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)

A soluble porcine H,K-ATPase preparation was obtained with the nonionic detergent, C12E8. ATP hydrolysis by the soluble H,K-ATPase was stimulated with respect to the native preparation at pH 6.1, while the K(+)-phosphatase activity was comparable to the native enzyme. The soluble enzyme demonstrated characteristic ligand-dependent effects on ATP hydrolysis, including ATP activation of K(+)-stimulated hydrolysis with a K0.5 of 28 +/- 4 microM ATP, and inhibition with an IC50 of 2.1 mM ATP. The activation and inhibition of ATP hydrolysis by K+ was also observed with a K0.5 for activation of 2.8 +/- 0.4 mM KCl at 2.0 mM ATP (pH 6.1) and inhibition with an IC50 of 135 mM KCl at 0.05 mM ATP. 2-Methyl-8-(phenylmethoxy)imidazo[1,2a]pyridine-3-acetonitrile (SCH 28080), a specific inhibitor of the native H,K-ATPase, competitively inhibited the K(+)-stimulated activity with a Ki of 0.035 microM. The soluble enzyme was stable with a t0.5 for ATPase activity of 6 h between 4 and 11 degrees C. The demonstration of these related ligand responses in the catalytic reactions of the soluble preparation indicates that it is an appropriate medium for investigation of the subunit associations of the functional H,K-ATPase. Subunit associations of the active soluble enzyme were assessed following treatment with the crosslinking reagent, glutaraldehyde. The distribution of crosslinked particles was independent of the soluble protein concentration in the crosslinking buffer within the protein range 0.3 to 2.0 mg/ml or the detergent to protein ratio varied from 1 to 15 (w/w). The crosslinked pattern was unaffected by the presence or absence of K during crosslinking or nucleotide concentration. These observations suggest that crosslinking occurs in associated subunits that do not undergo rapid associations dependent upon enzyme turnover. Phosphorylation of the soluble enzyme with 0.1 mM MgATP produced a phosphoprotein at 94 kDa. A phosphoprotein obtained after glutaraldehyde treatment exhibited identical electrophoretic mobility to the crosslinked particle identified by silver stain. Glutaraldehyde treatment of soluble protein fractions resolved on a linear 10-35% glycerol gradient revealed several smaller peptides partially resolved from the crosslinked pump particle, but no active fraction enriched in the monomeric H,K-ATPase. This data indicates that the functional porcine gastric H,K-ATPase is organized as a structural dimer.
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PMID:Glutaraldehyde crosslinking analysis of the C12E8 solubilized H,K-ATPase. 216 16

Stimulation of the gastric parietal cell requires massive membrane transformations as H(+)-pumps from the domain of cytoplasmic tubulovesicles are recruited into the apical plasma membrane domain. The recycling of membrane pools, through fusion and fission processes that accompany stimulation and inhibition of HCl secretion, also involves highly selective events of protein incorporation and segregation. This manuscript describes several proteins that have been identified with the apical plasma membrane from maximally stimulated parietal cells, and broadly characterizes them either as permanent resident proteins of the apical membrane, or transient proteins that move into and out of the apical membrane as the cell progresses through the secretory cycle. A typical example of transient association with the apical membrane concerns the pump proteins, including the 94 kDa catalytic alpha-subunit of the H+K(+)-ATPase and its newly discovered beta-subunit glycoprotein, which move between tubulovesicles. Proteins that remain associated with the apical plasma membrane during rest and secretion include actin, and an 80-kDa phosphoprotein, which has been variously called 80 K, ezrin, p81 and cytovillin, and whose phosphorylation is increased by the histamine/cAMP pathway of parietal cell stimulation. An example of a cytosolic protein that becomes associated with the apical plasma membrane after stimulation is a 120-kDa protein, which appears to have protein kinase activity. Note that the identification, localization and characterization of the K+ and Cl- transport proteins, which participate in net HCl secretion, are of immediate importance.
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PMID:Membrane and protein recycling associated with gastric HCl secretion. 216 24

