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1.
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
2.
In striated muscle, regulation of actin-myosin interactions depends on a series of conformational changes within the thin filament that result in a shifting of the tropomyosin-troponin complex between distinct locations on actin. The major factors activating the filament are Ca2+ and strongly bound myosin heads. Many lines of evidence also point to an active role of actin in the regulation. Involvement of the actin C-terminus in binding of tropomyosin-troponin in different activation states and the regulation of actin-myosin interactions were examined using actin modified by proteolytic removal of three C-terminal amino acids. Actin C-terminal modification has no effect on the binding of tropomyosin or tropomyosin-troponin + Ca2+, but it reduces tropomyosin-troponin affinity in the absence of Ca2+. In contrast, myosin S1 induces binding of tropomyosin to truncated actin more readily than to native actin. The rate of actin-activated myosin S1 ATPase activity is reduced by actin truncation both in the absence and presence of tropomyosin. The Ca2+-dependent regulation of the ATPase activity is preserved. Without Ca2+ the ATPase activity is fully inhibited, but in the presence of Ca2+ the activation does not reach the level observed for native actin. The results suggest that through long-range allosteric interactions the actin C-terminus participates in the thin filament regulation. 相似文献
3.
R Dabrowska D Aromatorio J M Sherry D J Hartshorne 《Biochemical and biophysical research communications》1977,78(4):1263-1272
The Ca2+-dependent protein kinase (ATP:myosin light chain phosphotransferase) from chicken gizzard smooth muscle requires two proteins for enzymatic activity. These have approximate molecular weights of 105,000 and 17,000 daltons. The isolation procedure for each component is described. Neither component alone markedly alters either the actin-moderated ATPase activity or the phosphorylation of myosin. Activation of ATPase activity by a combination of the two components occurred only in the presence of Ca2+ and was always accompanied by the phosphorylation of myosin. The simultaneous activation of ATPase activity and myosin phosphorylation establishes a direct correlation between the two events. 相似文献
4.
John E. Hesketh Noelle Virmaux Paul Mandel 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,542(1):39-46
Myosin has been isolated from bovine retinae and characterised by its ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, its mobility in sodium dodecyl sulphate polyacrylamide gels and by electron microscopy. The purified myosin shows high ATPase activity in the presence of EDTA or Ca2+ and a low activity in the presence of Mg2+. The Mg2+-dependent ATPase activity is stimulated by rabbit skeletal muscle actin. The presumptive retinal myosin possesses a major component which has a mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis similar to that of the heavy chain of bovine skeletal mucle myosin. Electron microscopy showed retinal myosin to form bipolar filaments in 0.1 M KCl. It is concluded that the retina possesses a protein with enzymic and structural properties similar to those of muscle myosin. 相似文献
5.
Summary (i) In human red cell membranes the sensitivity to N-ethylmaleimide of Ca2+-dependent ATPase and phosphatase activities is at least ten times larger than the sensitivity to N-ethylmaleimide of (Na++K+)-ATPase and K+-activated phosphatase activities. All activities are partially protected against N-ethylmaleimide by ATP but not by inorganic phosphate or byp-nitrophenylphosphate. (ii) Protection by ATP of (Na++K+)-ATPase is impeded by either Na+ or K+ whereas only K+ impedes protection by ATP of K+-activated phosphatase. On the other hand, Na+ or K+ slightly protects Ca2+-dependent activities against N-ethylmaleimide, this effect being independent of ATP. (iii) The sensitivity to N-ethylmaleimide of Ca2+-dependent ATPase and phosphatase activities is markedly enhanced by low concentrations of Ca2+. This effect is half-maximal at less than 1 m Ca2+ and does not require ATP, which suggests that sites with high affinity for Ca2+ exist in the Ca2+-ATPase in the absence of ATP. (iv) Under all conditions tested the response to N-ethylmaleimide of the ATPase and phosphatase activites stimulated by K+ or Na+ in the presence of Ca2+ parallels that of the Ca2+-dependent activities, suggesting that the Ca2+-ATPase system possesses sites at which monovalent cations bind to increase its activity. 相似文献
6.
S. L. Malyshev N. A. Freidina I. M. Vikhlyantsev D. A. Blejyants E. V. Karaduleva Yu. V. Shumilina S. N. Udal’tsov L. G. Marsagishvili A. G. Bobylev Z. A. Podlubnaya 《Biophysics》2010,55(5):707-717
The data of the study on Ca2+ sensitivity of ATPase activity of myosin from vertebrate striated muscles in the presence of actin and the conditions of
its manifestation and disappearance are presented. The role of Ca2+ sensitivity of actin-activated myosin ATPase in the regulation of contraction of vertebrate striated muscles is discussed. 相似文献
7.
