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1.
Modification of heart sarcolemmal Na+/K+-ATPase activity during development of the calcium paradox 总被引:3,自引:0,他引:3
This study examined the status of sarcolemmal Na+/K+-ATPase activity in rat heart under conditions of Ca2+-paradox to explore the existence of a relationship between changes in Na+/K+-pump function and myocardial Na+ as well as K+ content. One min of reperfusion with Ca2+ after 5 min of Ca2+-free perfusion reduced Na+/K+-ATPase activity in the isolated heart by 53% while Mg2+-ATPase, another sarcolemmal bound enzyme, retained 74% of its control activity. These changes in sarcolemmal ATPase activities were dependent on the duration and Ca2+ concentration of the initial perfusion and subsequent reperfusion periods; however, the Na+/K+-ATPase activity was consistently more depressed than Mg2+-ATPase activity under all conditions. The depression in both enzyme activities was associated with a reduction in Vmax without any changes in Km values. Low Na+ perfusion and hypothermia, which protect the isolated heart from the Ca2+-paradox, also prevented reperfusion-induced enzyme alterations. A significant relationship emerged upon comparison of the changes in myocardial Na+ and K+ content to Na+/K+-ATPase activity under identical conditions. At least 60% of the control enzyme activity was necessary to maintain normal cation gradients. Depression of the Na+/K+-ATPase activity by 60-65% resulted in a marked increase and decrease in intracellular Na+ and K+ content, respectively. These results suggest that changes in myocardial Na+ and K+ content during Ca2+-paradox are related to activity of the Na+/K+-pump; the impaired Na+/K+-ATPase activity may lead to augmentation of Ca2+-overload via an enhancement of the Na+/Ca2+-exchange system. 相似文献
2.
Ziegelhöffer Attila Ravingerová Tanya Styk Ján Šeboková Jana Waczulíkova Iveta Breier Albert Džurba Andrej Volkovová Katarína Čársky Jozef Turecký Ladislav 《Molecular and cellular biochemistry》1997,176(1-2):191-198
In diabetes the hearts exhibit impaired membrane functions, but also increased tolerance to Ca2+ (iCaT) However, neither the true meaning nor the molecular mechanisms of these changes are fully understood. The present study is devoted to elucidation of molecular alterations, particularly those induced by non-enzymatic glycation of proteins, that may be responsible for iCaT of the rat hearts in the stage of fully developed, but still compensated diabetic cardiomyopathy (DH). Insulin-dependent diabetes (DIA) was induced by a single i.v. dose of streptozotocin (45 mg.kg-1). Beginning with the subsequent day, animals obtained 6 U insulin daily. Glucose, triglycerides, cholesterol and glycohemoglobin were investigated in blood. ATPase activities, the kinetics of activation of (Na,K)-ATPase by Na+ and K+, further the fluorescence anisotropy of diphenyl-hexatriene as well as the order parameters of membranes in isolated heart sarcolemma (SL) were also investigated. In addition, the degree of glycation and glycation-related potency for radical generation in SL proteins were determined by investigating their fructosamine content. In order to study calcium tolerance of DH in a 'transparent' model, hearts were subjected to calcium paradox (Ca-Pa, 3 min of Ca2+ depletion; 10 min of Ca2+ repletion). In this model of Ca2+-overload, Ca2+ ions enter the cardiac cells in a way that is not mediated by receptors. Results revealed that more than 83% of the isolated perfused DH recovered, while the non-DIA control hearts all failed after Ca-Pa. DH exhibited well preserved SL ATPase activities and kinetics of (Na,K)-ATPase activation by Na+, even after the Ca-Pa. This was considered as a reason for their iCaT. Pretreatment and administration of resorcylidene aminoguanidine (RAG 4 or 8 mg.kg-1) during the disease prevented partially the pathobiochemical effects of DIA-induced glycation of SL proteins. DIAinduced perturbations in anisotropy and order parameters of SL were completely prevented by administration of RAG (4 mg.kg-1). Although, the latter treatment exerted little influence on the (Na,K)-ATPase activity, it decreased the calcium tolerance of the DH. Results are supporting our hypothesis that the glycation-induced enhancement in free radical formation and protein crosslinking in SL may participate in adaptive mechanisms that may be also considered as 'positive' and are responsible for iCaT of the DH. 相似文献
3.
