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)

Reactive disulfide reagents (RDSs) with a biotin moiety have been synthesized and found to cause Ca2+ release from sarcoplasmic reticulum (SR) vesicles. The RDSs oxidize SH sites on SR proteins via a thiol-disulfide exchange, with the formation of mixed disulfide bonds between SR proteins and biotin. Biotinylated RDSs identified a 106-kDa protein which was purified by biotin-avidin chromatography. Disulfide reducing agents, like dithiothreitol, reverse the effect of RDSs and thus promoted active re-uptake of Ca2+ and dissociated biotin from the labeled protein indicating that biotin was covalently linked to the 106-kDa protein via a disulfide bond. Several lines of evidence indicate that this protein is not Ca2+, Mg2+-ATPase and is not a proteolytic fragment or a subunit of the 400-kDa Ca2+-ryanodine receptor complex (RRC). Monoclonal antibodies against the ATPase did not cross-react with the 106-kDa protein, and polyclonal antibodies against the 106-kDa did not cross-react with either the ATPase or the 400-kDa RRC. RDSs did not label the 400-kDa RRC with biotin. Linear sucrose gradients used to purify the RRC show that the 106-kDa protein migrated throughout 5-20% linear sucrose gradients, including the high sucrose density protein fractions containing 400-kDa RRC. Protease inhibitors diisopropylfluorophosphate used to prevent proteolysis of 400-kDa proteins did not alter the migration of 106-kDa in sucrose gradients nor the patterns of biotin labeling of the 106-kDa protein. Incorporation of highly purified 106-kDa protein (free of RRC) in planar bilayers revealed cationic channels with large Na+ (gNa+ = 375 +/- 15 pS) and Ca2+ (gCa2+ = 107.7 +/- 12 pS) conductances which were activated by micromolar [Ca2+]free or millimolar [ATP] and blocked by micromolar ruthenium red or millimolar [Mg2+]. Thus, the SR contains a sulfhydryl-activated 106-kDa Ca2+ channel with apparently similar characteristics to the 400-kDa "feet" proteins.
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PMID:Disulfide linkage of biotin identifies a 106-kDa Ca2+ release channel in sarcoplasmic reticulum. 248 Sep 55

The microsomal fraction of normal human skeletal muscle was subfractionated by isopycnic sucrose-density centrifugation, using the procedure originally described by Saito et al. for rabbit fast muscle, and specific markers of the junctional face membrane of terminal cisternae (TC) (ryanodine receptor, high-molecular-weight feet proteins and membrane-associated calcium-binding protein calsequestrin), of the sarcoplasmic reticulum (SR) Ca-pump membrane (chicken antibody to rabbit Ca-ATPase), and of transverse tubules (TT) (dihydropiridine receptor, membrane cholesterol), respectively. The results show that isolated TC from human skeletal muscle share extensive morphological characteristics, protein composition, as well as Ca-release properties with rabbit TC, as tested with an inhibitor (Ruthenium red) and an activator (doxorubicin) of SR Ca-release. The Ca-pump membrane of human muscle SR, in distinction to rabbit fast muscle SR, showed a relatively low specific activity of the Ca-ATPase, as expected from the mixed fiber composition of human muscles, but shared the presence of minor protein components, such as a Con A binding protein of about 57 kDa and blue-staining peptides in the 170-120 kDa range of molecular weights. Human muscle TT, as isolated from the same sucrose gradient, demonstrated a high affinity (3H)-dihydropiridine binding activity in the range of previously reported values for purified TT from rabbit skeletal muscle.
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PMID:Biochemical characteristics of free and junctional sarcoplasmic reticulum and of transverse tubules in human skeletal muscle. 254 16

The different membrane systems and proteins involved in the control of intracellular calcium movements in the skeletal muscle cell are described. These include the sarcoplasmic reticulum, that Ca(++)-ATPase sarcoplasmic reticular calcium pump, transverse tubules, calcium channels, and the ryanodine receptor protein. The significance of these systems is shown clearly in the myopathies, where the main errors involved do not concern the contractile system, but the command and control mechanisms.
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PMID:[Contraction of skeletal muscles: regulation of calcium intracellular movements]. 256 Jun 11