I Cohen E Kaminski R Lamed A Oplatka A Mühlrad 《Archives of biochemistry and biophysics》1976,175(1):249-255
Heavy meromyosin subfragment-1 from human platelets and chicken gizzard exhibited an identical chromatographic pattern on agarose-ATP columns both in the absence and in the presence of Ca2+ and Mg2+. In the presence of Ca2+, the behavior differed from that of rabbit white skeletal muscle subfragment-1. The reaction of lysyl residues of platelet myosin with 2,4,6-trinitrobenzene sulfonate did not affect the K+- or Mg2+-stimulated ATPase activity. A similar behavior was exhibited by chicken gizzard myosin whereas trinitrophenylation of the more active lysyl residues in skeletal muscle myosin caused a marked increase in Mg2+-stimulated and a decrease in K+-stimulated ATPase activity. These features may point to a similar location of the essential lysyl residue in platelet and smooth muscle myosin, which is different from that of skeletal muscle. Alkylation of thiol groups by N-ethyl maleimide in the absence of added nucleotides resulted in a loss of K+-ATPase and in an increase in the Ca2+-ATPase in all three myosins, the increase for the skeletal myosin being much greater than for the platelet and chicken gizzard preparations. Alkylation of myosin in the presence of MgADP led to a decrease in K+-ATPase of all preparations whereas the Ca2+-ATPase as a function of time exhibited a maximum for the platelet and skeletal muscle proteins. These features may point to a certain similarity with respect to the active site of platelet and smooth muscle myosins and a difference between these and skeletal muscle myosin. 相似文献
8.
Okafor C Liao R Perreault-Micale C Li X Ito T Stepanek A Doye A de Tombe P Gwathmey JK 《Molecular and cellular biochemistry》2003,245(1-2):77-89
We investigated the effects of two purported calcium sensitizing agents, MCI-154 and DPI 201–106, and a known calcium sensitizer caffeine on Mg-ATPase (myofibrillar ATPase) and myosin ATPase activity of left ventricular myofibrils isolated from non-failing, idiopathic (IDCM) and ischemic cardiomyopathic (ISCM) human hearts (i.e. failing hearts). The myofibrillar ATPase activity of non-failing myofibrils was higher than that of diseased myofibrils. MCI-154 increased myofibrillar ATPase Ca2+ sensitivity in myofibrils from non-failing and failing human hearts. Effects of caffeine similarly increased Ca2+ sensitivity. Effects of DPI 201–106 were, however, different. Only at the 10–6 M concentration was a significant increase in myofibrillar ATPase calcium sensitivity seen in myofibrils from non-failing human hearts. In contrast, in myofibrils from failing hearts, DPI 201–106 caused a concentration-dependent increase in myofibrillar ATPase Ca2+ sensitivity. Myosin ATPase activity in failing myocardium was also decreased. In the presence of MCI-154, myosin ATPase activity increased by 11, 19, and 24% for non-failing, IDCM, and ISCM hearts, respectively. DPI 201–106 caused an increase in the enzymatic activity of less than 5% for all preparations, and caffeine induced an increase of 4, 11, and 10% in non-failing, IDCM and ISCM hearts, respectively. The mechanism of restoring the myofibrillar Ca2+ sensitivity and myosin enzymatic activity in diseased human hearts is most likely due to enhancement of the Ca2+ activation of the contractile apparatus induced by these agents. We propose that myosin light chain-related regulation may play a complementary role to the troponin-related regulation of myocardial contractility. 相似文献
9.
To examine the role of two light chains (LCs) of the myosin II on Ca2+ regulation, we produced hybrid heavy meromyosin (HMM) having LCs from Physarum and/or scallop myosin using the smooth muscle myosin heavy chain. Ca2+ inhibited motility and ATPase activity of hybrid HMMs with LCs from Physarum myosin but activated those of hybrid HMM with LCs from scallop myosin, indicating an active role of LCs. ATPase activity of hybrid HMMs with LCs from different species showed the same effect by Ca2+ even though they did not support motility. Our results suggest that communication between the original combinations of LC is important for the motor function. 相似文献
10.
Summary A modified histochemical technique is described for the improved detection of myosin Ca2+-ATPase activity in single muscle cells in culture. The method was used to demonstrate the increase in myosin Ca2+-ATPase activity in differentiating chick skeletal muscle cells. Functional muscle cells were also positively identified in the heterogeneous cell population of primary hamster heart cell cultures. An age-dependent increase in the number of cells with high levels of myosin ATPase activity in mitotically arrested heart cell cultures was shown. Maturation of individual muscle cells could thus be evaluated. 相似文献
11.