This study examines the role of endogenous dopamine (DA) for the regulation of renal tubular sodium (Na) transport. The enzyme L-amino acid decarboxylase (L-AADC) that converts L-dopa to DA has been localized to the proximal tubule cells with immunocytochemistry. Locally formed DA will inhibit the activity of Na-K-ATPase, the enzyme that yields energy to active Na transport. The effect is of physiological importance during high salt diet. The phosphoprotein DARPP-32, a DA1 receptor associated third messenger is abundant in the medullary thick ascending limb of Henle (mTAL). DARPP-32 is phosphorylated after activation of DA1 receptors. DARPP-32 is in its phosphorylated form a potent phosphatase inhibitor. Activation of the DA1 receptor in mTAL with the DA1 agonist SKF 82526 causes dose-dependent inhibition of Na-K-ATPase activity. The effect involves activation of cAMP protein kinase. It is likely that this effect is potentiated by DARPP-32.
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PMID:The significance of L-amino acid decarboxylase and DARPP-32 in the kidney. 216 32

The present work provides evidence for the existence in rat brainstem of a form of Na,K-ATPase catalytic subunit that displays a high affinity for ouabain (Kd about 10(-9) M). Its kinetic identification was made out from studies on dose response curves of ouabain inhibition of Na,K-ATPase activity, ouabain inhibition of Na(+)-dependent phosphorylation from ATP and ouabain stabilized phosphoenzyme formation from inorganic phosphate (Pi). In all these studies this isoform comprises around 11 percent of the total Na,K-ATPase enzyme. The PAGE electrophoretic mobility of its phosphoprotein obtained from Pi in the presence of ouabain is lower than that of the alpha-1 form but it cannot be distinguished from that of alpha-2. Whether this highly ouabain sensitive form corresponds to the alpha-3 isoenzyme or represents the translational product of one of the additional genes described for the large catalytic subunit remains at the moment an open question.
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PMID:Detection of a highly ouabain sensitive isoform of rat brainstem Na,K-ATPase. 216 50

An ATP-dependent calcium transport component from rat liver plasma membranes was solubilized by cholate and reconstituted into egg lecithin vesicles by a cholate dialysis procedure. The uptake of Ca2+ into the reconstituted vesicles was ATP-dependent and the trapped Ca2+ could be released by A23187. Nucleotides, including ADP, UTP, GTP, CTP, GDP, AMP, and adenyl-5'-yl beta, gamma-imidophosphate, and p-nitrophenylphosphate did not substitute for ATP. The concentration of ATP required for half-maximal stimulation of Ca2+ uptake into the reconstituted vesicles was 6.2 microM. Magnesium was required for calcium uptake. Inhibitors of mitochondrial calcium-sequestering activities, i.e. oligomycin, sodium azide, ruthenium red, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and valinomycin did not affect the uptake of Ca2+ into the vesicles. In addition, strophanthidin and p-chloromercuribenzoate did not affect the transport. Calcium transport, however, was inhibited by vanadate in a concentration-dependent fashion with a K0.5 of 10 microM. A calcium-stimulated, vanadate-inhibitable phosphoprotein was demonstrated in the reconstituted vesicles with an apparent molecular weight of 118,000 +/- 1,300. These properties of Ca2+ transport by vesicles reconstituted from liver plasma membranes suggest that this ATP-dependent Ca2+ transport component is different from the high affinity (Ca2+-Mg2+)-ATPase found in the same membrane preparation (Lotersztajn, S., Hanoune, J. and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215; Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020). When the entire reconstituted vesicle population was treated with ATP and 45Ca in a buffer containing oxalate, the vesicles with Ca2+ transport activity could be separated from other vesicles by centrifugation in a density gradient and the ATP-dependent Ca2+ transport component was purified approximately 9-fold. This indicates that transport-specific fractionation may be used to isolate the ATP-dependent Ca2+ transport component from liver plasma membrane.
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PMID:Novel ATP-dependent calcium transport component from rat liver plasma membranes. The transporter and the previously reported (Ca2+-Mg2+)-ATPase are different proteins. 240 77

