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Query: UNIPROT:P20020 (adenosine triphosphatase)
 3,299 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Developing nauplii (embryos) of the brine shrimp Artemia salina are an excellent model system for studying the biogenesis of the sodium- and potassium-activated adenosine triphosphatase (Na,K-ATPase). The nauplii exhibit a burst of Na,K-ATPase synthesis between 6 and 32 h of development (Peterson, G. L., Churchill, L., Fisher, J. A., and Hokin, L. E. (1982) J. Exp. Zool. 221, 295-308). We have now determined the sites of synthesis of the alpha and beta subunits of the Na,K-ATPase in developing A. salina nauplii. Membrane-bound and free polysomes were isolated from nauplii, and RNA was extracted from the polysomes. The polysomal RNA was translated in vitro in a rabbit reticulocyte lysate, and the translation products were immunoprecipitated by anti-subunit antisera. The immunoprecipitated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by fluorography. Our data show that the alpha subunit precursor is synthesized on membrane-bound polysomes and the beta subunit precursor is synthesized on free polysomes. In addition, the alpha subunit precursor appears as two separate peptides on sodium dodecyl sulfate-polyacrylamide gels, which suggests that the two alpha subunit forms seen in mature brine shrimp Na,K-ATPase are products of two distinct messenger RNAs. The beta subunit precursor appears as a single discrete band, unlike the mature beta subunit, which appears as a diffuse band.
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PMID:Site of synthesis of the alpha and beta subunits of the Na,K-ATPase in brine shrimp nauplii. 609 44

A procedure for the purification of Mg2+ adenosine triphosphatase (EC 3.6.1.3) from free-living and bacteroid forms of Rhizobium lupini NZP2257 is described. The enzyme was released from cell envelopes using Triton X-100 and purified by gel filtration on Ultrogel AcA 22, followed by preparative gel electrophoresis on agarose. The purified ATPase had a molecular weight of about 355,000, as determined from sedimentation coefficients on sucrose gradients. Kinetic analysis of activity of the enzyme from free-living R. lupini showed it to be typical of F1-type Mg2+ ATPases from bacteria. Mg stimulated activity at pH 7.0, although, when present as the free ion, Mg caused non-competitive inhibition (K1 = 1.5 mM). Maximum activity with ATP occurred over a broad pH range from 6.0 to 10.5. ATP, GTP, and UTP, and, to a much lesser degree, CTP and ADP, were hydrolyzed by the enzyme. Hydrolysis of glucose 6-phosphate was not observed. The Km for ATP at pH 7.0 was 0.67 and for GTP 1.4 mM. ATPase activity was inhibited by ADP, and competitive with ATP (KI = 0.18 mM). Azide also caused inhibition but fluoride and DCCD had no effect. Native and sodium dodecyl sulfate-gel electrophoretic analysis revealed no obvious differences between ATPases from free-living and bacteroid forms of R. lupini.
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PMID:Mg2+ adenosine triphosphatase from cell envelopes of free-living and bacteroid forms of Rhizobium lupini strain NZP2257. 614 93

Polyadenylated RNA prepared from neonatal rat muscle was translated in a rabbit reticulocyte cell-free system. Two sarcoplasmic reticulum proteins, the Ca2+ + Mg2+-dependent adenosine triphosphatase (ATPase) and calsequestrin, were isolated from the translation mixture by immunoprecipitation, followed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The [35S]methionine-labeled translation products were characterized by molecular weight, peptide mapping, and NH2-terminal sequence analysis. The ATPase synthesized in the cell-free system was found to have the same molecular weight (Mr = 100,000) and [35S]-methionine-labeled peptide map as the mature ATPase. The methionine residue present at the NH2 terminus of the mature ATPase was donated by initiator methionyl-tRNArMet and it became acetylated during translation. These results suggest that the ATPase was synthesized without an NH2-terminal signal sequence. Calsequestrin (Mr - 63,000) was synthesized as a higher molecular weight precursor (Mr = 66,000) that contained an additional [35S]methionine-labeled peptide when compared to mature calsequestrin. The NH2-terminal sequence of the precursor was different from the mature protein. The precursor was processed to a polypeptide with a molecular weight identical with mature calsequestrin when microsomal membranes prepared from canine pancreas were included during translation. These results show that calsequestrin is synthesized with an NH2-terminal signal sequence that is removed during translation. These data add to the evidence that the ATPase and calsequestrin follow distinctly different biosynthetic pathways, even though, ultimately, they are both located in the same membrane.
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PMID:Assembly of the sarcoplasmic reticulum. Cell-free synthesis of te Ca2+ + Mg2+-adenosine triphosphatase and calsequestrin. 616 Jan 54

