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)

Phospholamban, a putative protein regulator of the Ca2(+)-dependent ATPase in cardiac sarcoplasmic reticulum, was isolated from bovine cardiac microsomes using a low concentration of the detergent. The phospholamban was phosphorylated by cyclic AMP-dependent protein kinase to a level of 7.55 nmol of phosphate per mg of protein. This molecular weight was found to be 20,000 dalton based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monoclonal antibody against the bovine phospholamban was made by the hybridoma technique. The specificity of the monoclonal antibody was determined by immunoblotting experiments and the antibody was shown capable of blocking the phosphorylation of phospholamban by cyclic AMP-dependent protein kinase. The immunoglobulin subclass of the antibody was identified as IgG1. The distribution of phospholamban in muscular tissues was observed by the immunohistological method and the results showed that myocardial cells were strongly and specifically stained, while skeletal and smooth muscle cells were stained at the background level. The results showed the phospholamban in the ventricular muscle to be localized along the cellular surface and in a network over the myofilaments. The monoclonal antibody identified a reactive component in the myocardial cells of monkey, canine, mouse and man.
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PMID:Monoclonal antibody to phospholamban isolated from bovine cardiac muscle. 253 73

The activity of the Ca-pumping ATPase of cardiac sarcoplasmic reticulum (SR) is controlled by the phosphorylation of the intrinsic regulatory protein phospholamban (PLB), which affects both the apparent Km(Ca) and the Vmax of the transport process. We have investigated the correlation between phosphorylation of PLB and the surface potential of the SR membrane. This latter influences the local concentration of relevant ionic species near biological membranes and thus modulates the activity of ion pumps and channels. The partitioning of the anionic probe 8-anilino-1-naphthalenesulfonate (ANS-) into the SR membrane was found to be dependent on the phosphorylation level of PLB. Changes of the surface membrane potential up to 7 mV could be obtained by phosphorylation. The increase in the apparent affinity of the Ca pump for Ca2+ induced by PLB phosphorylation was clearly reduced at high ionic strength, i.e. under conditions known to reduce the surface membrane potential and all processes dependent on it. The results show that electrostatic phenomena can account, in good part, for the modulation of the Ca pump by PLB in cardiac SR.
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PMID:Involvement of electrostatic phenomena in phospholamban-induced stimulation of Ca uptake into cardiac sarcoplasmic reticulum. 253 50

Highly purified sarcoplasmic reticulum (SR) has been prepared from dog hearts and has been incubated with the triplet probe erythrosinyl isothiocyanate to specifically label the Ca2+-stimulated ATPase (Ca2+-ATPase) of the SR. The rotational mobility of the Ca2+-ATPase has been studied in this erythrosin-labelled SR using time-resolved phosphorescence polarization. Qualitatively, the mobility of the cardiac Ca2+-ATPase resembles that of skeletal muscle SR Ca2+-ATPase. Addition of Ca2+ to SR affects the mobility of the Ca2+-ATPase in a way consistent with a segment of the ATPase altering its orientation relative to the plane of the membrane. Phosphorylation of phospholamban in cardiac SR by the purified catalytic subunit of cAMP-dependent protein kinase, which is known to increase the activity of the Ca2+-ATPase by deinhibition, also alters measured anisotropy. The changes observed are not compatible with dissociation of the Ca2+-ATPase from phospholamban after the latter is phosphorylated. The data are more consistent with phospholamban associating with the Ca2+-ATPase following phosphorylation, or more complex models in which only the hydrophilic domain of phospholamban binds with and dissociates from the Ca2+-ATPase.
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PMID:The effects of calcium, temperature and phospholamban phosphorylation on the dynamics of the calcium-stimulated ATPase of canine cardiac sarcoplasmic reticulum. 254 Aug 39

Ca2+ transients in myocardial cells are modulated by cyclic AMP-dependent phosphorylation of a protein in the sarcoplasmic reticulum. This protein, termed phospholamban, serves to regulate the Ca2+ pump ATPase of this membrane, thus altering the mode of Ca2+ transients and the myocardial contractile response. Elucidating the structure of phospholamban and its intimate interaction with the Ca2+ pump ATPase should provide the basis for understanding, at the molecular level, how the cAMP system contributes to excitation-contraction coupling in muscle cells.
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PMID:Regulation of the Ca2+ pump ATPase by cAMP-dependent phosphorylation of phospholamban. 254 89