The Ca2+-ryanodine receptor complex is solubilized in functional form on treating sarcoplasmic reticulum (SR) vesicles from rabbit fast skeletal muscle with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) (1 mg/mg protein) and 1 M NaCl at pH 7.1 by shaking for 30 min at 5 degrees C. The heavy membrane preparations obtained from pyrophosphate homogenates frequently exhibit junctional feet and appear to be derived primarily from the terminal cisternae of the SR. The characteristics of [3H]ryanodine binding are similar for the soluble receptor and the heavy SR vesicles with respect to dependence on Ca2+, pharmacological specificity for inhibition by six ryanoids and ruthenium red, and lack of sensitivity to voltage-dependent Ca2+-channel blockers, inositol 1,4,5-trisphosphate, or doxorubicin. In contrast, the cation sensitivity is decreased on receptor solubilization. The soluble receptor is modulated by cyclic nucleotides and rapidly denatured at 50 degrees C. Saturation experiments reveal a single class of receptors (Kd = 9.6 nM), whereas kinetic measurements yield a calculated association constant of 5.5 X 10(6) min-1 M-1 and a dissociation constant of 5.7 X 10(-4) min-1, suggesting that the [3H]ryanodine receptor complex ages with time to a state which is recalcitrant to dissociation. Sepharose chromatography shows that the receptor complex consists primarily of two protein fractions, one of apparent Mr 150,000-300,000 and a second, the [3H]ryanodine binding component, of approximately Mr 1.2 X 10(6). Preliminary analysis of the soluble receptor preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals subunits of Mr greater than 200,000 and major bands of calsequestrin and Ca2+-transport ATPase. These findings indicate that [3H]ryanodine binds to the Ca2+-induced open state of the channel involved in the release of contractile Ca2+.
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PMID:Calcium-ryanodine receptor complex. Solubilization and partial characterization from skeletal muscle junctional sarcoplasmic reticulum vesicles. 372 65

It is generally accepted that the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor play major roles in the complex mechanisms by which agonists increase intracellular Ca2+ concentration. In these mechanisms, the endoplasmic reticulum Ca(2+)-ATPase has been attributed an accessory role of refilling the intracellular Ca2+ store. In the present study, the activity of the microsomal Ca(2+)-ATPase of bovine adrenal cortex was investigated. We show that the Ca(2+)-pumping activity of the Ca(2+)-ATPase is related to the ADP/ATP ratio. Our results also show that a brisk increase of the ADP/ATP ratio upon addition of exogenous ADP triggered a rapid release of Ca2+ from preloaded microsomes. ADP released Ca2+ in a dose-dependent manner with an EC50 of 2.98 +/- 0.78 mM. ADP-induced Ca2+ release was not prevented by heparin, ruling out the participation of the inositol 1,4,5-trisphosphate receptor. ADP-induced Ca2+ release could not be attributed to the mere inhibition of the Ca(2+)-ATPase, since the rate of ADP-induced Ca2+ release was 20 times faster than the rate of Ca2+ release induced by a maximal concentration of thapsigargin (2 microM). ADP-induced Ca2+ release experiments performed in the presence of [32P]PO4 revealed a concomitant production of [32P]ATP. ADP-induced [32P]ATP production was dose-dependent, with an EC50 of 5.50 +/- 0.70 mM. ADP-induced [32P]ATP production was prevented by ionomycin (10 microM) and by high concentrations of extramicrosomal Ca2+. These results demonstrate that the microsomal Ca(2+)-ATPase of adrenal cortex possesses a bidirectional activity that depends on ADP concentrations, the Ca2+ gradient across the microsomal membrane, and probably also ATP concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Bidirectional activity of the endoplasmic reticulum Ca(2+)-ATPase of bovine adrenal cortex. 762 46

The effect of different free Mg2+ and ATP concentrations on depolarization-induced Ca2+ release in isolated skeletal muscle triadic vesicles was examined by simultaneously monitoring direct effects on ryanodine receptors from either isolated or coupled terminal cisternae. Free Mg2+ was increased to concentrations of 11-14 microM, 81 microM, 175-181 microM, and 1 mM while total ATP concentration was kept constant or MgATP concentration was kept constant. We observed the following. 1) Increasing MgATP reduces the measurable Ca2+ release from isolated vesicles by stimulating the Ca(2+)-ATPase in the terminal cisternae. 2) Half-maximal inhibition of functionally coupled ryanodine receptors during depolarization-induced Ca2+ release is observed at 1 mM Mg2+, whereas half-maximal inhibition of the nondepolarized ryanodine receptor is seen at 75 microM Mg2+ at the same free ATP and MgATP concentrations. 3) Two separate time constants for Ca2+ release were obtained for nondepolarized ryanodine receptors with free Mg2+ at 14 microM and free ATP at 6.1 mM; this may represent triadic ryanodine receptors vs. isolated terminal cisternae ryanodine receptors.
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PMID:Effect of Mg2+ and ATP on depolarization-induced Ca2+ release in isolated triads. 763 62

