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1.
Heart failure is common among the elderly and an alteration in myocardial Ca2+ transport is believed to be involved in its depressed contractile performance. Although ATP-dependent sarcoplasmic reticular (SR) Ca2+ transport has been reported to decrease in old hearts, virtually nothing appears to be known about the Ca2+ pump activity of SR in aging myocardium in the presence of calmodulin, one of its endogenous activators. In this study, the activity of the Ca2+ pump of aging cardiac SR was assessed in the presence of this endogenous stimulator. This assessment was therefore designed to give additional information about the status of this enzyme in old hearts. Male Sprague-Dawley rats were used and were divided into 3 groups: young (4–6 months old); middle-aged (15–17 months old) and old age (24–25 months old). Purified SR membranes were isolated from ventricular tissues. ATP-dependent Ca2+ accumulation by membrane vesicles of middle-aged and old hearts was significantly depressed in comparison to young hearts at all Ca2+ concentrations employed in the absence and presence of calmodulin. The activity of this Ca2+ transporter was similar in middle-aged and old hearts even in the presence of calmodulin. These results suggest that the activity of the Ca2+ pump in SR of aging hearts is depressed even in the presence of calmodulin.C. E. Heyliger is a Scholar of the British Columbia Heart Foundation.  相似文献   

2.
Ca2+ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37°C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 μM CaCl2, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca2+ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca2+ uptake, a second phase of Ca2+ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca2+ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca2+ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca2+ influx of sarcoplasmic reticulum near steady state of Ca2+ uptake was measured by pulse labeling with 45Ca2+. The Ca2+ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca2+ uptake in normal medium, Ca2+ influx was balanced by Ca2+ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca2+ exchange rate at the first plateau of Ca2+ uptake was about half of that in normal medium. When the second phase of Ca2+ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca2+ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 μg/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca2+ uptake was also observed. These data suggest that the Ca2+ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca2+ efflux, which subsequently stimulates Ca2+ exchange.  相似文献   

3.
Oxidized low density lipoprotein (oxLDL) has been identified as a potentially important atherogenic factor. Atherosclerosis is characterized by the accumulation of lipid and calcium in the vascular wall. OxLDL plays a significant role in altering calcium homeostasis within different cell types. In our previous study, chronic treatment of vascular smooth muscle cells (VSMC) with oxLDL depressed Ca2+ i homeostasis and altered two Ca2+ release mechanisms in these cells (IP3 and ryanodine sensitive channels). The purpose of the present study was to further define the effects of chronic treatment with oxLDL on the smooth muscle sarcoplasmic reticulum (SR) Ca2+ pump. One of the primary Ca2+ uptake mechanisms in VSMC is through the SERCA2 ATPase calcium pump in the sarcoplasmic reticulum. VSMC were chronically treated with 0.005-0.1 mg/ml oxLDL for up to 6 days in culture. Cells treated with oxLDL showed a significant increase in the total SERCA2 ATPase content. These changes were observed on both Western blot and immunocytochemical analysis. This increase in SERCA2 ATPase is in striking contrast to a significant decrease in the density of IP3 and ryanodine receptors in VSMC as the result of chronic treatment with oxLDL. This response may suggest a specific adaptive mechanism that the pump undergoes to attempt to maintain Ca2+ homeostasis in VSMC chronically exposed to atherogenic oxLDL.  相似文献   