The native form of phospholamban in cardiac sarcoplasmic reticulum membranes was investigated using photosensitive heterobifunctional cross-linkers, both cleavable and noncleavable, and common protein modifiers. The photosensitive heterobifunctional cleavable cross-linker ethyl 4-azidophenyl-1, 4-dithiobutyrimidate was used in native SR vesicles and it cross-linked phospholamban into an apparent phospholamban-phospholamban dimer and into an approximately 110,000-Da species. The phospholamban dimer migrated at approximately 12,000 Da on sodium dodecyl sulfate-polyacrylamide gels, and upon cleavage of the cross-linker before electrophoresis the dimer disappeared. The approximately 110,000-Da cross-linked species was not affected by boiling in sodium dodecyl sulfate prior to electrophoresis. This cross-linked form of phospholamban migrated approximately 5500 Da above the Ca2(+)-ATPase, which was visualized using fluorescein 5'-isothiocynate, a fluorescent marker that binds specifically to the Ca2(+)-ATPase. p-Azidophenacyl bromide, iodoacetic acid, and N-ethylmaleimide, all of which react with sulfhydryl groups, were also employed to further characterize phospholamban in native sarcoplasmic reticulum membranes. Cross-linking with p-azidophenacyl bromide resulted in only monomeric and dimeric forms of phospholamban as observed on sodium dodecyl sulfate-polyacrylamide gels. Iodoacetic acid and N-ethylmalemide were found to be effective in disrupting the pentameric form of phospholamban only when reacted with sodium dodecyl sulfate solubilized sarcoplasmic reticulum. In view of these findings, the amino acid sequence of phospholamban was examined for possible protein-protein interaction sites. Analysis by hydropathic profiling and secondary structure prediction suggests that the region of amino acids 1-14 may form an amphipathic alpha helix and the hydrophobic surface on one of its sites could interact with the reciprocal hydrophobic surface of another protein, such as the Ca2(+)-ATPase.
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PMID:Structural characterization of phospholamban in cardiac sarcoplasmic reticulum membranes by cross-linking. 263 36

Maturation has been shown to enhance the ability of the myocardium to contract. Whenever developmental changes in the systems that control or modulate myocardial contractility have been sought, they have indeed been found. These include an increase in the amount and organization of the myofilaments, an increase in SR amount, organization, differentiation, Ca-ATPase, phospholamban, and sensitivity to calcium and ryanodine, changes in the sarcolemmal pumps and channels, and changes in the expression of the contractile proteins. The characterization of the control systems that integrate these changes remains to be achieved. The overall gradual increase in myocardial contractility has superimposed on it an acute, sudden increase in ventricular contractility in the hours surrounding birth. In the subsequent neonatal days, the level of inotropy falls, and the reserves in contractility are replenished and expanded. Consequently, disease states that demand an increased use of or negatively affect mechanisms that bring about the neonatal enhancement in ventricular function will have their most malignant effect during the first days of neonatal life.
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PMID:Maturation and cardiac contractility. 265 71

Sequestration of calcium into an intracellular storage site is an important mechanism in helping to maintain a low cytoplasmic Ca2+ level in many cells. In platelets, increasing cytoplasmic cAMP lowers the free calcium level in correlation with the phosphorylation of a 22 kD protein. This protein has been thought to enhance uptake of calcium into a platelet membrane bound storage site by activating a calcium-ATPase activity by analogy with phospholamban in cardiac muscle. The evidence for an analogue of phospholamban in platelets is unclear. A pathway involving cAMP dependent kinase also seems unlikely to account for the transience of the calcium signal following agonists in platelets, some of which inhibit the cAMP dependent kinase. Here we discuss the issue of whether activation of protein kinase C, which follows agonist action, leads to enhanced calcium sequestration in platelets and if so, what indications there are for a mechanism. The evidence from our experiments with phorbol myristate acetate treated platelets shows that such an enhancement can be produced by activating protein kinase C. Phosphorylation studies suggest the involvement of a polypeptide or polypeptides distinct from the 22 kD polypeptide. Further work to test this idea is necessary. A brief overview of research on the role of phosphoproteins in calcium regulation in platelets and comparison with their role in cardiac muscle is also presented.
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PMID:Calcium sequestration in human platelets: is it stimulated by protein kinase C? 267 Feb 38