Vincenzo Panagia Vijayan Elimban Pallab K. Ganguly Naranjan S. Dhalla 《Molecular and cellular biochemistry》1987,78(1):65-71
Heart sarcolemma has been shown to possess three catalytic sites (I, II and III) for methyl transferase activity (Panagia V, Ganguly PK and Dhalla NS. Biochim Biophys Acta 792: 245–253, 1984). In this study we examined the effect of phosphatidylethanolamine N-methylation on ATP-independent Ca2+ binding and ATPase activities in isolated rat heart sarcolemma. Both low affinity (1.25 mM Ca2+) and high affinity (50 µM Ca2+) Ca2+ binding activities were decreased following incubation of sarcolemmal membranes with AdoMet under optimal conditions for site II and III. Similarly, Ca2+ ATPase activities measured at 1.25 mM and 4 mM Ca2+ were depressed by phospholipid N-methylation. S-adenosyl homocysteine, a specific inhibitor of phospholipid N-methylation, prevented the depression of low affinity Ca2+ binding and Ca2+ ATPase activities, whereas the methylation-induced effect on the high affinity Ca2+ binding was not influenced by this agent. Pretreatment of sarcolemma with methyl acetimidate hydrochloride, an amino group blocking agent, also prevented the methylation-induced inhibition of both Ca2+ binding and Ca2+ ATPase. A further decrease in Ca2+ binding and Ca2+ ATPase activities together with a marked increase in the intramembranal level of PC was seen when membranes were methylated under the site III conditions in the presence of phosphatidyldimethylethanolamine as exogenous substrate. There was no effect of phospholipid methylation on sarcolemmal Na+-K+ ATPase and Mg2+ ATPase activities. These results indicate a role of phospholipid N-methylation in the regulation of sarcolemmal Ca2+ ATPase and low affinity ATP-independent Ca2+ binding. 相似文献
4.
N Vrbjar A Breier A Ziegelh?ffer A Dzurba J Soós 《General physiology and biophysics》1986,5(5):545-549
Calcium-induced changes in (Na+ + K+)-ATPase activity and structural changes of membrane bound proteins in rat heart sarcolemma were investigated. Increasing concentrations of Ca2+ (0.1-8.0 mmol.l-1) gradually inhibited the (Na+ + K+)-ATPase activity and decreased the alpha-helix content of sarcolemmal proteins. Mathematical and graphical analysis of observed data yielded a quantitative relationship between Ca2+-induced changes in (Na+ + K+)-ATPase activity and the secondary structure of membrane proteins in cardiac sarcolemma. 相似文献
5.
Vijayan Elimban Dayuan Zhao Naranjan S. Dhalla 《Molecular and cellular biochemistry》1987,77(2):143-152
In order to gain some information regarding Ca2+-dependent ATPase, the enzyme was purified from cardiac sarcolemma and its properties were compared with Ca2+-ATPase activity of myosin purified from rat heart. Both Ca2+-dependent ATPase and myosin ATPase were stimulated by Ca2+ but the maximal activation of Ca2+-dependent ATPase required 4 mM Ca2+ whereas that of myosin ATPase required 10 mM Ca2+. These ATPases were also activated by other divalent cations in the order of Ca2+ > Mn2+ > Sr2+ > Br2+ > Mg2+; however, there was a marked difference in the pattern of their activation by these cations. Unlike the myosin ATPase, the ATP hydrolysis by Ca2+-dependent ATPase was not activated by actin. The pH optima of Ca2+-dependent ATPase and myosin ATPase were 9.5 and 6.5 respectively. Na+ markedly inhibited Ca2+-dependent ATPase but had no effect on the myosin ATPase activity. N-ethylmaleimide inhibited Ca2+-dependent ATPase more than myosin ATPase whereas the inhibitory effect of vanadate was more on myosin ATPase than Ca2+-dependent ATPase. Both Ca2+-dependent ATPase and myosin ATPase were stimulated by K-EDTA and NH4-EDTA. When myofibrils were treated with trypsin and passed through columns similar to those used for purifying Ca2+-ATPase from sarcolemma, an enzyme with ATPase activity was obtained. This myofibrillar ATPase was maximally activated at 3–4 mM Ca2+ and 3 to 4 mM ATP like sarcolemmal Ca2+-dependent ATPase. K+ stimulated both ATPase activities in the absence of Ca2+ and inhibited in the presence of Ca2+. Both enzymes were inhibited by Na+, Mg2+, La3+, and azide similarly. However, Ca2+ ATPase from myofibrils showed three peptide bands in SDS polyacrylamide gel electrophoresis whereas Ca2+ ATPase from sarcolemma contained only two bands. Sarcolemmal Ca2+-ATPase had two affinity sites for ATP (0.012 mM and 0.23 mM) while myofibrillar Ca2+-ATPase had only one affinity site (0.34 mM). Myofibrillar Ca2+-ATPase was more sensitive to maleic anhydride and iodoacetamide than sarcolemmal Ca2+-ATPase. These observations suggest that Ca2+-dependent ATPase may be a myosin like protein in the heart sarcolemma and is unlikely to be a tryptic fragment of myosin present in the myofibrils. 相似文献
6.