Pressure overload (PO)-induced cardiac hypertrophy in rabbits has been utilized extensively to study alterations in systolic and diastolic functions of the heart. In earlier studies we showed that the levels of mRNA encoding two important sarcoplasmic reticulum (SR) proteins, the cardiac/slow-twitch muscle Ca(2+)-ATPase (SERCA2a) and phospholamban, were decreased in PO rabbit hearts. In this study, we analyzed the expression of the Ca(2+)-release channel (ryanodine receptor), calsequestrin, SERCA2a, and phospholamban in PO-induced cardiac hypertrophy after 2, 4, 8, and 16 days of pulmonary artery banding. Northern blot and slot blot analyses showed that the steady-state level of mRNA encoding the cardiac ryanodine receptor, SERCA2a, and phospholamban was decreased significantly as early as 2 days after PO. In 16-day PO hearts, SERCA2a mRNA was reduced to 7.9 +/- 3.4% (P < 0.05), phospholamban mRNA was reduced to 15.9 +/- 6.5% (P < 0.05), and ryanodine receptor mRNA was reduced to 49.2 +/- 23.6% (P < 0.05). In this study, calsequestrin mRNA levels were also reduced to 29.9 +/- 15.2% by day 16 (P < 0.05). ATP-dependent Ca2+ uptake was reduced to 78% (P < 0.05); in contrast, the steady-state formation of ATPase phosphoenzyme was reduced to 81% of control (P < 0.05) and Ca(2+)-ATPase protein was reduced to 78% of control (P < 0.05) in crude SR vesicles or total muscle homogenate obtained from 16-day PO hearts. On the basis of these data, we propose that decreases in the expression of SR proteins may contribute to dysfunctions seen in systolic and diastolic properties of the hypertrophied myocardium.
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PMID:Sarcoplasmic reticulum gene expression in pressure overload-induced cardiac hypertrophy in rabbit. 784 Jan 54

Marlins, sailfish, spearfishes, and swordfish have extraocular muscles that are modified into thermogenic organs beneath the brain. The modified muscle cells, called heater cells, lack organized myofibrils and are densely packed with sarcoplasmic reticulum (SR), transverse (T) tubules, and mitochondria. Thermogenesis in the modified extraocular muscle fibers is hypothesized to be associated with increased energy turnover due to Ca2+ cycling at the SR. In this study, the proteins associated with sequestering and releasing Ca2+ from the SR (ryanodine receptor, Ca2+ ATPase, calsequestrin) of striated muscle cells were characterized in the heater SR using immunoblot and immunofluorescent techniques. Immunoblot analysis with a monoclonal antibody that recognizes both isoforms of nonmammalian RYRs indicates that the fish heater cells express only the alpha RYR isoform. The calcium dependency of [3H]ryanodine binding to the RYR isoform expressed in heater indicates functional identity with the non-mammalian alpha RYR isoform. Fluorescent labeling demonstrates that the RYR is localized in an anastomosing network throughout the heater cell cytoplasm. Measurements of oxalate supported 45Ca2+ uptake, Ca2+ ATPase activity, and [32P]phosphoenzyme formation demonstrate that the SR contains a high capacity for Ca2+ uptake via an ATP dependent enzyme. Immunoblot analysis of calsequestrin revealed a significant amount of the Ca2+ binding protein in the heater cell SR. The present study provides the first direct evidence that the heater SR system contains the proteins necessary for Ca2+ release, re-uptake and sequestration, thus supporting the hypothesis that thermogenesis in the modified muscle cells is achieved via an ATP-dependent cycling of Ca2+ between the SR and cytosolic compartments.
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PMID:Characterization of the sarcoplasmic reticulum proteins in the thermogenic muscles of fish. 796 89

