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)

Energy coupling between ATP hydrolysis and other enzyme reactions requires the phosphorylation of substrate-derived intermediates, or the existence of enzyme-derived intermediates capable of storage and transfer of energy. Salmonella typhimurium nicotinic acid phosphoribosyltransferase (NAPRTase, EC 2.4.2.11) couples net ATP hydrolysis to formation of NAMN and PPi from alpha-PRPP and nicotinic acid [Vinitsky, A., & Grubmeyer, C (1993) J. Biol. Chem. 268, 26004-26010]. In the current work, we have determined that the enzyme reacts with ATP to produce a covalently phosphorylated form of the enzyme (E-P), which is common to both the ATPase and NAMN synthesis functions of NAPRTase. We have isolated E-P and verified its catalytic competence. E-P showed acid lability and base stability, diagnostic of a phosphoramidate linkage. Pyridine and hydroxylamine-catalyzed hydrolysis of E-P gave second-order rate constants consistent with published values for phosphohistidine. Two-dimensional thin-layer chromatography of alkaline-hydrolyzed E-32P showed that the phosphorylated residue co-migrated with authentic 1-phosphohistidine. Chymotrypsin and trypsin proteolysis followed by HPLC and peptide sequencing localized the phosphopeptide to Ala-210 to Phe-222 of the 399-residue protein. This peptide contains a single histidine residue, His-219. NAPRTase phosphorylated at His-219 is an intermediate in the energy transduction mechanism of NAPRTase.
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PMID:Energy coupling in Salmonella typhimurium nicotinic acid phosphoribosyltransferase: identification of His-219 as site of phosphorylation. 867 22

4-Hydroxynonenal binds rapidly to Na(+)-K(+)-ATPase, and this was accompanied by a decrease in measurable sulfhydryl groups and a loss of enzyme activity. The I50 value for Na(+)-K(+)-ATPase inhibition by 4-hydroxynonenal was found to be 120 microM. Although the sulfhydryl groups could be completely restored with beta-mercaptoethanol during the reaction of the Na(+)-K(+)-ATPase-HNE-adduct, the Na(+)-K(+)-ATPase activity was only partially restored by this reducing agent. A combination of hydroxylamine and beta-mercaptoethanol yielded the greatest recovery of enzyme activity, 85% of original. Thus, 4-hydroxynonenal binding to Na(+)-K(+)-ATPase led to an irreversible decrease of enzyme activity under the conditions employed. It is hypothesized that 4-hydroxynonenal reacts with sulfhydryls at sites on the enzyme that are inaccessible by beta-mercaptoethanol. Furthermore, evidence was obtained that 4-hydroxynonenal reacts with other amino acids such as lysine to form adducts that also interfere with protein function.
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PMID:4-hydroxynonenal inhibits Na(+)-K(+)-ATPase. 874 42

Monoclonal antibodies to isoforms of the Na,K-ATPase have become important tools in the study of the enzyme's distribution, physiological roles, and gene regulation, and when their epitopes are defined, they are useful in the study of enzyme structure as well. Evidence is presented that the alpha3-specific antibody McBX3 recognizes an unusual epitope that is not present on alpha3 in the heart. The epitope, which is also found in kidney alpha1 from some species, was mapped to a site on the large intracellular loop near the ATP binding site. DNA sequencing of reverse transcribed-PCR products encompassing the corresponding regions from alpha3 from brain (where McBX3 recognizes alpha3) and heart demonstrated that the tissue difference in epitope is not due to alternative splicing of the mRNA. Instead, hydroxylamine sensitivity indicated that the antibody recognizes a post-translational modification. The epitope for a new antibody for alpha3, XVIF9-G10, was mapped to a site near the N terminus, a location analogous to the sites for the well-characterized antibodies McK1 (alpha1) and McB2 (alpha2). The antibody XVIF9-G10 reacted with the alpha3 of the heart as well as that of the brain; however, McBX3 and XVIF9-G10 both stained the same cellular structures in sections of the rat retina. A new alpha1-specific antibody, 6F, was characterized and mapped to another site near the N terminus; this antibody has broader species specificity than the other well-characterized alpha1 antibody, McK1.
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PMID:Isoform-specific monoclonal antibodies to Na,K-ATPase alpha subunits. Evidence for a tissue-specific post-translational modification of the alpha subunit. 879 46