The preparation and characterization of a Ca2+-sensitive actomyosin from chicken gizzard is described. The pH curve of the Mg2+ ATPase activity of the actomyosin was dominated by the activity of the myosin component, and this gave rise to the acid and alkaline optima. Skeletal muscle myosin showed a similar curve. Both the activation of myosin ATPase by actin, and the Ca2+ sensitivity were confined to the neutral pH region. The subunit composition of the Ca2+-sensitive actomyosin was interesting in that no components corresponding to skeletal muscle troponin were obvious. It is suggested that the activity of gizzard actomyosin is regulated by a protein on the thin filaments with a subunit weight of ~130,000. 相似文献
12.
Whereas dissociation of rabbit skeletal muscle myosin light chains occurs at an increased temperature (25°) and in the obsence of divalent cations, reassociation of the myosin oligomer requires a low temperature (4°C) and the presence of divalent cations, thus resulting in the original light to heavy chain stoichiometry. With a 5–10 per cent release of alkali light chains, LC1 and LC3, and a 50 per cent dissociation of the Ca2+ binding light chain, LC2, there is no significant decrease in myosin ATPase activity irrespective of the cation activator, however, there is an approximate 15–20 per cent decrease in actomyosin ATPase activity. With reassociation of the myosin oligomer, actomyosin ATPase activity is partially restored as well as the original number of Ca2+ binding sites. 相似文献
13.
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
14.
The ATPase activities of cardiac myosin from thyrotoxic and euthyroid rabbits have been compared. The Ca2+-ATPase activity of myosin from thyrotoxic animals was elevated by 200%, while the K+(EDTA)-ATPase activity was the same as in euthyroid animals. Modification by N-ethyl-maleimide of the most rapidly reacting class of sulfhydryls (SH1) in myosin from euthyroid animals increased Ca2+-ATPase activity about 177% over the unreacted value. Modification of the SH1 groups in myosin from thyrotoxic animals had no effect on CA2+-ATPase activity. We conclude that thyroxin may increase cardiac myosin ATPase activity by a conformational change in the same region as the SH1 thiols. 相似文献
15.
The Ca2+/Mg2+ ATPase of the rat heart sarcolemmal particles was solublized with Triton X-100 after treating the membranes with trypsin and purified by high speed centrifugation, ammonium sulfate fractionation, hydrophobic chromatography and gel filtration. The purified enzyme was seen as a single protein band in nondenaturing polyacrylamide gel electrophoresis and its molecular weight by gel filtration was found to be about 240000. The enzyme utilized Ca-ATP or Mg-ATP as a substrate with high affinity sites (Km = 0.12 – 0.16 mM) and low affinity sites (Km = 1 mM). The enzyme also utilized CTP, GTP, ITP, UTP and ADP as substrates but at a lower rate in comparison to ATP. The enzyme was activated by Ca2+ (Ka = 0.4 mM) and Mg2+ (Ka = 0.2 mM) as well as by other cations in the order Ca2– > Mg2+ > Mn2+ > Sr2+ > Ba2+ > Ni2+ > Cu2+. The ATPase activity in the presence of Ca2+ was markedly inhibited by Mg2+, Mn2+, Ni2+ and Cu2+ whereas the monovalent cations such as Na+ and K+ were without effect. The enzyme did not exhibit Ca2+ stimulated Mg2+ dependent ATPase activity and was insensitive to calmodulin, ouabain, verapamil, D-600, oligomycin, azide and vanadate. Optimum pH for Ca2+ or Mg2+ ATPase activity was 8.5 – 9.0. In view of the possible ectoenzyme nature of the ATPase, its role in adenine nucleotide and Ca2+ metabolism in the myocardium is discussed. 相似文献
16.
D. E. Richards J. C. Vidal P. J. Garrahan A. F. Rega 《The Journal of membrane biology》1977,35(1):125-136
Summary Treatment of human red cell membranes with pure phospholipase A2 results in a progressive inactivation of both Ca2+-dependent and (Ca2++K+)-dependent ATPase and phosphatase activities. When phospholipase C replaces phospholipase A2, Ca2+-dependent ATPase activity and Ca2+-dependent phosphorylation of red cell membranes are lost, while Ca2+-dependent phosphatase activity is enhanced and its apparent affinity for Ca2+ is increased about 20-fold. Activation of Ca2+-dependent phosphatase following phospholipase C treatment was not observed in sarcoplasmic reticulum preparation. Phospholipase C increases the sensitivity of the phosphatase to N-ethylmaleimide but has little effect on the kinetic parameters relating the phosphatase activity to substrate and cofactors, suggesting that no extensive structural disarrangement of the Ca2+-ATPase system has occurred after incubation with phospholipase C. 相似文献
17.