We have demonstrated recently that in cardiac sarcoplasmic reticulum (SR), a membrane-associated Ca2+/calmodulin-dependent protein kinase (CaM kinase) phosphorylates and activates the Ca(2+)-pumping ATPase (Ca(2+)-ATPase) in addition to phosphorylating the previously characterized substrates, phospholamban, and Ca2+ release channel (ryanodine receptor) (Xu, A., Hawkins, C., and Narayanan, N. (1993) J. Biol. Chem. 268, 8394-8397). The present study shows that a CaM kinase regulatory system capable of modulating SR Ca2+ pump activity through direct phosphorylation of the Ca(2+)-ATPase is functional in slow twitch but not fast twitch skeletal muscle. Incubation of SR vesicles isolated from rabbit slow twitch (soleus) and fast twitch (adductor magnus) skeletal muscles in the presence of Ca2+ and calmodulin resulted in phosphorylation of the Ca(2+)-ATPase in slow twitch muscle SR but not in fast twitch muscle SR. Exogenous CaM kinase II, which stimulated phosphorylation of the cardiac and slow twitch muscle SR Ca(2+)-ATPase, failed to phosphorylate fast twitch muscle SR Ca(2+)-ATPase. These observations demonstrate that CaM kinase-catalyzed phosphorylation of the Ca2+ pump is isoform-specific since heart and slow twitch muscle express the same Ca(2+)-ATPase isoform (SERCA2a), which is distinct from that of fast twitch muscle (SERCA1). As in the case of cardiac SR Ca(2+)-ATPase, phosphorylation of the slow twitch muscle SR Ca(2+)-ATPase (occurring at a serine residue) resulted in a 2-fold increase in catalytic activity of the enzyme without alteration in its Ca2+ sensitivity. In addition, Ca2+/calmodulin-dependent prephosphorylation of slow twitch muscle SR resulted in a greater than 2-fold increase in its Ca2+ transport activity. In both cardiac and slow twitch muscle SR, phosphorylation of the Ca(2+)-ATPase by the endogenous CaM kinase occurred rapidly (maximum within 2 min at 37 degrees C), had similar pH optimum (8.5-9.0), temperature optimum (30 degrees C), and calmodulin concentration-dependence (k0.5 50-60 nM). cAMP-dependent protein kinase did not phosphorylate the Ca(2+)-ATPase appreciably in either cardiac or slow twitch muscle SR. These findings suggest a muscle-specific role for the membrane-associated CaM kinase in the modulation of Ca2+ uptake and release functions of the SR. In cardiac and slow twitch muscle, phosphorylation of the SR Ca(2+)-ATPase by CaM kinase might provide a novel mechanism for the modulation of the enzymatic and Ca2+ transport functions of this enzyme.
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PMID:Sarcoplasmic reticulum calcium pump in cardiac and slow twitch skeletal muscle but not fast twitch skeletal muscle undergoes phosphorylation by endogenous and exogenous Ca2+/calmodulin-dependent protein kinase. Characterization of optimal conditions for calcium pump phosphorylation. 798 62

In birds, prolonged cold exposure induces the development of a nonshivering thermogenesis (NST) of muscular origin. NST is characterized by an increased heat production, which may be achieved by an increased ATP-dependent cycling of Ca2+ between the sarcoplasmic reticulum (SR) and cytosolic compartments in muscle. In this study, the effects of prolonged cold exposure on SR function were assessed by determining the contents of the SR Ca(2+)-ATPase and Ca2+ release channel (ryanodine receptor) in the gastrocnemius muscle of ducklings (Cairina moschata) kept at thermoneutrality (25 degrees C) or cold acclimated (4 degrees C, 5 wk). Measurement of oxalate-supported 45Ca2+ uptake by whole muscle homogenates revealed that the SR Ca(2+)-ATPase activity, and fraction of vesicles containing a ryanodine-sensitive Ca2+ release channel were increased by 30-50% in response to prolonged cold exposure. Sodium dodecyl sulfate-polyacrylamide gel and immunoblot analysis, 45Ca2+ uptake, Ca(2+)-ATPase activity and [3H]ryanodine binding measurements with unfractionated and "heavy" SR membrane fractions also indicated an elevated Ca(2+)-ATPase and Ca2+ release channel content in cold-acclimated ducklings. These results showed that the contents of two components directly involved in Ca2+ cycling by the SR are increased by cold acclimation, and we suggest that this is related to NST.
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PMID:Sarcoplasmic reticulum Ca(2+)-ATPase and ryanodine receptor in cold-acclimated ducklings and thermogenesis. 817 61


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