Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P20020 (adenosine triphosphatase)
 3,299 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A particulate fraction of rat intestinal mucosal homogenates, termed the "calcium-binding complex," contains three vitamin D-dependent activities: calcium binding of high affinity, calcium-dependent adenosine triphosphatase, and p-nitrophenylphosphatase. These particulate activities vary concordantly with intestinal calcium transport, suggesting that they represent membrane components of the translocation mechanism. The particulate was solubilized with 1-butanol and the activities were resolved partially by gel filtration and by DEAE-cellulose and spheroidal hydroxyl-apatite column chromatography. The Ca-binding activity was separated from the enzymes and isolated as a protein of molecular weight approximately 200,000, as estimated by gel filtration in 0.1% Triton X-100. The membrane protein, named IMCal (intestinal membrane calcium-binding protein), was dissociated with sodium dodecyl sulfate to yield a monomer of molecular weight 20,500 which is clearly distinguishable from the soluble calcium-binding protein (molecular weight 11,500) of rat mucosa. The apparent dissociation constants of Ca2+ of IMCal and of the soluble calcium-binding protein were estimated as 0.37 microM and 2.25 microM, respectively. The vitamin D-dependent activities of the calcium-binding complex are present in isolated intestinal microvillus membranes and may mediate the translocation of calcium from the intestinal lumen to the cytosol.
 ...
PMID:Intestinal membrane calcium-binding protein. Vitamin D-dependent membrane component of the intestinal calcium transport mechanism. 625 88

The catalytic subunit of sodium and potassium ion transport adenosine triphosphatase was isolated by sodium dodecyl sulfate-polyacrylamide electrophoresis and was subjected to isoelectric focussing on 3.5% acrylamide in 2% Triton X-100, 9 M urea, and 2% Bio-Lyte 3/10 from Bio-Rad Laboratories. At 20 degrees C this resolved 2 equal and closely spaced bands centered at pH 5.5 about 0.04 pH unit apart. The distribution of the polypeptide between the 2 bands came to a temperature-dependent equilibrium during focussing. At 15 degrees C predominantly the acidic band and at 25 degrees C predominantly the alkaline band appeared. Perhaps association of the nonionic detergent with the polypeptide resulted in its partitioning into bands corresponding to different physical states. A change of phase in a polypeptide-detergent complex might have altered its charge. To test functional homogeneity of the subunit in the native enzyme, the active center for ATP binding was covalently labeled with fluorescein isothiocyanate, an acidic ligand. Isoelectric focussing of the derivatized subunit at 20 degrees C showed displacement of all of the alkaline band to the position of the acidic band, which was fluorescent. Isoelectric focussing at 25 degrees C showed displacement of almost half of the alkaline band to the position of the acidic band, and both bands were fluorescent. The results suggest that all of the subunit accepted the fluorescent label and that derivatization slightly raised the temperature at which the polypeptide equilibrated between the 2 states. A few experiments on the calcium-dependent ATPase of sarcoplasmic reticulum indicated that it responded similarly.
 ...
PMID:Isoelectric focussing of the catalytic subunit of (Na,K)-ATPase from pig kidney. 630 Jan 24

Three techniques for the disruption/recovery of tegumental free-surface plasmalemma were compared by (i) morphological examination of carcasses and centrifugally-derived isolates, (ii) specific enrichment of bound surface tags (lectin) and of "marker" enzymes for membrane, and (iii) assessment of total protein and lectin recovered by each procedure. Procedures compared included the use of Triton X-100, freezing and thawing, and high ionic strength calcium. Triton X-100 consistently provided the greatest amounts of recovered surface membrane on a per worm basis, whereas calcium retained the highest amounts of alkaline p-nitrophenyl phosphatase, adenosine triphosphatase, and adenosine monophosphatase activity. Ultrastructural examination of membrane isolates and worm carcasses prepared by freezing and thawing indicated that significant amounts of parenchymal material contaminated the membrane fractions. Thus results based on the freeze-thaw technique can be difficult to interpret.
 ...
PMID:Comparison of calcium, freeze-thaw, and Triton X-100 tegumental disruption/recovery techniques applied to Schistosoma mansoni. 631 92

