Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Isolated sarcoplasmic reticulum (SR) vesicles with polymerized calcium pump protein were freeze-dried and rotary shadowed following uranyl acetate stabilization. This technique allows direct observation of a single side of the vesicle without requiring optical filtering. The heads of individual ATPase molecules, projecting above the cytoplasmic surface, are clearly resolved in the replicas. Ca ATPase molecules form extensive arrays in vanadate-treated, rabbit SR vesicles and in gently isolated, native SR vesicles from scallop. Gentle isolation results in limited areas of orderly structure in native SR isolated from vertebrate muscles. Special attention is given to the effect of various shadow thicknesses on the appearance of the heads. This information is essential to the interpretation of images in the accompanying paper (Franzini-Armstrong, C., and D.J. Ferguson, 1985, Biophys. J., 48:607-615).
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PMID:Ordered arrays of Ca2+-ATPase on the cytoplasmic surface of isolated sarcoplasmic reticulum. 293 70

The effects of extracellular Na+ (Na+o) on cytosolic ionized calcium (Ca2+i) and on calcium and sodium fluxes were measured in monkey kidney cells (LLC-MK2). Ca2+i was measured with aequorin and the ion fluxes with 45Ca and 22Na. Na+-free media rapidly increased Ca2+i from 60 to a maximum of about 700 nM in 2-3 min. After the peak, Ca2+i declined and reached a plateau of about twice the resting Ca2+i. The peak Ca2+i was inversely proportional to Na+o and directly proportional to the extracellular calcium concentration (Ca2+o). On the other hand, a pH of 6.8 reduced and Ca2+o substitution with Sr2+ completely blocked the Ca2+i response to low Na+o. A Na+-free medium stimulated calcium efflux from the cells 4-5-fold, a response which was abolished in the absence of extracellular Ca2+. Na+-free media also stimulated calcium influx and sodium efflux. The cell calcium content, however, was not increased. These results indicate that removal of extracellular Na+ increases Ca2+i by stimulating calcium influx and not by inhibiting calcium efflux; the increased calcium influx takes place on the Na+-Ca2+ antiporter operating in the reverse mode in exchange for sodium efflux. The increased calcium efflux occurs as a consequence of the rise in Ca2+i and presumably takes place on the (Ca2+-Mg2+) ATPase-dependent calcium pump.
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PMID:Effects of low extracellular sodium on cytosolic ionized calcium. Na+-Ca2+ exchange as a major calcium influx pathway in kidney cells. 293 97

Titration of the specific calcium binding sites of sarcoplasmic reticulum ATPase was carried out by measurements of intrinsic fluorescence in the absence and in the presence of vanadate. The previous finding that vanadate binding to the enzyme inhibits high-affinity calcium binding was confirmed. In addition, taking advantage of the slow kinetics of vanadate association and dissociation from the enzyme, we were able to titrate the fraction of sites remaining in the high affinity state in the presence of non-saturating vanadate. These sites were demonstrated to retain the characteristics displayed by the high-affinity sites in the absence of vanadate, and yielded information consistent with a competitive inhibition between vanadate and calcium. Reversal of the vanadate effect and reconversion of the binding sites to the high-affinity state was demonstrated by adding appropriate calcium concentrations to the enzyme-vanadate complex, and showing the appearance of the intrinsic fluorescence signal which is indicative of calcium occupancy of the sites in the high-affinity state. Partial or total reversal of the vanadate effect was obtained with very slow kinetics following addition of micromolar calcium or, at a somewhat faster rate, following addition of millimolar calcium. The latter experiments yielded titration of the binding sites in the low-affinity state, with a dissociation constant of approx. 2 mM at neutral pH and 10 mM Mg2+. The time course of the fluorescence rise following addition of calcium in the presence of vanadate was more rapid in 'leaky' than in native sarcoplasmic reticulum vesicles, suggesting an intravesicular orientation of the low-affinity calcium sites involved in the reversal of the vanadate effect. Our observations provide experimental support for the postulated mechanism of high- and low-affinity interconversion of the ATPase calcium binding sites, and its dependence on the occupancy of the phosphorylation site by vanadate.
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PMID:Fluorometric titration of the sarcoplasmic reticulum adenosinetriphosphatase calcium sites in the presence of vanadate. 293 92