Phosphorylation of myofibrillar and sacroplasmic-reticulum (SR) proteins was studied in Langendorff-perfused rabbit hearts subjected to various inotropic interventions. Stimulation of hearts with isoprenaline resulted in the phosphorylation of both troponin I (TnI) and C-protein in myofibrils and phospholamban in SR. Phosphorylation of phospholamban could be reversed by a 15 min perfusion with drug-free buffer, after a 1 minute pulse perfusion with isoprenaline, at which time the mechanical effects of isoprenaline stimulation had also been reversed. However, both TnI and C-protein remained phosphorylated at this time. Moreover, the inhibition of Ca2+ activation of the Mg2+-dependent ATPase (Mg-ATPase) activity associated with myofibrillar phosphorylation persisted in myofibrils prepared from hearts frozen after 15 min of washout of isoprenaline. To assess the contribution of C-protein phosphorylation in the decrease of Ca2+ activation of the myofibrillar Mg-ATPase activity, we reconstituted a regulated actomyosin system in which only C-protein was phosphorylated. In this system, C-protein phosphorylation did not contribute to the decrease in Ca2+ activation of Mg-ATPase activity, indicating that TnI phosphorylation is responsible for the diminished sensitivity of the myofibrils to Ca2+. These observations support the hypothesis that phospholamban phosphorylation plays a more dominant role than TnI or C-protein phosphorylation in the mechanical response of the mammalian heart to beta-adrenergic stimulation.
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PMID:Phosphorylation of C-protein, troponin I and phospholamban in isolated rabbit hearts. 289 34

Membrane vesicles capable of energized Ca2+ pumping have been reconstituted from cardiac sarcoplasmic reticulum (SR). Cardiac SR was solubilized with Triton X-100 in a detergent to protein weight ratio of 0.8, and membranous vesicles were reconstituted by removal of detergent with Bio-Beads SM-2 (a neutral porous styrene-divinylbenzene copolymer). The reconstituted vesicles exhibited ATP-dependent oxalate-facilitated Ca2+ accumulation with rates and efficiency comparable to the best reconstituted skeletal muscle preparation (Ca2+-loading rate = 1.65 +/- 0.31 mumol mg-1 min-1, Ca2+-activated ATPase activity = 2.39 +/- 0.25 mumol mg-1 min-1, efficiency (Ca2+/ATP) = 0.69 +/- 0.09). Phospholamban in the reconstituted vesicles was phosphorylated with added catalytic subunit of cAMP-dependent protein kinase to almost the same extent as that in original vesicles. However, phosphorylation of phospholamban had no effect on the Ca2+ accumulation of the reconstituted vesicles. This is to be contrasted with a decrease in the half-maximal concentration of Ca2+ for Ca2+ accumulation (KCa) in the original vesicles from 1.35 +/- 0.08 microM to 0.75 +/- 0.12 microM by cAMP-dependent phosphorylation of phospholamban. On the other hand KCa for the reconstituted vesicles was about 0.5 microM and remained unchanged by phosphorylation, indicating that the Ca2+ pump in the reconstituted vesicles is already fully activated. These results suggest that in normal cardiac SR, phospholamban in the dephosphorylated state acts as a suppressor of the Ca2+ pump and that phosphorylation of phospholamban serves to reverse the suppression.
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PMID:The nature of the modulation of Ca2+ transport as studied by reconstitution of cardiac sarcoplasmic reticulum. 293 32

We describe the 50fold purification of phospholamban, the production of antibodies against it and preliminary experiments of reconstitution of purified phospholamban into vesicles of skeletal sarcoplasmic reticulum (SR). Purified phospholamban migrates in a modified Laemmli SDS polyacrylamide gel electrophoresis (PAGE) system with a relative molecular mass (Mr) of 22 kD and 6 kD. The higher Mr form is detectable by immunoreaction on Western blots only in heart SR preparations, whereas the low Mr form is also present in sarcolemmal preparations. No immunoreactivity was found in SR of skeletal muscle. Reconstituted skeletal SR vesicles were not influenced by phospholamban in respect to their Ca++-ATPase activity and calcium accumulation rate, but the obtained data suggest that phospholamban alters the calcium storage capacity of SR.
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PMID:The putative role of phospholamban in the regulation of the heart muscle. 293 76


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