Two different Mg2+-dependent adenosine 5'-triphosphate-hydrolyzing activities were detected in membranes of Vibrio costicola, a novel 5'-nucleotidase and an N,N'-dicyclohexylcarbodiimide-sensitive adenosine triphosphatase. The former and the latter had different requirements for Mg2+ and were selectively assayed in the membranes by using, respectively, 20 and 2 mM Mg2+. The two enzymes were extracted with a combination of Triton X-100 and octylglucoside, separated on a diethylaminoethyl cellulose column, and purified on glycerol gradients. The purified 5'-nucleotidase consisted of one major polypeptide of 70,000 daltons when analyzed on polyacrylamide gels in the presence of sodium dodecyl sulfate. The purified 5'-nucleotidase was similar in substrate specificities, divalent cation specificities, and pH profiles to the membrane-bound N,N'-dicyclohexylcarbodiimide-insensitive nucleotide-phosphohydrolyzing activity. The enzyme hydrolyzed nucleoside 5'-tri, 5'-di, and 5'-monophosphates at comparable rates. Inorganic pyrophosphate, p-nitrophenyl phosphate, glucose 6-phosphate, beta-glycerophosphate, adenosine 5'-diphosphate glucose, adenosine 3'-monophosphate, and cyclic adenosine 3',5'-monophosphate were not hydrolyzed, either im membranes or by the purified 5'-nucleotides. Actions of NaCl and KCl on the activity of the 5'-nucleotidase were studied. The enzyme was activated by both NaCl and KCl; the activation profiles however, were different for the membrane-bound and purified 5'-nucleotidase. The purified enzyme, unlike the membrane-bound enzyme, was markedly stimulated by high concentrations of NaCl (up to 3 M).
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PMID:Purification and properties of 5'-nucleotidase from the membrane of Vibrio costicola, a moderately halophilic bacterium. 616 62

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.
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PMID:Intestinal membrane calcium-binding protein. Vitamin D-dependent membrane component of the intestinal calcium transport mechanism. 625 88

The molecular weight and stoichiometry of the subunits of the sodium- and potassium-activated adenosine triphosphatase. (Na,K)-ATPase, have been examined in four highly purified preparations of this enzyme: dog kidney, eel electroplax, dogfish rectal gland, and brine shrimp nauplius. The molecular weights of the subunits were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 10 different total acrylamide concentrations (%T) and by a gradient SDS-PAGE system, standardized with 16 molecular weight marker proteins of known primary structure. The molecular weight of the large (Na,K)-ATPase subunit (alpha subunit) was found to be reliably estimated by SDS-PAGE, whereas the small subunit (beta subunit) was not. The molecular weight of the alpha subunit was 97,000, 97,700, 104,200, and 97,800 for the dog, eel, dogfish, and brine shrimp (Na,K)-ATPase, respectively. The molecular weight of the beta subunit is overestimated by SDS-PAGE. Mass ratio analysis of the alpha and beta subunits separated by SDS-PAGE was determined by three techniques: total amino acid quantitation, gel scanning and dye elution of Coomassie blue-stained gels, and UV gel scanning. The former method, which provides the most reliable estimate of the mass ratio, gives an alpha/beta mass ratio of 2.41, 2.33, 2.91, and 2.44 for the dog, eel, dogfish, and brine shrimp preparations, respectively. Results of the mass ratio studies, the molecular weight analysis, and prior cross-linking studies clearly demonstrate that the quaternary structure of the (Na,K)-ATPase is an alpha 2 beta 2 tetramer in all four species. Based on all of the above data, a more reliable molecular weight estimate of the beta subunit is calculated as 40,200, 43,000, 35,800, and 40,100 for the dog, eel, dogfish, and brine shrimp preparations, respectively. These results now confirm that the molecular weight of the protein portion of the alpha subunit is near 100,000 and that of the beta subunit is near 40,000 and that the (Na,K)-ATPase holoenzyme is an alpha 2 beta 2 tetramer of molecular weight 274,000 to 280,000, regardless of species.
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PMID:Molecular weight and stoichiometry of the sodium- and potassium-activated adenosine triphosphatase subunits. 626 Jul 77