Isothiocyanates are potent modifiers of thiol groups, and they have been successfully applied in studying the active site structure of renal (Na+ + K+)-ATPase. However, very little has been known on interactions of isothiocyanates with myocardial sarcolemmal ATPases. In the present study the mode of interaction and inhibitory effect of p-bromophenyl isothiocyanate (BPITC) on isolated rat heart sarcolemmal preparation ATPase activities not exhibiting (Mg-Ca)-ATPase activity was investigated. BPITC in concentrations of 10(-7)-10(-4) mol . l-1 inhibited selectively and non-competitively the (Na+ + K+)-ATPase activity in the sarcolemma with an ID50 around 2.10(-7) mol . l-1. The non-specific interaction of BPITC with bivalent cations, namely with Mg2+ and Ca2+, in the reaction system was eliminated by preincubation of membranes with BPITC keeping the ratio of inhibitor to membrane protein concentration constant. Under these conditions no considerable inhibitory effects were observed on Mg2+-ATPase or the low-affinity Ca2+-ATPase of sarcolemma. Preincubation of membranes with 2 mmol . l-1 ATP protected (Na+ + K+)-ATPase activity against inhibition by BPITC. The interaction of BIPTC with the sarcolemma proved to be reversible in the presence of beta-mercaptoethanol or dithiothreitol. 相似文献
7.
Rat hearts were depleted of Ca2+ (less than 10(-9) M) for 10 min, followed by 15 min of Ca2+-repletion. The calcium paradox injury occurs during Ca2+-repletion, after a period of calcium depletion. The calcium paradox injury was assessed by percent recovery (hemodynamics, [Ca2+]i, and energy levels) during Ca2+-repletion. A decrease in Na+ concentration during Ca2(+)-depletion did not allow for recovery during Ca2(+)-repletion, however 2.5% and 5% ethanol during Ca2(+)-depletion allowed for an approximate 50% recovery during Ca2(+)-repletion. A combination of ethanol (2.5% or 5%) with a low extracellular Na+ concentration (88 mM) allowed for complete recovery. Ethanol prevented a depletion of diastolic [Ca2+]i during Ca2(+)-depletion, and allowed for a return of normal diastolic [Ca2+]i during Ca2(+)-repletion. Ethanol modulates the activity of the Na+/Ca2+ exchanger and protects against the Ca2(+)-paradox injury. 相似文献
8.
Effects of endotoxin administration on the ATP-dependent Ca2+ transport in canine cardiac sarcolemma were investigated. The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca2+ transport in cardiac sarcolemma was decreased by 22 to 46% (p < 0.05) at 4 h following endotoxin administration. The kinetic analysis indicates that the Vmax for ATP and for Ca2+ were decreased by 50% (p < 0.01) and 32% (p < 0.01), respectively, while the Km values for ATP and Ca2+ were not significantly affected after endotoxin administration. Magnesium (1–5 mM) stimulated while vanadate (0.25–3.0 M) inhibited the ATP-dependent Ca2+ transport, but the Mg2+-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals. These data demonstrate that endotoxin administration impairs the ATP-dependent Ca2+ transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca2+. Additional experiments show that the Ca2+ sensitivity of the Ca2+-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca2+ pumping without affecting Ca2+-ATPase activity. Since sarcolemmal ATP-dependent Ca2+ transport plays an important role in the regulation of cytosolic Ca2+ homeostasis, an impairment in the sarcolemmal ATP-dependent Ca2+ transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock. 相似文献
9.