The role of six negatively charged residues located in or around the fifth and sixth transmembrane domain of the catalytic subunit of gastric H+,K+-ATPase, which are conserved in P-type ATPases, was investigated by site-directed mutagenesis of each of these residues. The acid residues were converted into their corresponding acid amides. Sf9 cells were used as the expression system using a baculovirus with coding sequences for the alpha- and beta-subunits of H+,K+-ATPase behind two different promoters. Both subunits of all mutants were expressed like the wild type enzyme in intracellular membranes of Sf9 cells as indicated by Western blotting experiments, an enzyme-linked immunosorbent assay, and confocal laser scan microscopy studies. The mutants D824N, E834Q, E837Q, and D839N showed no 3-(cyanomethyl)-2-methyl-8(phenylmethoxy)-imidazo[1, 2a]pyridine (SCH 28080)-sensitive ATP dependent phosphorylation capacity. Mutants E795Q and E820Q formed a phosphorylated intermediate, which, like the wild type enzyme, was hydroxylamine-sensitive, indicating that an acylphosphate was formed. Formation of the phosphorylated intermediate from the E795Q mutant was similarly inhibited by K+ (I50 = 0.4 mM) and SCH 28080 (I50 = 10 nM) as the wild type enzyme, when the membranes were preincubated with these ligands before phosphorylation. The dephosphorylation reaction was K+-sensitive, whereas ADP had hardly any effect. Formation of the phosphorylated intermediate of mutant E820Q was much less sensitive toward K+ (I50 = 4.5 mM) and SCH 28080 (I50 = 1.7 microM) than the wild type enzyme. The dephosphorylation reaction of this intermediate was not stimulated by either K+ or ADP. In contrast to the wild type enzyme and mutant E795Q, mutant E820Q did not show any K+-stimulated ATPase activity. These findings indicate that residue Glu820 might be involved in K+ binding and transition to the E2 form of gastric H+,K+-ATPase.
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PMID:Role of negatively charged residues in the fifth and sixth transmembrane domains of the catalytic subunit of gastric H+,K+-ATPase. 893 13

The energy-rich compound acetyl phosphate (ACP) was examined as a substrate for energy-linked reactions by the yeast plasma membrane H+-ATPase. The hydrolysis of ACP was sensitive to inhibition by vanadate with an IC50 approximately 1 microM, which is comparable to the level obtained in the presence of ATP. A Km of 8.29 +/- 0.65 mM for the hydrolysis of ACP was approximately 10-fold higher than that obtained for ATP, while Vmax values of 8.66 +/- 0.29 and 7.23 +/- 0.34 micromol Pi mg(-1) min(-1) were obtained with ATP and ACP, respectively. ACP formed a phosphorylated intermediate that was efficiently chased with hydroxylamine. Both ACP and ATP effectively protected the enzyme from trypsin-induced inactivation and formed identical tryptic digestion patterns, suggesting that ACP mimics the formation of conformational intermediates induced by ATP. However, unlike ATP, ACP was unable to drive proton transport by H+-ATPase. In addition, a pma1-S368F mutant enzyme that is highly insensitive to inhibition by vanadate in the presence of ATP was largely sensitive to vanadate in the presence of ACP. These results are interpreted in terms of a reverse, short-circuit pathway of the normal P-type ATPase kinetic pathway, in which the formation of E2P by-passes the E1P high-energy intermediate. In this pathway, ACP favors the formation of an E2P conformational state, which can interact with classical inhibitors like vanadate, but possesses insufficient free energy to drive proton transport by the H+-ATPase.
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PMID:Probing energy coupling in the yeast plasma membrane H+-ATPase with acetyl phosphate. 926 44

The position in the acyl phosphate linkage of the phosphorylated intermediate of (Na+, K+)-ATPase that is cleaved by N-methylhydroxylamine was compared with that of the model compound acetylphosphate. The products of the cleavage of the phosphoenzyme by methylhydroxylamine were the active enzyme and a N-P compound, not the inhibited enzyme and inorganic phosphate. This means that the bond cleaved by methylhydroxylamine was the O-P bond, not the C-O bond. In contrast, methylhydroxylamine did not cleave the O-P bond of acetylphosphate in solution, at pH values from 0.3 to 7.0, whether or not the phosphoryl group formed a complex with magnesium. Acetylphosphate and hydroxylamine formed acetohydroxamic acid. Therefore, the state of the acyl phosphate bond in the native phosphoenzyme and in acetylphosphate in solution was different, and the difference was not due to different dissociation states of their phosphoryl groups or the binding of magnesium to the phosphoenzyme. Molecular orbital calculations for acetylphosphate revealed that the phosphorus atom charge is more positive than the carbon atom, irrespective of the dissociation state of the phosphoryl group. Similarly, the overlapping electron population of the O-P bond is always smaller than that of the C-O bond. Thus, the electronic structure of the acyl phosphate linkage of acetylphosphate under vacuum supports the results obtained with the native phosphoenzyme, rather than those obtained with acetylphosphate in solution. The linkage in the active site of the phosphorylated intermediate of (Na+,K+)-ATPase appeared to be equivalent to the non-hydrated state of the model compound acetylphosphate. The phosphoenzyme with bound ouabain, or without a tightly bound divalent cation was insensitive to methylhydroxylamine. The native phosphoenzyme of (Ca2+)-ATPase was not susceptible to methylhydroxylamine.
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PMID:Non-hydrated state of the acyl phosphate group in the phosphorylated intermediate of (Na+,K+)-ATPase. 934