Using the reconstituted Ca-ATPase vesicles as a model system, we demonstrated that the presence of 1,2-dioleoyl-sn-glycerol (diolein) in the membrane introduces a pronounced enhancement in the Ca-transport function of Ca-ATPase, while the 1,2-dipalmitoyl-sn-glycerol (dipalmitin) does not. We also found by both 31P NMR and freeze-fraction electron microscopy that diolein destabilized lipid bilayers to a greater extent than did dipalmitin. We conclude that the tendency of diacylglycerols to destabilize the phospholipid bilayer is related to their capacity to enhance the activity of the membrane calcium pump.
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PMID:Correlation between bilayer destabilization and activity enhancement by diacylglycerols in reconstituted Ca-ATPase vesicles. 293 4

The decomposition of 32P phosphorylated enzyme intermediate formed by incubation of sarcoplasmic reticulum ATPase with [gamma-32P]ATP was studied following dilution of the reaction medium with a large excess of nonradioactive ATP. The phosphoenzyme decomposition includes two kinetic components. The fraction of intermediate undergoing slower decomposition is minimal in the presence of low (microM) Ca2+ and maximal in the presence of high (mM) Ca2+. A large fraction of phosphoenzyme undergoes slow decomposition when the Ca2+ concentration is high inside the vesicles, even if the Ca2+ concentration in the medium outside the vesicles is low. Parallel measurements of ATPase steady state velocity in the same experimental conditions indicate that the apparent rate constant for the slow component of phosphoenzyme decomposition is inadequate to account for the steady state ATPase velocity observed under the same conditions and cannot be the rate-limiting step in a single, obligatory pathway of the catalytic cycle. On the contrary, the steady state enzyme velocity at various Ca2+ concentrations is accounted for by the simultaneous contribution of both phosphoenzyme fractions undergoing fast and slow decomposition. Contrary to its slow rate of decomposition in the forward direction of the cycle, the phosphoenzyme pool formed in the presence of high Ca2+ reacts rapidly with ADP to form ATP in the reverse direction of the cycle. Detailed analysis of these experimental observations is consistent with a branched pathway following phosphoryl transfer from ATP to the enzyme, whereby the phosphoenzyme undergoes an isomeric transition followed by ADP dissociation, or ADP dissociation followed by the isomeric transition. The former path is much faster and is prevalent when the intravesicular Ca2+ concentration is low. When the intravesicular Ca2+ concentration rises, a pool of phosphoenzyme is formed by reverse equilibration through the alternate path. In the absence of ADP this intermediate decays slowly in the forward direction, and in the presence of ADP it decays rapidly in the reverse direction of the cycle.
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PMID:Kinetic effects of calcium and ADP on the phosphorylated intermediate of sarcoplasmic reticulum ATPase. 293 32

Calcium release from the ADP-sensitive phosphoenzyme intermediate of the sarcoplasmic reticulum ATPase was investigated at 6 degrees C under a variety of conditions using the purified ATPase protein and the rapid membrane filtration system. The rate of calcium release measured in the presence of [ethylene bis-(oxyethylenenitrilo)]tetraacetic acid increased monotonically with increasing pH of the medium, the time at which 50% of the bound calcium was released being reduced to one third when the pH was raised from 5.5 to 9.0. Dimethyl sulfoxide at 10 or 20% (v/v) also was very effective in accelerating the calcium release. ATP at a millimolar concentration range also was stimulatory, but millimolar concentrations of Mg2+ were found to be inhibitory. Using an indirect method, i.e. by measuring the overall rate of calcium transport by the reconstituted vesicles under conditions where calcium release from the ADP-sensitive phosphoenzyme was presumably rate-limiting, the calcium release was shown to be accelerated up to 1.5-fold by the inside-negative potential imposed across the membrane using the K+-valinomycin system. As evidence was presented suggesting that the observed calcium release primarily reflects the phosphoenzyme isomerization which leads to reduction in calcium affinity of the phosphoenzyme, the results strongly suggest that this phosphoenzyme isomerization was affected significantly by each of the factors described above.
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PMID:Factors influencing calcium release from the ADP-sensitive phosphoenzyme intermediate of the sarcoplasmic reticulum ATPase. 294 34