Intracellular Ca2+ concentrations in cardiac cells are dependent on trans-sarcolemmal Ca2+ fluxes and the ability of sarcoplasmic reticulum to release and take up Ca2+. Ca2+ accumulation by sarcoplasmic reticulum membranes causes muscle to relax, whereas Ca2+ release from sarcoplasmic reticulum initiates contraction. Ca2+ transport by the sarcoplasmic is mediated by a Ca2+-dependent ATPase enzyme. Ca2+ release from sarcoplasmic reticulum may be mediated by a ligant-gated Ca2+ channel. The physiological role of sarcoplasmic reticulum in developing muscle is not well established. In this report we investigated the composition and function of sarcoplasmic reticulum membranes during cardiac myogenesis. Phospholamban, a major phosphoprotein in mature sarcoplasmic reticulum membranes was present during early stages of cardiac myogenesis. The embryonic form of phospholamban was phosphorylated by cAMP-dependent protein kinase but not in the presence of Ca2+ and calmodulin. Ca2+ uptake and Ca2+-dependent ATPase activity were low in fetal sarcoplasmic reticulum compared to adult control membranes, although the apparent affinities of the enzyme for Ca2+ were similar. Sarcoplasmic reticulum vesicles used in these studies had very low levels of plasma membrane and mitochondrial contamination. The amounts of both 110-kDa Ca2+-ATPase and 55-kDa calsequestrin in the sarcoplasmic reticulum membrane were lower in fetal sarcoplasmic reticulum vesicles compared to mature membranes. Ca2+-ATPase and calsequestrin were identified in the isolated sarcoplasmic reticulum vesicles using specific antibodies produced against these membrane proteins. Age-related differences in Ca2+ transport properties of cardiac sarcoplasmic reticulum and in the amount of Ca2+-ATPase and calsequestrin may explain alterations in the regulation of intracellular Ca2+ concentrations in fetal heart muscle. This may relate to the developmental changes observed in myocardial function.
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PMID:Sarcoplasmic reticulum membrane and heart development. 244 May 34

During ATP hydrolysis the K+-translocating Kdp-ATPase from Escherichia coli forms a phosphorylated intermediate as part of the catalytic cycle. The influence of effectors (K+, Na+, Mg2+, ATP, ADP) and inhibitors (vanadate, N-ethylmaleimide, bafilomycin A1) on the phosphointermediate level and on the ATPase activity was analyzed in purified wild-type enzyme (apparent Km = 10 microM) and a KdpA mutant ATPase exhibiting a lower affinity for K+ (Km = 6 mM). Based on these data we propose a minimum reaction scheme consisting of (i) a Mg2+-dependent protein kinase, (ii) a Mg2+-dependent and K+-stimulated phosphoprotein phosphatase, and (iii) a K+-independent basal phosphoprotein phosphatase. The findings of a K+-uncoupled basal activity, inhibition by high K+ concentrations, lower ATP saturation values for the phosphorylation than for the overall ATPase reaction, and presumed reversibility of the phosphoprotein formation by excess ADP indicated similarities in fundamental principles of the reaction cycle between the Kdp-ATPase and eukaryotic E1E2-ATPases. The phosphoprotein was tentatively characterized as an acylphosphate on the basis of its alkali-lability and its sensitivity to hydroxylamine. The KdpB polypeptide was identified as the phosphorylated subunit after electrophoretic separation at pH 2.4, 4 degrees C of cytoplasmic membranes or of purified ATPase labeled with [gamma-32P]ATP.
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PMID:Characterization of the phosphorylated intermediate of the K+-translocating Kdp-ATPase from Escherichia coli. 252 40

Inhibition of platelet function by cAMP is due at least in part to a reduction in the agonist stimulated increase in cytoplasmic calcium during cell activation. This inhibition is also associated with cAMP-dependent phosphorylation of thrombolamban, a 22 kDa phosphoprotein which is present in the same membrane fraction as the calcium-dependent ATPase. Phosphorylation of this protein has been correlated with increased uptake of calcium by microsomal membranes. The present study was undertaken to examine the interaction of thrombolamban with the Ca++-ATPase in order to assess the possibility that the increased calcium uptake was by a direct effect of thrombolamban on Ca++-ATPase activity or that thrombolamban was a component of the Ca++-ATPase. Several approaches were utilized to assess the interaction of thrombolamban with the microsomal Ca++-ATPase. Gel filtration of labeled microsomes solubilized under non-denaturing conditions showed a major peak of radioactivity (Kav 0.64) corresponding to thrombolamban which was well separated from the Ca++-ATPase activity (Kav 0.09). Chemical cross-linking studies using partially purified thrombolamban and intact microsomes showed incorporation of the phosphoprotein into a 147,000 dalton complex. Indirect immunostaining with an anti-Ca++-ATPase antibody failed to demonstrate the Ca++-ATPase in the 147,000 dalton complex. Recombination of the phosphorylated thrombolamban with the Ca++-ATPase had no effect on Ca++-ATPase activity. These results indicate that, under the conditions used in these experiments, there was no apparent interaction between thrombolamban and the microsomal Ca++-ATPase. We conclude that thrombolamban is covalently bound to the Ca++-ATPase.
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PMID:cAMP-dependent protein kinase substrates in platelets. Evidence that thrombolamban, a 22,000 dalton substrate, and the Ca++-ATPase are not associated proteins. 252 71

