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 cDNA for a rat vesicular monoamine transporter, designated MAT, was isolated by expression cloning in a mammalian cell line (CV-1). The cDNA sequence predicts a protein of 515 amino acids with 12 putative membrane-spanning domains. The characteristics of [3H]serotonin accumulation by CV-1 cells expressing the cDNA clone suggested sequestration by an intracellular compartment. In cells permeabilized with digitonin, uptake was ATP dependent with an apparent Km of 1.3 microM. Uptake was abolished by the proton-translocating ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone and with tri-(n-butyl)tin, an inhibitor of the vacuolar H(+)-ATPase. The rank order of potency to inhibit uptake was reserpine > tetrabenazine > serotonin > dopamine > norepinephrine > epinephrine. Direct comparison of [3H]monoamine uptake indicated that serotonin was the preferred substrate. Photolabeling of membranes prepared from CV-1 cells expressing MAT with 7-azido-8-[125I]iodoketanserin revealed a predominant tetrabenazine-sensitive photolabeled glycoprotein with an apparent molecular mass of approximately 75 kDa. The mRNA that encodes MAT was present specifically in monoamine-containing cells of the locus coeruleus, substantia nigra, and raphe nucleus of rat brain, each of which expresses a unique plasma membrane reuptake transporter. The MAT cDNA clone defines a vesicular monoamine transporter representing a distinct class of neurotransmitter transport molecules.
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PMID:Expression cloning of a reserpine-sensitive vesicular monoamine transporter. 143 4

After treatment of human platelets by a sulfhydryl-dependent bacterial protein cytolysin, a glycoprotein was reproducibly purified by a one-step affinity chromatography using 6-fluorotryptamine as ligand and elution by serotonin (5-HT), cyanoimipramine, citalopram, or a Na(+)-free buffer. The purified fraction migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 68 kDa. The purified glycoprotein bound the 5-HT uptake blockers 3H-paroxetine, 3H-cyanoimipramine, and 3H-citalopram with Kds similar to the ones observed for intact human platelets. No binding was detected with 3H-hydroxytetrabenazine, 3H-ouabain, 3H-gamma aminobutyric acid or 3H-BTCP, the respective markers of the granular monoamine transporter, the plasma membrane Na+, K(+)-ATPase, the gamma aminobutyric acid and dopamine carriers. The purified 68-kDa glycoprotein is therefore likely to correspond at least to the paroxetine and imipramine binding domains of the 5-HT transporter located at the human platelet plasma membrane. Finally a 68-kDa protein was purified in the same conditions from the human megakaryocytic cell line Dami and to a lesser extent from the human megakaryoblastic cell line MEG-01 but not from the human erythroleukaemic cell line HEL.
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PMID:[Platelet plasma membrane: characterization of the serotonin transporter]. 149 76

Rat seminal vesicles and the lateral prostate secrete a glycoprotein designated as SVS II in an androgen-dependent manner. SVS II, which has a M(r) of 49,000 and a pI of 10.5, is an actin-binding protein. G- and F-actins cosediment with SVS II at a ratio of 2:1 (actin:SVS II). SVS II affects the kinetics of actin polymerization in the same way as do barbed end capping proteins. Interaction with actin is specific for the skeletal and cardiac muscle isoforms and there is no corresponding interaction with cytoplasmic actins. The binding site is close to the C-terminus of actin. Monospecific polyclonal antibodies directed against the N-terminus of actin cross-react with SVS II, but there is no cross-reaction by a monoclonal antibody directed against a C-terminal epitope on actin. Recent sequence analysis of SVS II shows a sequence of about 14 residues that is repeated 13 times between residues 86 and 298. The consensus sequence based on these repeats is homologous to residues 10 to 25 of actin; this may account for the immunological cross-reactivity. Like actin, SVS II binds and inhibits the activity of DNase I, but SVS II has no effect on the ATPase activity of myosin subfragment 1. Thus, SVS II is an actin-binding protein which retains some properties of actin itself.
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PMID:SVS II--an androgen-dependent actin-binding glycoprotein in rat semen. 151 6