4.
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   

5.
The effect of Hg2+ and Ch3-Hg+ on the passive and active transport properties of the Ca2+-Mg2+-ATPase-rich fraction of skeletal sarcoplasmic reticulum (SR) is reported. The agents abolish active transport, at 10–5 and 10–4 M concentrations, respectively. Addition of the mercurials was also shown to release actively accumulated Ca2+. The mercurials increase the passive Ca2+ and Mg2+ permeability in the absence of ATP at the same concentrations at which they inhibit transport. It is proposed that both effects are the result of direct binding of the mercurials to the SH groups of the Ca2+-Mg2+-ATPase pump, altering the conformational equilibria of the pump. The agents were also shown to increase the passive KCl permeability. The SR preparation consists of two vesicle populations with respect to K+ permeability, one with rapid KCl equilibration faciliated by a monovalent cation channel function and one with slow KCl equilibration. The mercurials increase the rates of KCl equilibration in both fractions, but produce higher rates in the fraction containing the channel function. The results are discussed in terms of pump and channel function and are compared with results for the electrical behavior of the Ca2+-Mg2+-ATPase and other SR proteins in black lipid membranes, as presented by others.  相似文献   

6.
Oxygen free radicals and calcium homeostasis in the heart   总被引:10,自引:0,他引:10  
Many experiments have been done to clarify the effects of oxygen free radicals on Ca2+ homeostasis in the hearts. A burst of oxygen free radicals occurs immediately after reperfusion, but we have to be reminded that the exact levels of oxygen free radicals in the hearts are yet unknown in both physiological and pathophysiological conditions. Therefore, we should give careful consideration to this point when we perform the experiments and analyze the results. It is, however, evident that Ca2+ overload occurs when the hearts are exposed to an excess amount of oxygen free radicals. Though ATP-independent Ca2+ binding is increased, Ca2+ influx through Ca2+ channel does not increase in the presence of oxygen free radicals. Another possible pathway through which Ca2+ can enter the myocytes is Na+?Ca2+ exchanger. Although, the activities of Na+?K+ ATPase and Na+?H+ exchange are inhibited by oxygen free radicals, it is not known whether intracellular Na+ level increases under oxidative stress or not. The question has to be solved for the understanding of the importance of Na+?Ca2+ exchange in Ca2+ influx process from extracellular space. Another question is ‘which way does Na+?Ca2+ exchange work under oxidative stress? Net influx or efflux of Ca2+?’ Membrane permeability for Ca2+ may be maintained in a relatively early phase of free radical injury. Since sarcolemmal Ca2+-pump ATPase activity is depressed by oxygen free radicals, Ca2+ extrusion from cytosol to extracellular space is considered to be reduced. It has also been shown that oxygen free radicals promote Ca2+ release from sarcoplasmic reticulum and inhibit Ca2+ sequestration to sarcoplasmic reticulum. Thus, these changes in Ca2+ handling systems could cause the Ca2+ overload due to oxygen free radicals.  相似文献   

7.
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   

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.
The Ca2+ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca2+ in intracellular membrane bound compartments, thereby lowering cytosolic Ca2+ to induce relaxation. The stored Ca2+ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca2+, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca2+. We review here biochemical and biophysical evidence demonstrating that release of bound Ca2+ into the lumen of SR requires Ca2+/H+ exchange at the low affinity Ca2+ sites. Rise of lumenal Ca2+ above its dissociation constant from low affinity sites, or reduction of the H+ concentration by high pH, prevent Ca2+/H+ exchange. Under these conditions Ca2+ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca2+pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.  相似文献   

10.
Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. Here, we consider the well characterized Ca2+ ATPase of sarcoplasmic reticulum (SERCA) and describe a series of Ca2+ binding isotherms obtained by equilibrium measurements in the presence of various H+ and Mg2+ concentrations. We subject the isotherms to statistical mechanics analysis, using a model based on a minimal number of mechanistic steps. The analysis allows satisfactory fits and yields information on occupancy of the specific Ca2+ sites under various conditions. It also provides a fundamental method for analysis of binding specificity to transport sites under equilibrium conditions that lead to tightly coupled catalytic activation.  相似文献   