Donald M. Bers 《生物化学与生物物理学报:生物膜》1979,555(1):131-146
A procedure was developed for the isolation of cardiac sarcolemmal vesicles. These vesicles are enriched about ten-fold (with respect to the tissue homogenate) in K+-stimulated p-nitrophenylphosphatase, (Na+ + K+)-ATPase, 5'-nucleotidase activities and sialic acid content, all of which are believed to be components of the sarcolemma. The sarcolemma of tissue culture cardiac cells were radioiodinated and the distribution of this radioiodine paralleled the distribution of the other membrane markers above. There was very little contamination of the sarcolemmal fraction by sarcoplasmic reticulum (as judged by Ca2+-ATPase and glucose-6-phosphatase activities) or inner mitochondrial membranes (as judged by succinate dehydrogenase activity). There may, however, be some contamination by outer mitochondrial membranes (as judged by monoamine oxidase and rotenone-insensitive NADH cytochrome c reductase activities) which have rarely been monitored in cardiac sarcolemmal preparations. The purity of this preparation is good when compared with other cardiac sarcolemmal preparations. This preparation should be very useful in studying the roles of the cardiac sarcolemma (e.g. in excitation contraction coupling and Ca2+ binding). 相似文献
10.
Enn K. Seppet Frantisek Kolar Ian M. C. Dixon Tomoji Hata Naranjan S. Dhalla 《Molecular and cellular biochemistry》1993,129(2):145-159
In order to examine the regulatory role of thyroid hormone on sarcolemmal Ca2+-channels, Na+–Ca2+ exchange and Ca2+-pump as well as heart function, the effects of hypothyroidism and hyperthyroidism on rat heart performance and sarcolemmal Ca2+-handling were studied. Hyperthyroid rats showed higher values for heart rate (HR), maximal rates of ventricular pressure development+(dP/dt)max and pressure fall–(dP/dt)max, but shorter time to peak ventricular pressure (TPVP) and contraction time (CT) when compared with euthyroid rats. The left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), as well as aortic systolic and diastolic pressures (ASP and ADP, respectively) were not significantly altered. Hypothyroid rats exhibited decreased values of LVSP, HR, ASP, ADP, +(dP/dt)max and –(dP/dt)max but higher CT when compared with euthyroid rats; the values of LVEDP and TPVP were not changed. Studies with isolated-perfused hearts showed that while hypothyroidism did not modulate the inotropic response to extracellular Ca2+ and Ca2+ channel blocker verapamil, hyperthyroidism increased sensitivity to Ca2+ and decreased sensitivity to verapamil in comparison to euthyroid hearts. Studies of [3H]-nitrendipine binding with purified cardiac sarcolemmal membrane revealed decreased number of high affinity binding sites (Bmax) without any change in the dissociation constant for receptor-ligand complex (Kd) in the hyperthyroid group when compared with euthyroid sarcolemma; hypothyroidism had no effect on these parameters. The activities of sarcolemmal Ca2+-stimulated ATPase, ATP-dependent Ca2+ uptake and ouabain-sensitive Na+–K+ ATPase were decreased whereas the Mg2+-ATPase activity was increased in hypothyroid hearts. On the other hand, sarcolemmal membranes from hyperthyroid samples exhibited increased ouabain-sensitive Na+–K+ ATPase activity, whereas Ca2+-stimulated ATPase, ATP-dependent Ca2+ uptake, and Mg2+-ATPase activities were unchanged. The Vmax and Ka for Ca2+ of cardiac sarcolemmal Na+–Ca2+ exchange were not altered in both hyperthyroid and hypothyroid states. These results indicate that the status of sarcolemmal Ca2+-transport processes is regulated by thyroid hormones and the modification of Ca2+-fluxes across the sarcolemmal membrane may play a crucial role in the development of thyroid state-dependent contractile changes in the heart. 相似文献
11.