A 68-kDa glycoprotein bearing the biological activity of the plasma membrane serotonin (5-hydroxytryptamine, 5-HT) transporter has been purified from human blood platelets, a classical cell model for the study of 5-HT uptake. After treatment of the whole platelet population or its plasma membrane fraction by sulfhydryl-dependent bacterial protein toxins or by digitonin, purification was reproducibly obtained by a one-step affinity chromatography using two different columns with 5-HT or 6-fluorotryptamine as ligands and elution by 5-HT or Na(+)-free buffer. The purified fraction migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 68 kDa and exhibited an apparent isoelectric point of 5.6-6.2. Two sialic acid residues were detected in the purified material. The purified glycoprotein bound the 5-HT uptake blocker [3H]paroxetine with a Kd (0.25 nM) similar to the one observed for intact human platelets. It also bound [3H] 5-HT but neither [3H]hydroxytetrabenazine nor [3H] ouabain, the respective markers of the granular monoamine transporter and of the Na+,K(+)-ATPase associated to the plasma membrane 5-HT transporter. 5-HT derivatives and 5-HT uptake inhibitors exhibited similar Ki values for 5-HT uptake and paroxetine binding in intact human platelets and in the purified glycoprotein. Under laser UV irradiation, 40% of this purified glycoprotein could be labeled by either [3H]paroxetine or [3H]cyanoimipramine. No labeling was detected with either [3H] gamma-aminobutyric acid or [3H]GBR 12783, the respective markers of gamma-aminobutyric acid and dopamine carriers. The purified 68-kDa protein is therefore likely to correspond at least to the binding domain of the 5-HT transporter located at the human platelet plasma membrane.
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PMID:One-step purification of the serotonin transporter located at the human platelet plasma membrane. 153 59

Histamine stimulation of gastric acid secretion has for a long time been known to be mediated by an H2-type receptor located on the parietal cell surface, but the biochemical nature of this receptor has only very recently been elucidated. It is a 70-kDa glycoprotein showing structural analogies with the beta 2-adrenergic receptor and the other seven membrane-spanning domains/G protein-coupled receptors. It activates adenylated cyclase through a cholera toxin-sensitive, pertussis toxin-insensitive, guanosine 5'-triphosphatase-binding regulatory Gs protein. The cAMP thereby produced is believed to play a crucial role in the opening of the Cl- channel associated with the (H+,K+)-ATPase in the secretory membrane. However, other sites of action are likely to be involved, since several histamine- or cAMP-dependent phosphoproteins have been detected in the parietal cell. In addition to its action on cAMP production, histamine was found to produce a transient increase in the intracellular Ca2+ concentration, but this effect remains unexplained. On the other hand, the intervention of an H3-type histamine receptor in the regulation of gastric acid secretion has recently been documented, but the cellular location of this new receptor has not been yet investigated.
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PMID:Receptors regulating acid secretion. 164 88

Murine autoimmune gastritis, induced by neonatal thymectomy, bears a striking similarity in pathology to the human autoimmune disease, pernicious anemia. Autoantibodies to parietal cells are found in both murine and human diseases. Monoclonal immunoglobulin G autoantibodies, obtained from neonatally thymectomized mice, have previously been shown to recognize two groups of gastric parietal cell antigens. In the present study, it is shown that two of these monoclonal autoantibodies, designated 1H9 and 2B6, are directed against the alpha subunit and beta subunit, respectively, of the gastric hydrogen-potassium-stimulated adenosine triphosphatase (H+,K(+)-ATPase; proton pump). Monoclonal antibody 1H9 showed reactivity by immunoblotting with a 95-kilodalton component of dog gastric tubulovesicular membranes and with a fusion protein containing the hydrophilic domain of the alpha subunit of the H+,K(+)-ATPase. Monoclonal antibody 2B6 reacted by immunoblotting with the 60-90-kilodalton glycoprotein (beta subunit) of the tomato lectin-purified dog H+,K(+)-ATPase and with the 60-90-kilodalton autoantigen purified with human parietal cell autoantibodies. Monoclonal antibody 2B6 also reacted with the deglycosylated 35-kilodalton core protein of the tomato lectin-purified 60-90-kilodalton beta subunit and of the purified 60-90-kilodalton autoantigen. Parietal cell autoantibody-positive sera from 20 mice with experimentally induced gastritis showed reactivity predominantly with the alpha and/or beta subunit of the gastric H+,K(+)-ATPase. Therefore, it is concluded that the major molecules targeted by parietal cell autoantibodies from mice with neonatal thymectomy-induced murine autoimmune gastritis and from humans with pernicious anemia are identical.
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PMID:The parietal cell autoantigens recognized in neonatal thymectomy-induced murine gastritis are the alpha and beta subunits of the gastric proton pump [corrected]. 164 25