The effect of hydrostatic pressure on the self-association of sarcoplasmic reticulum ATPase solubilized by nonionic detergent was studied in the pressure range of 1 atm up to 2 kilobars. Polarization of intrinsic tryptophan fluorescence or of fluorescence of a pyrene probe covalently attached to the ATPase was measured. An increase in hydrostatic pressure promoted dissociation of the protein into monomers. For a midpoint dissociation pressure of 1.3 kilobars, the standard volume change in the dissociation reaction was delta Vop = -167 ml/mol. Full reversibility of the pressure effects was shown to occur, as seen by recovery of polarization. An increase in Ca2+ concentration from 50 microM to 5 mM and of pH from 6.9 to 8.6 were found to increase the midpoint dissociation pressure, indicating that these factors stabilize the dimeric state. The hydrolytic activity of the ATPase was measured under pressure. The activity was inhibited by pressure increase. It was found that an irreversible inactivation of the solubilized enzyme occurred during turnover and that increasing pressure added to this instability. Reversibility of the activity was critically dependent on the presence of 10 mM Ca2+ in the assay medium.
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PMID:Pressure-induced dissociation of solubilized sarcoplasmic reticulum ATPase. 294 35

We have investigated the kinetics of the intrinsic fluorescence drop observed when ATP is added to purified sarcoplasmic reticulum ATPase in a potassium-free medium containing magnesium and calcium, at pH 6 and 20 degrees C. Under these conditions, analysis of the fluorescence drop is complex. Several events contributed to the rate of the fluorescence drop initiated by turnover, including phosphorylation, conformational transition of the phosphorylated complex, and dephosphorylation. On the other hand, when 75% of total fluorescence was quenched by energy transfer to the membrane-bound ionophore A23187, the observed turnover-dependent drop in residual fluorescence mainly reflected the conformational transition of the phosphorylated ATPase. Combination of fast kinetics with the quenching of selected tryptophan residues is suggested to be a promising tool for the study of proteins containing many of these residues.
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PMID:Does intrinsic fluorescence reflect conformational changes in the Ca2+-ATPase of sarcoplasmic reticulum? 294 63

A great controversy has been set for several years related to an indirect versus a direct effect of cholesterol upon the muscle calcium pump. Employing an enriched cardiac sarcolemma preparation and a (Ca2+, Mg2+)-ATPase fraction isolated from this preparation, this study demonstrates that cholesterol directly interacts with the sarcolemmal calcium pump importantly inhibiting its enzyme activity. It was discovered that this inhibition can be in part explained by a total sensitivity loss of the pump to calmodulin. These results can be considered of importance in the correlation of plasma membrane cholesterol levels with deficiencies in calcium transport and cardiac muscle cell damage.
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PMID:Direct regulatory effect of cholesterol on the calmodulin stimulated calcium pump of cardiac sarcolemma. 294 64

Long-chain acylcarnitines are membrane-active intermediates of fatty acid metabolism whose intracellular accumulation has been implicated in the myocardial injury associated with both streptozotocin-induced diabetes and acute ischemia. In the present study, rats treated with streptozotocin (50 mg/kg i.v.) exhibited increases in myocardial long-chain acylcarnitines comparable to those previously reported to occur in moderate to severe ischemic injury. With the exception of a reduction in the sedimentable (lysosome-associated) fraction of myocardial N-acetyl-beta-glucosaminidase and a decrease in sarcoplasmic reticulum K+, Ca++-stimulated ATPase activity, other characteristic indices of myocardial ischemic damage, notably inhibition of sarcolemmal and mitochondrial ATPase activities as well as alterations in the ionic composition of myocardial tissue, were not apparent in the hearts of the streptozotocin-diabetic animals. On the basis of in vitro studies using palmitylcarnitine, it does not seem that differential sensitivity to long-chain acylcarnitine inactivation can explain the preferential inhibition of the sarcoplasmic reticulum ATPase enzyme observed in vivo. Our data are consistent with the findings of others suggesting that long-chain acylcarnitines are unlikely to be the most important or sole mediators of myocardial ischemic injury. However, a modulatory role of these substances in myocardial ischemic injury or in determining the increased susceptibility of diabetics to the complications of ischemic heart disease cannot be excluded at present.
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PMID:Subcellular myocardial abnormalities in experimental diabetes: role of long-chain acylcarnitines. 294 27


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