Helen M. Garnett R.B. Kemp Ute Gröschel-Stewart 《Archives of biochemistry and biophysics》1976,172(2):419-424
The effect of mersalyl and of antibodies, directed against smooth-muscle myosin and skeletal muscle myosin, on the (Ca2+ + Mg2+)-activated adenosine triphosphatase (Ca,Mg)ATPase) system of mouse liver plasma membranes has been studied. Antismooth-muscle myosin inhibited by 38.6% at optimum substrate concentration the (Ca,Mg)ATPase with a Km of 0.88 × 10?3m. Mersalyl (0.5 mm) also inhibited this enzyme, the percentage inhibition being 44.6% at optimal substrate concentration. These results suggest the presence of a smooth-muscle myosin-like protein in the plasma membrane of mouse liver cells which has an associated (Ca,Mg)ATPase activity. 相似文献
18.
Jing Hui Xue Hiroko Takahashi Masayoshi Yamaguchi 《Journal of cellular biochemistry》2001,80(2):285-292
The effect of regucalcin, which is a regulatory protein of Ca2+ signaling, on Ca2+‐ATPase activity in isolated rat renal cortex mitochondria was investigated. The presence of regucalcin (50, 100, and 250 nM) in the enzyme reaction mixture led to a significant increase in Ca2+‐ATPase activity. Regucalcin significantly stimulated ATP‐dependent 45Ca2+ uptake by the mitochondria. Ruthenium red (10−6 M) or lanthunum chloride (10−6 M), an inhibitor of mitochondrial Ca2+ uptake, markedly inhibited regucalcin (100 nM)‐increased mitochondrial Ca2+‐ATPase activity and 45Ca2+ uptake. The effect of regucalcin (100 nM) in elevating Ca2+‐ATPase activity was completely prevented by the presence of digitonin (10−2%), a solubilizing reagent of membranous lipids, vanadate, an inhibitor of phosphorylation of ATPase, or dithiothreitol (50 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme. The activating effect of regucalcin (100 nM) on Ca2+‐ATPase activity was not further enhanced by calmodulin (0.30 μM) or dibutyryl cyclic AMP (10−4 M), which could increase Ca2+‐ATPase activity. Trifluoperazine (TFP; 50 μM), an antagonist of calmodulin, significantly decreased Ca2+‐ATPase activity. The activating effect of regucalcin on the enzyme was also seen in the presence of TFP, indicating that regucalcin's effect is not involved in mitochondrial calmodulin. The present study demonstrates that regucalcin can stimulate Ca2+‐pump activity in rat renal cortex mitochondria, and that the protein may act on an active site (SH group) related to phosphorylation of mitochondrial Ca2+‐ATPase. J. Cell. Biochem. 80:285–292, 2000. © 2000 Wiley‐Liss, Inc. 相似文献
19.
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
20.
The effect of tropomyosin on the adenosine triphosphatase activity of desensitized actomyosin 总被引:7,自引:4,他引:3
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1. Tropomyosin preparations of the Bailey type, and those prepared in the presence of dithiothreitol to prevent oxidation of protein thiol groups, inhibit the Ca2+-activated adenosine triphosphatase (ATPase) of desensitized actomyosin by up to 60%. 2. The inhibitory activity of myofibrillar extracts and tropomyosin survives various agents known to denature proteins but to the action of which tropomyosin is unusually stable, namely heating at 100° and mild tryptic digestion. It is destroyed by prolonged treatment with trypsin. 3. The ethylenedioxybis-(ethyleneamino)tetra-acetic acid (EGTA)-sensitizing factor present in extracts of natural actomyosin and myofibrils could be selectively destroyed, leaving unchanged the inhibitory effect on the Ca2+-activated ATPase. There was no correlation between the EGTA-sensitizing and the Ca2+-activated inhibitory activities of tropomyosin prepared under different conditions. 4. Optimum inhibition was achieved when tropomyosin and the myosin of desensitized actomyosin were present in approximately equimolar proportions. Tropomyosin had no effect on the Ca2+-activated ATPase of myosin measured under similar conditions. 5. Evidence is presented showing that the tropomyosin binds to desensitized actomyosin under the conditions in which the ATPase is inhibited. 相似文献