S Takeo P Duke G M Taam P K Singal N S Dhalla 《Canadian journal of physiology and pharmacology》1979,57(5):496-503
Effects of lanthanum on Ca2+-ATPase, Mg2+-ATPase, Na+-K+-ATPase, and calcium binding activities were studied in rat heart sarcolemma. Ten to 100 micrometers lanthanum depressed significantly the Ca2+-ATPase activity and 50--200 micrometers lanthanum inhibited the calcium binding activity. Lineweaver-Burk plots of the Ca2+-ATPase activity showed that the inhibition by lanthanum was competitive with calcium concentration. Neither Mg2+-ATPase nor Na+-K+-ATPase activities were affected by lanthanum when the assay medium contained 1 mM EDTA; however, in the absence of EDTA, these enzyme activities were significantly decreased by 10--100 micrometers lanthanum. Rat hearts perfused with HEPES buffer containing 0.5 mM lanthanum showed electron-dense deposits restricted to the outer cell surface and the sarcolemma obtained from these hearts also had the deposits, indicating that the membrane fraction isolated by the hypotonic shock--LiBr treatment method is of sarcolemmal origin. The Ca2+-ATPase activity of the sarcolemma isolated from lanthanum-perfused hearts, unlike the Mg2+-ATPase, Na+-K+-ATPase, and calcium binding activities, was significantly less than the control value. From these observations it is suggested that lanthanum may influence calcium movement across the sarcolemma by affecting sarcolemmal ATPase and calcium binding activities. 相似文献
12.
Electrical stimulation of the rat heart sarcolemmal membranes with a square wave current was found to increase Ca2+-ATPase activity. This activation of the enzyme was dependent upon the voltage of the electric current, frequency of stimulation and duration of stimulation of the sarcolemmal membranes. The increase in ca2+-ATPase was reversible upon terminating the electrical stimulation. The activation of sarcolemmal Ca2+-ATPase due to electrical stimulation was markedly depressed when the reaction was carried out at high pH (7.8 to 8.2), low pH (6.6 to 7.0), high temperatures (45 to 50°C) and low temperatures (17 to 25°C) of the incubation medium. Ca2+-antagonists, verapamil and D-600, unlike other types of inhibitors such as propranolol and ouabain, were found to reduce the activation of sarcolemmal Ca2+-ATPase by electrical stimulation. These results support the view that Ca2+/Mg2+ ATPase may be involved in the gating mechanism for opening Ca2+-channels in the sarcolemmal membrane upon excitation of the cardiac muscle. 相似文献
13.
N.Charle Morcos 《生物化学与生物物理学报:生物膜》1982,688(3):747-756
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 ( 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. 相似文献
14.
《Comparative biochemistry and physiology. C: Comparative pharmacology》1993,104(2):225-229
1. Langendorff-perfusion of rat hearts with either 10 mM caffeine or 1 mM 2,4-dinitrophenol (DNP) caused severe ultrastructural damage to the myofilaments and mitochondria that was similar to that found in a standard Ca2+-paradox.2. This damage occurred in the presence and absence of extracellular Ca2+3. Creatine kinase (CK) release (indicative of sarcolemma breakdown) was not recorded unless the caffeine- or DNP-perfusion was preceded by Ca2+0-depletion.4. It is concluded that: (i) the pathways leading to damage to the myofilaments and sarcolemma are independent; (ii) the CK release mechanism requires dual activation of Ca2+0-depletion plus a rise in [Ca2+]i; and (iii) current theories concerning the mechanisms underlying the genesis of the Ca2+-paradox are incorrect or incomplete. 相似文献
15.
Xu KY Kuppusamy SP Wang JQ Li H Cui H Dawson TM Huang PL Burnett AL Kuppusamy P Becker LC 《The Journal of biological chemistry》2003,278(43):41798-41803
Nitric oxide (NO.) generated from nitric oxide synthase (NOS) isoforms bound to cellular membranes may serve to modulate oxidative stresses in cardiac muscle and thereby regulate the function of key membrane-associated enzymes. Ischemia is known to inhibit the function of sarcolemmal enzymes, including the (Na+ + K+)-ATPase, but it is unknown whether concomitant injury to sarcolemma (SL)-associated NOS isoforms may contribute to this process by reducing the availability of locally generated NO. Here we report that nNOS, as well as eNOS (SL NOSs), are tightly associated with cardiac SL membranes in several different species. In isolated perfused rat hearts, global ischemia caused a time-dependent irreversible injury to cardiac SL NOSs and a disruption of SL NO. generation. Pretreatment with low concentrations of the NO. donor 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7) markedly protected both SL NOS and (Na+ + K+)-ATPase functions against ischemia-induced inactivation. Moreover, ischemia impaired SL Na+/K+ binding, and NOC-7 significantly prevented ischemic injury to the ion binding sites on (Na+ + K+)-ATPase. These novel findings indicate that NO. can protect cardiac SL NOSs and (Na+ + K+)-ATPase against ischemia-induced inactivation and suggest that locally generated NO. may serve to regulate SL Na+/K+ ion active transport in the heart. 相似文献
16.