The Mg(2+)-ATPase present in rabbit skeletal-muscle transverse tubules is an integral membrane enzyme which has been solubilized and purified previously in this laboratory [Kirley (1988) J. Biol. Chem. 263, 12682-12689]. The present study indicates that, in addition to the approx. 100 kDa protein (distinct from the sarcoplasmic-reticulum Ca(2+)-ATPase) seen previously to co-purify with the Mg(2+)-ATPase activity, there are also proteins having molecular masses of 160, 70 and 43 kDa. The 70 and 43 kDa glycosylated proteins (50 and 31 kDa after deglycosylation) are difficult to detect by SDS/PAGE before deglycosylation, owing to the broadness of the bands. Additional purification procedures, cross-linking studies and chemical and enzymic deglycosylation studies were undertaken to determine the structure and relationship of these proteins. Both the 97 and 160 kDa proteins were demonstrated to be N-glycosylated at multiple sites, the 97 kDa protein being reduced to a peptide core of 84 kDa and the 160 kDa protein to a peptide core of 131 kDa after deglycosylation. Although the Mg(2+)-ATPase activity is resistant to a number of chemical modification reagents, cross-linking inactivates the enzyme at low concentrations. This inactivation is accompanied by cross-linking of two 97 kDa molecules to one another, suggesting that the 97 kDa protein is involved in ATP hydrolysis. The existence of several proteins along with the inhibition of ATPase activity by cross-linking is consistent with the interpretation of the susceptibility of this enzyme to inactivation by most detergents as being due to the disruption of a protein complex of associated subunits by the inactivating detergents. The 160 kDa glycoprotein can be partially resolved from the Mg(2+)-ATPase activity, and is identified by its N-terminal amino acid sequence as angiotensin-converting enzyme.
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PMID:The Mg(2+)-ATPase of rabbit skeletal-muscle transverse tubule is a highly glycosylated multiple-subunit enzyme. 165 80

We have demonstrated for the first time the isolation of sarcoplasmic reticulum (SR) membranes from adult rat ventricular myocytes obtained from a single rat heart. The myocyte SR preparation exhibits similar Ca(2+)-transport and Ca2+/K(+)-ATPase activity as well as a similar protein profile to SR membranes isolated from intact rat heart tissue. This SR preparation exhibited a Ca2+/K(+)-ATPase activity of 371 +/- 55 nmol/min/mg protein (mean +/- S.E.M.; n = 5) and an oxalate-stimulated Ca(2+)-uptake activity of 103 +/- 4 nmol/min/mg protein (mean +/- S.E.M.; n = 6). Pretreatment of the SR vesicles with 5 microM ruthenium red increased the oxalate-stimulated Ca(2+)-uptake to 204 +/- 12 nmol/min/mg protein demonstrating the presence of junctional SR membranes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis shows that the isolated SR membranes contained protein bands at 430 (Ca(2+)-release channel), 100 (Ca2+/K(+)-ATPase), 55 (calsequestrin and/or calreticulin) and 53 kDa (glycoprotein). Western blots of myocyte SR membranes stained with ruthenium red detected 2 major Ca(2+)-binding protein bands in this preparation at 53-55 kDa (calsequestrin and/or calreticulin) and 97-100 kDa (Ca2+/K(+)-ATPase). The presence of phospholamban, a regulatory protein of the Ca2+/K(+)-ATPase of cardiac SR, was confirmed in the myocyte SR membranes by western blots probed with a monoclonal antibody to phospholamban. Isoproterenol stimulation of intact [32P]orthophosphate equilibriated myocytes was associated with an increase in the phosphorylation of 3 distinct proteins (27, 31 and 152 kDa) in myocyte homogenates. The 27 kDa phosphorylated protein was identified in purified SR membranes as phospholamban my migration on electrophoretic gels and by immunoblotting. The ability to prepare SR membranes from intact isolated adult rat ventricular myocytes makes this system a potentially useful model for the study of SR regulation by protein phosphorylation.
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PMID:Isolation and characterization of purified sarcoplasmic reticulum membranes from isolated adult rat ventricular myocytes. 166 Sep 35