Homogeneous preparations of cytoplasmic membrane isolated from Staphylococcus aureus 6538P exhibited membrane-associated adenosine triphosphatase (ATPase) activity. Membrane ATPase activity was activated by divalent cations (4.0 mM: Mg2+ greater than Mn2+ greater than Co2+ greater than Zn2+), and ATP was hydrolyzed more readily than other nucleoside triphosphates and phosphorylated substrates. The pH optimum for the membrane ATPase was 6.5. The ATPase could not be released from the membrane by differential osmotic treatments, but detergent treatment effectively solubilized active enzyme. The nonionic detergent Triton X-100 (1%) released a protein with ATPase activity, after substrate-dependent staining in polyacrylamide gels, that differed slightly in electrophoretic migration when compared to the active enzyme solubilized with sodium dodecyl sulfate (0.1%). Membrane-associated ATPase activity was inhibited by N,N'-dicyclohexylcarbodiimide (0.001 to 1 mM) and NaF (50% inhibition at 5 mM NaF). Azide and trypsin inhibited activity, whereas ouabain had a slight inhibitory effect. Diethylstilbestrol showed appreciable activation of the membrane ATPase over the range employed (0.001 to 1 mM).
 ...
PMID:Staphylococcus aureus adenosine triphosphatase: inhibitor sensitivity and release from membrane. 645 44

Cytoplasmic membranes were isolated from late-exponential phase Staphylococcus aureus 6539 P and the membrane proteins examined under non-denaturing conditions by thin-layer isoelectric focusing (TLIEF) in a pH 3.5-9.5 gradient. Isolated membrane preparations retained protein integrity as judged by the demonstration of membrane bound adenosine triphosphatase (ATPase) activity in addition to four other solubilized membrane enzyme markers. Membranes were effectively solubilized with 2.5% Triton X-100 (final concentration). Examination of Triton X-100 solubilized membrane preparations established the presence of 22 membrane proteins with isoelectric points between 3.7 and 6.0. The focused proteins displayed the following enzymatic activities and isoelectric points by zymogram methods: ATPase (EC 3.6.1.3), 4.20; malate dehydrogenase (EC 1.1.1.37), 3.90; lactate dehydrogenase (EC 1.1.1.27), 3.85; two membrane proteins exhibited multiple bands upon enzymatic staining NADH dehydrogenase (EC 1.6.99.3), 4.25, 4.35; succinate dehydrogenase (EC 1.3.99.1), 4.85, 5.10, 5.35.
 ...
PMID:Analysis of Staphylococcus aureus cytoplasmic membrane proteins by isoelectric focusing. 645 26

Phosphorylation of sodium and potassium ion-activated adenosine triphosphatase (Na,K-ATPase) by protein kinase A (PKA) and protein kinase C (PKC) was investigated in vitro, where substrate conformation, kinase activity, and consequent effects on Na,K-ATPase activity could be controlled. With Na, K-ATPase from rat kidney, optimal stoichiometries were close to 1 mol 32P/mol Na,K-ATPase for both kinases. Addition of Na+, K+, P(i), or ouabain is known to stabilize the Na,K-ATPase in different states and was found to affect phosphorylation by the two kinases in a reciprocal way. This indicates that exposure of the phosphorylation sites varies with conformation and suggests a structural basis for the variable responses to kinase activation in intact cells. Further evidence for the importance of Na,K-ATPase conformation in its interaction with kinase came from the autophosphorylation of PKC, which varied in proportion to both the concentration and conformation of rat Na,K-ATPase. With pig and dog Na,K-ATPase, little phosphorylation by PKC was detected, and yet the PKC phosphorylated itself when the Na,K-ATPase was in the optimal conformation. The location of the PKA phosphorylation site was confirmed to be Ser-938 by sequence analysis of a tryptic peptide. Effects of PKA on Na,K-ATPase activity could not be measured because of inhibition by the Triton X-100 needed to obtain phosphorylation. Phosphorylation by PKC, even in optimal conditions, failed to result in inhibition of Na,K-ATPase activity. This suggests that any physiological role of phosphorylation either entails a subtle modulation of enzyme properties, or requires additional regulatory proteins.
 ...
PMID:Conformation-dependent phosphorylation of Na,K-ATPase by protein kinase A and protein kinase C. 798 58

Glycoprotein IV of bovine adrenal chromaffin granule membranes was purified by membrane fractionation with Triton X-114 and lectin affinity chromatography. An antiserum raised against this protein recognized the same component as one directed against subunit Ac45 of the proton-translocating adenosine triphosphatase in the granule membrane. Amino acid sequencing confirmed that glycoprotein IV and Ac45 are identical proteins, and also showed that they are derived from a larger precursor by removal of a 246-amino acid N-terminal sequence. Enzymatic deglycosylation indicated an apparent polypeptide molecular mass of 29 kDa for the mature Ac45/glycoprotein IV. Blue Native electrophoresis confirmed that this protein is a component of the membrane sector of the V-ATPase.
 ...
PMID:Chromaffin granule membrane glycoprotein IV is identical with Ac45, a membrane-integral subunit of the granule's H(+)-ATPase. 896 Dec 92