11.
Heart sarcolemma has been shown to contain an ATPase hydrolizing system which is activated by millimolar concentrations of divalent cations such as Ca2+ or Mg2+. Although Ca2+-dependent ATPase is released upon treating sarcolemma with trypsin, a considerable amount of the divalent cation dependent ATPase activity was retained in the membrane. This divalent cation dependent ATPase was solubilized by sonication of the trypsin-treated dog heart sarcolemma with 1% Triton X-100. The solubilized enzyme was subjected to column chromatography on a Sepharose-6B column, followed by ion-exchange chromatography on a DEAE cellulose column. The enzyme preparation was found to be rather labile and thus the purity of the sample could not be accurately assessed. The solubilized ATPase preparations did not show any cross-reactivity with dog heart myosin antiserum or with Na+ + K+ ATPase antiserum. The enzyme was found to be insensitive to inhibitors such as ouabain, verapamil, oligomycin and vanadate. The enzyme preparation did not exhibit any Ca2+-stimulated Mg2+ dependent ATPase activity. Furthermore, the low affinity of the enzyme for Ca2– (Ka = 0.3 mM) rules out the possibility of its involvement in the Ca2+ pump mechanism located in the plasma membrane of the cardiac cell.  相似文献   

12.
Two groups of weanling Sprague-Dawley rats were fed a low-selenium basal diet (Se 0.009 mg/kg) and the same diet supplemented with sodium selenite (Se 0.25 mg/kg), respectively, for 1, 2, and 3 months. At each feeding time, the Ca2+-ATPase activity, Ca2+ uptake rate and the capacity of Ca2+ uptake in isolated cardiac sacroplasmic reticulum from the Se-deficient rats were decreased significantly compared to those from the Se-supplemented rats, the contents of lipid peroxide in postmitochondrial supernatant and isolated sarcoplasmic reticulum from the Se-deficient rats were significantly higher than that from Se-supplemented rats. Compared to the Se-supplemented rats, the cytosolic glutathione peroxidase activity in Se-deficient rats decreased significantly. In addition, significant linear negative correlations of lipid peroxide in postmitochondrial supernatant to sarcoplasmic reticular Ca2+-ATPase activity, Ca2+ uptake rate and to whole blood selenium concentration were observed. The results suggest that the enhancement of lipid peroxidation via the depressed glutathione peroxidase activity might be responsible for the decrease of Ca2+-ATPase and Ca2+ uptake activities in sarcoplasmic reticulum in Se-deficient animals.  相似文献   

13.
Two kinds of ATP binding sites were found on the ATPase molecule in deoxycholic acid-treated sarcoplasmic reticulum. One was the catalytic site (1 mol/mol active site) and its affinity was high. Upon addition of Ca2+, all the ATP bound to the catalytic site disappeared at 75 mM KCl, while a significant amount of ATP remained bound to the site at 0–2 mM KCl. The latter binding was found to be due to the formation of a slowly exchanging enzyme-ATP complex, which is in equilibrium with phosphoenzyme + ADP. The other binding site was the regulatory one (1 mol/mol active site) and its affinity was low, changing only insignificantly upon addition of Ca2+. The ATP binding to the regulatory site shifted the equilibrium between the slowly exchanging complex and EP toward EP.  相似文献   

14.
Store-operated Ca2+ entry (SOCE) is an important mechanism for Ca2+ influx in smooth muscle cells; however the activation and regulation of this influx pathway are incompletely understood. In the present study we have examined the effect of several protein kinases in regulating SOCE in pulmonary artery smooth muscle cells (PASMCs) of the rat. Inhibition of protein kinase C with chelerythrine (3 μM) potentiated SOCE by 47 ± 2%, while the tyrosine kinase inhibitors genistein (100 μM) and tyrphostin 23 (100 μM) caused a significant reduction in SOCE of 55 ± 9% and 43 ± 7%, respectively. It has been proposed that Ca2+-insensitive phospholipase A2 (iPLA2) is involved in the activation of SOCE in many different cell types. The iPLA2 inhibitor, bromoenol lactone had no effect on SOCE, suggesting that this mechanism was not involved in the activation of the pathway. The calmodulin antagonists, calmidazolium (CMZ) (10 μM) and W-7 (10 μM) appeared to potentiate SOCE in PASMCs. Further investigation established that CMZ was actually activating a Ca2+ influx pathway that was independent of the filling state of the sarcoplasmic reticulum. The CMZ-activated Ca2+ influx was blocked by Gd3+ (10 μM), but unaffected by 2-APB (75 μM), indicating a pharmacological profile distinct from the classical SOCE pathway.  相似文献   