The chronic stimulation of predominantly fast-twitch mammalian skeletal muscle causes a transformation to physiological characteristics of slow-twitch skeletal muscle. Here, we report the effects of chronic stimulation on the protein components of the sarcoplasmic reticulum and transverse tubular membranes which are directly involved in excitation-contraction coupling. Comparison of protein composition of microsomal fractions from control and chronically stimulated muscle was performed by immunoblot analysis and also by staining with Coomassie blue or the cationic carbocyanine dye Stains-all. Consistent with previous experiments, a greatly reduced density was observed for the fast-twitch isozyme of Ca(2+)-ATPase, while the expression of the slow-twitch Ca(2+)-ATPase was found to be greatly enhanced. Components of the sarcolemma (Na+/K(+)-ATPase, dystrophin-glycoprotein complex) and the free sarcoplasmic reticulum (Ca(2+)-binding protein sarcalumenin and a 53-kDa glycoprotein) were not affected by chronic stimulation. The relative abundance of calsequestrin was slightly reduced in transformed skeletal muscle. However, the expression of the ryanodine receptor/Ca(Ca2+)-release channel from junctional sarcoplasmic reticulum and the transverse tubular dihydropyridine-sensitive Ca2+ channel, as well as two junctional sarcoplasmic reticulum proteins of 90 kDa and 94 kDa, was greatly suppressed in transformed muscle. Thus, the expression of the major protein components of the triad junction involved in excitation-contraction coupling is suppressed, while the expression of other muscle membrane proteins is not affected in chronically stimulated muscle.
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PMID:Analysis of excitation-contraction-coupling components in chronically stimulated canine skeletal muscle. 166 14

Detergent-solubilization of hog gastric microsomal membrane proteins followed by affinity chromatography using wheat germ agglutinin or Ricinus communis I agglutinin resulted in the isolation of five glycoproteins with the apparent molecular masses on sodium dodecyl sulfate polyacrylamide gels of (in kDa): 60-80 (two glycoproteins sharing this molecular mass); 125-150; and 190-210. In the nonionic detergent Nonidet P-40 (NP-40), the 94 kDa H+/K(+)-ATPase was recovered exclusively in the lectin-binding fraction; however, in the cationic detergent dodecyltrimethylammonium bromide, most of the ATPase was recovered in the nonbinding fraction. Detection of glycoproteins either by periodic acid-dansyl hydrazine staining of carbohydrate in polyacrylamide gels or by Western blots probed with lectins indicated that the majority of the ATPase molecules are not glycosylated. In addition, in the absence of microsomal glycoproteins, the NP-40-solubilized ATPase does not bind to a lectin column. Taken together, these results suggest that the recovery of NP-40-solubilized ATPase in the lectin-binding fraction is due to its noncovalent interaction with a gastric microsomal glycoprotein. Immunoprecipitation of the ATPase from NP-40-solubilized microsomal membrane proteins resulted in the co-precipitation of a single 60-80 kDa glycoprotein. Characterization of the 60-80 kDa glycoprotein associated with the ATPase revealed that: it is a transmembrane protein; it has an apparent core molecular mass of 32 kDa; and, it has five asparagine-linked oligosaccharide chains. Given its similarity to the glycosylated beta-subunit of the Na+/K(+)-ATPase, this 60-80 kDa gastric microsomal glycoprotein is suggested to be a beta-subunit of the H+/K(+)-ATPase.
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PMID:Isolation and characterization of gastric microsomal glycoproteins. Evidence for a glycosylated beta-subunit of the H+/K(+)-ATPase. 169 26


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