AIF4- inhibits the (Ca2+ + Mg2+)-ATPase activity of the plasma-membrane and the sarcoplasmic-reticulum Ca2+-transport ATPase [Missiaen, Wuytack, De Smedt, Vrolix & Casteels (1988) Biochem. J. 253, 827-833]. The aim of the present work was to investigate this inhibition further. We now report that AIF4- inhibits not only the (Ca2+ + Mg2+)-ATPase activity, but also the ATP-dependent 45Ca2+ transport, and the formation of the phosphoprotein intermediate by these pumps. Mg2+ potentiated the effect of AIF4-, whereas K+ had no such effect. The plasma-membrane Ca2+-transport ATPase from erythrocytes was 20 times less sensitive to inhibition by AIF4- as compared with the Ca2+-transport ATPase from smooth muscle. The endoplasmic-reticulum Ca2+-transport ATPase from smooth muscle was inhibited to a greater extent than the sarcoplasmic-reticulum Ca2+-transport ATPase of slow and fast skeletal muscle.
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PMID:AIF4-induced inhibition of the ATPase activity, the Ca2+-transport activity and the phosphoprotein-intermediate formation of plasma-membrane and endo(sarco)plasmic-reticulum Ca2+-transport ATPases in different tissues. Evidence for a tissue-dependent functional difference. 252 47

The Ca2+-transport ATPases of smooth muscle were studied. It was concluded that smooth muscle expresses at least two different Ca2+-transport ATPases. One is present in the plasma membrane with an Mr of 140-130 kDa it is stimulated by calmodulin and it could be purified by affinity chromatography on immobilized calmodulin. This ATPase could be reconstituted in artificial membrane vesicles that were then able to catalyze an ATP-dependent Ca2+-uptake. This Ca2+-transport ATPase could also be stimulated by partial proteolysis and by negatively charged phospholipids. Polyclonal and monoclonal antibodies were found to inhibit this ATPase and concomitantly the Ca2+-transport specifically in plasma membranes and not in the endoplasmic reticulum. This plasma-membrane Ca2+ pump from smooth muscle is controlled by cGMP via phosphorylation of a phosphatidylinositol kinase which phosphorylates phosphatidylinositol to phosphatidylinositol-monophosphate for which a specific binding site exists on the Ca2+-transport ATPase. The catalytic phosphoprotein intermediate of this ATPase can be easily demonstrated and this forms a highly sensitive method to detect the presence of the ATPase in different smooth muscles and even in non-muscle sources as the kidney. A second type of Ca2+ pump with an Mr of 100 kDa is found in smooth-muscle endoplasmic reticulum. By means of its catalytic phosphointermediate this pump could be characterized as similar to the cardiac/slow muscle isoform of the sarcoplasmic reticulum Ca2+ pumps, but different from the fast skeletal-muscle isoform. Immunological studies confirmed this conclusion. This endoplasmic reticulum Ca2+ pump in smooth muscle is regulated by cAMP and cGMP via phosphorylation of phospholamban. Once Ca2+ is accumulated in the lumen of the endoplasmic reticulum, it can be bound to calsequestrin. The calsequestrin of smooth muscle appears to be a similar isoform as that found in cardiac muscle. The Ca2+-transport ATPases were found to be inhibited by fluoroaluminate complexes without the involvement of GTP-binding proteins.
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PMID:Ca2+ transport in muscle. A study of the Ca2+-transport ATPases in smooth muscle. 253 11


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