of a vessel and its location. The purpose of the present investigation was to determine if the biochemical characteristics of the Na⁺–Ca²⁺ exchanger and the Ca²⁺ channel differ in sarcolemmal membrane preparations isolated from a large conduit vessel (thoracic aorta) or from large and small coronary arteries. We also investigated the possibility of differences between sarcolemmal membranes isolated from coronary arteries dissected from the right and left ventricles. The purification of the sarcolemmal membranes was of a similar magnitude amongst the different groups. Contamination of the sarcolemmal membranes with other membranous organelles was negligible and similar amongst the groups. The K_m and V_max of Na⁺-dependent Ca²⁺ uptake in sarcolemmal vesicles was similar amongst the groups. Calcium channel characteristics were examined by measuring [³H]PN200-110 binding to sarcolemmal vesicles. The right coronary artery membranes from both large and small caliber vessels exhibited a higher K_d and the small right coronary artery sarcolemmal preparation had a lower maximal binding density for [³H] PN200-110. The results suggest that the right coronary artery, and in particular the small diameter right coronary artery, possesses altered Ca²⁺ channel characteristics in isolated sarcolemmal membranes.     

Glycoprotein characteristics of the sodium channel saxitoxin-binding component from mammalian sarcolemma

The saxitoxin-binding component of the excitable membrane sodium channel exhibits glycoprotein characteristics as evidenced by its specific interaction with various agarose-immobilized lectins. The detergent-solubilized saxitoxin-binding component interacts quantitatively with immobilized wheat germ agglutinin and concanavalin A and fractionally with immobilized Lens culinaris hemagglutinin and Ricinus communis agglutinin. These lectins preferentially bind $\text{N-}\text{acetylglucosamine}$ and sialic acid (wheat germ agglutinin), mannose (concanavalin A and Lens cunilaris and galactose (Ricinus communis). Removal of terminal sialic acid residues by neuraminidase markedly decreases binding to immobilized wheat germ agglutinin but uncovers sites capable of interacting with lectins specific for galactose and $\text{N-}\text{acetylgalactosamine}$. $\text{β-N-}\text{acetylglucosaminidase}$, an exoglycosidase has no effect on the binding of the channel protein to wheat germ agglutinin. Similarly, phospholipase C has no effect on binding of the solubilized toxin binding component to this lectin. Neither wheat germ agglutinin nor concanavalin A free in solution alters the number of toxin binding sites or their affinity for toxin. The sodium channel saxitoxin-binding component appears to be a glycoprotein containing terminal sialic acid residues and internal mannose, galactose, $\text{N-}\text{acetylglucosamine}$, and $\text{N-}\text{acetylgalactosamine}$ residues. The toxin binding site is spatially separated from the binding sites for the lectins studied. The effect of these sugar moieties must be considered when evaluating the biophysical parameters of the sodium channel.     

Changes of sodium and ATP affinities of the cardiac (Na,K)-ATase during and after nitric oxide deficient hypertension

In the cardiovascular system, NO is involved in the regulation of a variety of functions. Inhibition of NO synthesis induces sustained hypertension. In several models of hypertension, elevation of intracellular sodium level was documented in cardiac tissue. To assess the molecular basis of disturbances in transmembraneous transport of Na⁺, we studied the response of cardiac (Na,K)-ATPase to NO-deficient hypertension induced in rats by NO-synthase inhibition with 40 mg/kg/day N^G-nitro-L-arginine methyl ester (L-NAME) for 4 four weeks. After 4-week administration of L-NAME, the systolic blood pressure (SBP) increased by 36%. Two weeks after terminating the treatment, the SBP recovered to control value. When activating the (Na,K)-ATPase with its substrate ATP, no changes in K_m and V_max values were observed in NO-deficient rats. During activation with Na⁺, the V_max remained unchanged, however the K_Na increased by 50%, indicating a profound decrease in the affinity of the Na⁺-binding site in NO-deficient rats. After recovery from hypertension, the activity of (Na,K)-ATPase increased, due to higher affinity of the ATP-binding site, as revealed from the lowered K_m value for ATP. The K_Na value for Na⁺ returned to control value. Inhibition of NO-synthase induced a reversible hypertension accompanied by depressed Na⁺-extrusion from cardiac cells as a consequence of deteriorated Na⁺-binding properties of the (Na,K)-ATPase. After recovery of blood pressure to control values, the extrusion of Na⁺ from cardiac cells was normalized, as revealed by restoration of the (Na,K)-ATPase activity. (Mol Cell Biochem 000: 000-000, 1999)