Enzyme activity that represents ouabain-insensitive, potassium-dependent p-nitrophenylphosphatase (p-NPPase) was assessed in rat atrial myocytes by biochemical and cytochemical procedures. No activity was detected in parallel experiments with ventricular myocytes. Fixed tissues were incubated in a reaction medium containing Tricine buffer, p-nitrophenylphosphate (p-NPP), KCl, MgCl2, CaCl2, CeCl3. Triton X-100, levamisole, and ouabain. Final pH was adjusted to 7.5. Biochemical studies showed that accumulation of p-nitrophenol in the medium was increased proportionally in accordance with the amount of incubated tissue. This activity was optimal with incubation at pH 7.5 and in the presence of KCl. Approximately 70% of the enzyme was inhibited by 2 mM CeCl3. Electron microscopic observations revealed reaction product (RP) at sites of ouabain-insensitive, potassium-dependent p-NPPase activity as electron-dense precipitate localized at the inner surface of the plasma membrane and at the T-tubules of atrial myocytes. Control experiments indicated that the activity was strongly inhibited by sodium orthovanadate and was repressed by omeprazole and 1,3-dicyclohexylcarbodiimide. X-ray microanalysis confirmed the presence of cerium within the cytochemical RP. The ouabain-insensitive, K-dependent p-NPPase activity detected in the present study is considered to be an isoform of a P-type, H-transporting, K-dependent adenosine triphosphatase (H,K-ATPase).
 ...
PMID:Biochemical properties and cytochemical localization of ouabain-insensitive, potassium-dependent p-nitrophenylphosphatase activity in rat atrial myocytes. 901 8

Membranes prepared from various members of the genus Halobacterium contained a Triton X-100 activated adenosine triphosphatase. The enzyme from Halobacterium saccharovorum was unstable in solutions of low ionic strength (< 3 M NaCl) and maximally active in the presence of 3.5 M NaCl. A variety of nucleotide triphosphates was hydrolyzed. MgADP, the product of ATP hydrolysis, was not hydrolyzed and was a competitive inhibitor with respect to MgATP. The enzyme from H. saccharovorum was composed of at least 2 and possibly 4 subunits. The 83-kDa and 60-kDa subunits represented about 90% of total protein. The 60-kDa subunit reacted with dicyclohexylcarbodiimide (DCCD) when inhibition was carried out in an acidic medium. The significance of the two minor components (28 kDa and 12 kDa is not established. The enzyme from H. saccharovorum, which differs from previously described halobacterial ATPases, possesses properties of an F1F0 as well as an E1E2 ATPase.
 ...
PMID:Halobacterial adenosine triphosphatases and the adenosine triphosphatase from Halobacterium saccharovorum. 1154 91

Experiments were conducted to determine conditions essential for electrophoretic characterization of a detergent-extracted plasma membrane fraction from corn (Zea mays L.) roots. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) initially gave poor resolution of polypeptides in the plasma membrane fraction and, upon detergent treatment for purification of the proton-pumping adenosine triphosphatase (ATPase), showed no enrichment for a 100 kilodalton catalytic subunit characteristic of the ATPase. In contrast to SDS-PAGE, phenol urea acetic acid (PAU)-PAGE clearly resolved two polypeptides in the 100 kilodalton region that were enriched during detergent treatment and indicated at least one polypeptide forms a phosphorylated intermediate characteristic of the ATPase. Problems with SDS-PAGE were found to be caused, in part, by a combination of endogenous proteases and heat-induced aggregation of high molecular weight proteins. The usually standard procedure of boiling the sample prior to SDS-PAGE caused the aggregation of the 100 kilodalton polypeptides. By controlling for proteases using chymostatin and/or phenylmethane sulfonyl floride, and not boiling the sample prior to electrophoresis, two polypeptides were clearly resolved by SDS-PAGE in the 100 kilodalton region of Triton X-114-extracted membranes from corn, oat, barley, and tomato.
 ...
PMID:Electrophoretic characterization of a detergent-treated plasma membrane fraction from corn roots. 1666 34


<< Previous 1 2 3 4