15.
Summary The Ca2+ channel blockers felodipine and bepridil are known to affect selectively functions of calmodulin. We studied their effects on calmodulin binding and ATPase activities of calmodulin-containing and calmodulin-depleted rabbit heart sarcolemma. Both drugs as well as the specific anti-calmodulin drug calmidazolium at a concentration of 50 µM, inhibited the Ca2+-stimulated calmodulin binding to calmodulin-depleted sarcolemma. Within the concentration range of 3 to 100 µM all three drugs also progressively inhibited Ca2+ pumping ATPase in calmodulin containing sarcolemma, although the enzyme was assayed at saturating Ca2+ (100 µM). The inhibitory potency of calmidazolium and bepridil, but not that of felodipine, increased when the membrane protein concentration in the ATPase assay was lowered. At low membrane protein concentration 30 µM calmidazolium completely blocked calmodulin-dependent Ca2+ pumping ATPase, whereas the inhibition caused by 30 µM felodipine or bepridil remained partially. A similar inhibition pattern of the drugs was found in the calmodulin binding experiments. Within a concentration range of 3 to 30 µM, all three drugs had negligible effects on the basal Ca2+ pumping ATPase which was measured in calmodulin-depleted sarcolemma. In conclusion, the characteristics of the anti-calmodulin action of felodipine on the rabbit heart sarcolemmal Ca2+ pumping ATPase are not different from those of bepridil. Both drugs may inhibit the enzyme by interference with the Ca2+-stimulated binding of calmodulin.Abbreviations Ca2+ pumping ATPase Ca2+ stimulated Mg2+-dependent ATP hydrolyzing activity - Na+ pumping ATPase Na+-stimulated K+- and Mg2+-dependent ATP hydrolyzing activity - Tris-maleate tris (hydroxymethyl) aminomethane hydrogen maleate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Mes 2-(N-morpholino) ethane sulfonic acid and Egta, ethylene glycol bis (p-amino ethylether)-N,N,N,N tetraacetic acid  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.
This review will focus on the recent advance in the study of effect of transmembrane Ca2+ gradient on the function of membrane proteins. It consits of two parts: 1. Transmembrane Ca2+ gradient and sarcoplasmic reticulum Ca2+-ATPase; 2. Effect of transmembrane Ca2+ gradient on the components and coupling of cAMP signal transduction pathway. The results obtained indicate that a proper transmembrane Ca2+ gradient may play an important role in modulating the conformation and activity of SR Ca2+-ATPase and the function of membrane proteins involved in the cAMP signal transduction by mediating the physical state change of the membrane phospholipids.Abbreviations Cai Ca2+ inside vesicles - Ca0 Ca2+ outside vesicles - SR sarcoplasmic reticulum - PC phosphatidylcholine - PS phosphatidylserine - PG phosphatidylglycerol - PE phosphatidylethanolamine - DPH 1,6-diphenyl-1,3,5-hexatriene - n-AS n-(9-anthroyloxy) fatty acids - TMA-DPH 1-(4-trimethylammoniumphenyl)-6)-phenyl-1,3,5-hexatriene - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - -AR -adrenergic receptors - DHA dihydroalprenolol - AC adenylate cyclase - AC·Lca+– higher Ca2+ inside vesicles - AC·Lca– – lower Ca2+ on both side of vesicles - AC·Lca++ higher Ca2+ on both side of vesicles - AC·Lca– + higher Ca2+ outside vesicles - cAMP cyclic adenosine monophosphate - Gs stimulatory GTP-binding protein - GTP guanosine triposphate - GTPS guanosine 50-(3-thiotriphosphate)  相似文献   