Effect of various inhibitors on the (NH4+ + Na+)-activated ATPase of an anaerobic alkaliphile, Ep01(a strain of Amphibacillus xylanus), was examined. Among the chemicals tested, the enzyme was drastically inactivated by p-chloromercuribenzoic acid and diethyl pyrocarbonate. The ATPase activity of the enzyme, which was inactivated by p-chloromercuribenzoic acid and diethyl pyrocarbonate, was remarkably restored by beta-mercaptoethanol and hydroxylamine, respectively, suggesting the involvement of cysteine and histidine residues in the enzyme activity. Analysis of the inhibition kinetics by diethyl pyrocarbonate indicated that modification of a single histidine residue per ATPase molecule was sufficient to inactivate the enzyme.
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PMID:Possible involvement of cysteine and histidine residues in the (NH4+ + Na+)-activated ATPase of an anaerobic alkaliphile, Amphibacillus xylanus. 940 39

Plasma membranes isolated from the marine unicellular alga Tetraselmis (Platymonas) viridis were phosphorylated by [gamma-32P]ATP, and membrane proteins were then analyzed by PAGE in SDS, under acidic conditions. Three radioactive components with apparent molecular masses of 100 kDa, 76 kDa, and 26 kDa were detected. The phosphorylation of one of them, the 100 kDa polypeptide, was specifically stimulated by Na+. Vanadate almost completely inhibited the Na+-mediated phosphorylation of the peptide. The phosphate bound to this peptide underwent rapid turnover and was discharged by hydroxylamine. The 100 kDa phosphopeptide was sensitive to ADP. The conclusion is drawn that the 100 kDa phosphopeptide is a phosphorylated intermediate of the Na+-transporting ATPase in the T. viridis plasma membrane.
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PMID:Na+-ATPase from the plasma membrane of the marine alga Tetraselmis (Platymonas) viridis forms a phosphorylated intermediate. 959 99

The properties of Na,K-ATPase phosphoenzymes formed either from ATP in the presence of Mg2+ and Na+ or from Pi in the absence of alkali cations were investigated by biochemical methods and spectrofluorometry employing the styryl dye RH421. We characterized the phosphoenzyme species by their reaction to N-methyl hydroxylamine, which attacks specifically the protein-phosphate bond. We studied reactions of the phospho- and dephospho-enzymes with vanadate, which is a transition-state analogue of phosphate in this enzyme. On the basis of substantial differences in the properties of the phosphoenzyme species formed either from ATP or Pi, especially in their reactivity to N-methyl hydroxylamine, it is suggested that the two phosphoenzyme species are two subconformations of the E2P phosphoform. Analysis of the RH421 fluorescence responses under a variety of experimental conditions and comparing different enzyme sources suggested that the increase of RH421 fluorescence induced by inorganic phosphate in the absence of alkali cations is associated with the formation of the covalent acyl-phosphate bond.
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PMID:E2P phosphoforms of Na,K-ATPase. I. Comparison of phosphointermediates formed from ATP and Pi by their reactivity toward hydroxylamine and vanadate. 975 50

We purified Cl- pump in the rat brain and obtained 520 or 580 kDa protein complexes which consisted of 62, 60, 55 and 51 kDa proteins. An antiserum against 520 kDa protein complex recognized 51 kDa protein in both 520 and 580 kDa complexes, and reduced both Cl(-)-ATPase and Cl(-) pump activities. Such an immunoreactive 51 kDa protein was found in the brain, spinal cord and kidney. When incubated with [gamma-(32)P]ATP, the protein complex yielded phosphorylated 51 kDa protein, the label being hydroxylamine-sensitive and increased in the presence of Cl- and/or an inhibitor of Cl- pump, ethacrynic acid. Thus, the antibody appears to recognize a possible catalytic subunit of Cl- pump, 51 kDa protein, in the rat.
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PMID:Antiserum against Cl- pump complex recognizes 51 kDa protein, a possible catalytic unit in the rat brain. 987 33


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