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. 相似文献
17.
Leslie J. Reinlib Arthur F. Clark Ernesto Carafoli 《Journal of bioenergetics and biomembranes》1984,16(5-6):611-622
A detergent extract of dog or beef heart sarcolemmal vesicles was prepared and found to have a stimulatory effect on the Ca++-ATPase of plasma membranes from human erythrocyte and cardiac sarcolemma. A procedure is described which enriches the activating fraction. The protein nature of the preparation is illustrated by its sensitivity to boiling and to the proteolytic enzyme(s) trypsin and chymotrypsin. SDS polyacrylamide gels indicate that the protein(s) involved have a molecular weight of 56 and 60 kDa. The sarcolemmal activator can stimulate the Ca++-ATPase activity of the isolated enzyme more than 100% in the presence of saturating amounts of calmodulin. The activation is calcium dependent, being greatest at approximately 10µm Ca++, free, but does not change theK
m
for Ca++. A possible physiological role for the activator is discussed. 相似文献
18.
Inhibition of Na+-Ca2+ exchange in heart sarcolemmal vesicles by phosphatidylethanolamine N-methylation 总被引:1,自引:0,他引:1
The effect of phosphatidylethanolamine N-methylation on Na+-Ca2+ exchange was studied in sarcolemmal vesicles isolated from rat heart. Phosphatidylethanolamine N-methylation following incubation of membranes with S-adenosyl-L-methionine, a methyl donor for the enzymatic N-methylation, inhibited Nai+-dependent Ca2+ uptake by about 50%. The N-methylation reaction did not alter the passive permeability of the sarcolemmal vesicles to Na+ and Ca2+ and did not modify the electrogenic characteristics of the exchanger. The depressant effect of phosphatidylethanolamine N-methylation on Nai+-dependent Ca2+ uptake was prevented by S-adenosyl-L-homocysteine, an inhibitor of the N-methylation. Pretreatment of sarcolemma with methyl acetimidate hydrochloride, an amino-group-blocking agent, also prevented methylation-induced inhibition of Ca2+ uptake. In the presence of exogenous phospholipid substrate, the phospholipid N-methylation process in methyl-acetimidate-treated sarcolemmal vesicles was restored and the inhibitory effect on Ca2+ uptake was evident. These results suggest that phosphatidylethanolamine N-methylation influences the heart sarcolemmal Na+-Ca2+ exchange system. 相似文献
19.
Localization of (Ca2+ + Mg2+)-ATPase, Ca2+ pump and other ATPase activities in cardiac sarcolemma 总被引:2,自引:0,他引:2
N C Morcos 《Biochimica et biophysica acta》1982,688(3):747-756
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 (K1/2 for inhibition approx. 1.5 microM) 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. 相似文献
20.
A Dzurba A Ziegelh?ffer L Schmidtová A Breier N Vrbjar J Okolicány 《General physiology and biophysics》1985,4(3):257-264
Beta-adrenoceptor blocking agents may have, in addition to their primary action, also ancillary effects on the cell membrane. In the present paper the non-specific interaction of exaprolol with the ATPase systems in isolated rat heart sarcolemmal membranes was investigated. When preincubated with sarcolemmal membranes in vitro, exaprolol in concentrations below 10(-4) mol.l-1 had no significant effect on sarcolemmal Mg2+-, Ca2+- and (Na+ + K+)-ATPase activities. At exaprolol concentration of 10(-4) mol.l-1 the Mg2+- and Ca2+-ATPase activities became inhibited whereas the (Na+ + K+)-ATPase activity was markedly stimulated. A kinetic analysis of these interactions revealed a non-competitive inhibition of Mg2+- and Ca2+-ATPase. In the case of (Na+ + K+)-ATPase a synergistic type of stimulation characterized by an exaprolol-induced conversion of an essential sulfhydryl group in the active site of the enzyme to the more reactive [S-] form has been observed thus increasing the affinity of the enzyme to ATP. Exaprolol concentrations exceeding 5 X 10(-4) mol.l-1 induced an overall depression of the investigated enzyme activities. 相似文献