18.
The sarcoplasmic reticulum (SR) of skeletal muscle controls the contraction-relaxation cycle by raising and lowering the myoplasmic free-Ca2+ concentration. The coupling between excitation, i.e., depolarization of sarcolemma and transvers tubule (TT) and Ca2+ release from the terminal cisternae (TC) of SR takes place at the triad. The triad junction is formed by a specialized region of the TC, the junctional SR, and the TT. The molecular architecture and protein composition of the junctional SR are under active investigation. Since the junctional SR plays a central role in excitation-contraction coupling and Ca2+ release, some of its protein constituents are directly involved in these processes. The biochemical evidence supporting this contention is reviewed in this article.  相似文献   

19.
The thermodynamic efficiency of the Ca2+-Mg2+-ATPase of skeletal sarcoplasmic reticulum has been evaluated by comparing the Ca2+ gradient established with the ATP/(ADP*Pi) ratio. The evaluation was made at an external Ca2+ level (4.7 × 10–8 M) which is below theK m value of 7 × 10–8 M. The Mg-ATP and phosphate concentrations were held constant (0.1 mM) and the ADP concentration was varied. Maximal uptake to an internal free Ca2+ concentration of 17 mM was observed at infinite ATP/(ADP*Pi) ratio (absence of ADP). This corresponds to a [Ca2+]i/[Ca2+]0 gradient of 3.6 × 105. A Ca2+ gradient one-half as large was observed at an ATP/(ADP*Pi) ratio of 3.5 × 103 M–1. The square of the Ca2+ gradient is shown to be proportional to the ATP/(ADP*Pi) ratio, for finite values of the latter. The proportionality constant is identical to the equilibrium constant for hydrolysis of ATP (9.02 × 106 M) under these conditions (0.1 mM Mg2+, 30°C). The intrinsic thermodynamic efficiency of the pump is shown to be 100%, with a maximal uncertainty of 3%. The efficiency is lower under less optimal conditions, when the pump is inhibited and passive leak processes compete.Dedicated to Prof. Philip George, University of Pennsylvania, whose instruction, research, and example made this contribution possible.  相似文献   

20.
In order to determine whether polymorphic forms of the Ca2+ + Mg2+-dependent ATPase exist, we have examined the cross-reactivity of five monoclonal antibodies prepared against the rabbit skeletal muscle sarcoplasmic reticulum enzyme with proteins from microsomal fractions isolated from a variety of muscle and nonmuscle tissues. All of the monoclonal antibodies cross-reacted in immunoblots against rat skeletal muscle Ca2+ + Mg2+-dependent ATPase but they cross-reacted differentially with the enzyme from chicken skeletal muscle. No cross-reactivity was observed with the Ca2+ + Mg2+-dependent ATPase of lobster skeletal muscle. The pattern of antibody cross-reactivity with a 100,000 dalton protein from sarcoplasmic reticulum and microsomes isolated from various muscle and nonmuscle tissues of rabbit demonstrated the presence of common epitopes in multiple polymorphic forms of the Ca2+ + Mg2+-dependent ATPase. One of the monoclonal antibodies prepared against the purified Ca2+ + Mg2+-dependent ATPase of rabbit skeletal muscle sarcoplasmic reticulum was found to cross-react with calsequestrin and with a series of other Ca2+-binding proteins and their proteolytic fragments. Its cross-reactivity was enhanced in the presence of EGTA and diminished in the presence of Ca2+. Its lack of cross-reactivity with proteins that do not bind Ca2+ suggests that it has specificity for antigenic determinants that make up the Ca2+-binding sites in several Ca2+-binding proteins including the Ca2+ + Mg2+-dependent ATPase.This paper is dedicated to the memory of Dr. David E. Green.  相似文献   

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