A plasma membrane-enriched vesicle fraction has been prepared from Trypanosoma brucei by sonication and differential centrifugation on sucrose gradients. This fraction is enriched 5-fold in the plasma membrane marker enzymes adenyl cyclase (EC 4.6.1.1) and ouabain-inhibitable, (Na+ +K+)-dependent adenosine triphosphatase (EC 3.6.1.3). It is also enriched up to 14-fold in iodinated surface proteins, and up to 4-fold in (3H-mannose-labeled glycoproteins, of which the major variable surface coat glycoprotein is the main constituent. Proteins of the plasma membrane fraction and other subcellular fractions have been identified by electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gradient slab gels. Several high molecular weight surface glycopeptides have been selectively investigated and partially characterized by a combination of metabolic labeling with [3H]mannose, lactoperoxidase-catalyzed surface iodination, and affinity chromatography on Con A-Sepharose. In addition to the major variable surface coat glycoprotein (estimated Mr = 58000), there are several minor surface glycopeptides (Mr = 76000, 86000 and 92000-100000) which are apparent extrinsic membrane components, and two surface glycopeptides (Mr = 42000 and 130000) which are intrinsic membrane components.
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PMID:Identification and partial characterization of plasma membrane polypeptides of Trypanosoma brucei. 628 66

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.
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PMID:Isoelectric focussing of the catalytic subunit of (Na,K)-ATPase from pig kidney. 630 Jan 24

The inhibitory subunit (epsilon) of the F1 adenosine triphosphatase (ATPase) was purified to homogeneity from the ML 308-225 and K12 (lambda) strains of Escherichia coli. No tryptophan or cysteine was detected in the subunit from either strain. The highly active epsilon from both strains was found to be a globular protein with a Stokes' radius of 18--19 A. Circular dichroism spectra suggested an alpha-helix content of approximately 40%. The molecular weight of epsilon was approximately 15000--16000 by sedimentation equilibrium centrifugation in the presence and absence of guanidinium hydrochloride, molecular sieve chromatography, and gel electrophoresis in the presence of sodium dodecyl sulfate and 8 M urea. The s20,w of epsilon was approximately 1.6 s-1. Inhibition of the purified F1 ATPase by epsilon displayed noncompetitive kinetics with a Ki of approximately 10 nM. The inhibition of the ATPase was rapidly reversed by diluting the enzyme--epsilon mixture. [125I]epsilon which was incorporated into ECF1 was readily displaced by unlabeled epsilon. epsilon had no significant effect on the ATPase activity of "native" or reconstituted everted membrane vesicles under a variety of assay conditions. Combining the epsilon-inhibited F1 ATPase with its hydrophobic portion in everted membrane vesicles reconstituted the reversible proton-translocating ATPase and restored nearly full ATPase activity. These results suggest that epsilon inhibits the enzyme only when the F1 ATPase becomes detached from its hydrophobic subunits.
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PMID:Characterization of the inhibitory (epsilon) subunit of the proton-translocating adenosine triphosphatase from Escherichia coli. 644 14

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).
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PMID:Staphylococcus aureus adenosine triphosphatase: inhibitor sensitivity and release from membrane. 645 44


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