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)

Trypsin treatment of N-[p-(2-benzimidazolyl)phenyl]maleimide modified enzyme caused a marked reduction in Na+,K+-ATPase activity and in the amount of the alpha-chain, which contains the phosphorylation and ouabain binding sites. However, these preparations retained nearly 90% of the ouabain binding capacity and showed ouabain sensitive dynamic fluorescence changes accompanying the hydrolysis of ATP. The data showed that the three dimensional structure of Na+,K+-ATPase, which is important in the dynamic fluorescence change, is little affected in spite of extensive covalent bond splitting in the alpha-chain of Na+,K+-ATPase.
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PMID:Effect of peptide bond splitting on ouabain sensitive conformational changes in Na+,K+-ATPase treated with N-[p-(2-benzimidazolyl)phenyl]maleimide. 303 63

Since Na+,K+-ATPase (EC 3.6.1.3) of pig kidney modified with a fluorescent sulfhydryl reagent, N-[p-(2-benzimidazolyl) phenyl]maleimide, at Cys-964 of the alpha-chain showed ATP-dependent, reversible, and dynamic fluorescence changes (Nagai, M., Taniguchi, K., Kangawa, K., Matsuo, S., Nakamura, S., and Iida, S. (1986) J. Biol. Chem. 261, 13197-13202), we studied the conformational change during Na+,K+-ATPase reaction using the modified enzyme. The addition of K+ to the enzyme increased the fluorescence intensity to 2% in the presence of 160 mM Na+ and 3 mM Mg2+ (K0.5 = 16.4 mM). Addition of low concentrations of ATP immediately increased the intensity to 3.2% (K0.5 less than 0.1 microM) to accumulate fully K+-bound enzyme in the presence of 43 mM K+ with Na+ and Mg2+, but further addition of higher concentrations of ATP diminished the increase (K0.5 = 120 microM). After exhaustion of ATP, the fluorescence intensity decreased to -0.4% (K0.5 = 0.3 microM) and -2% (K0.5 = 20 microM), respectively, in the presence of low and high concentrations of ADP produced from ATP. High concentrations of ATP accelerated Na+,K+-ATPase activity with a simultaneous increase in the amount of ADP-sensitive phosphoenzyme irrespective of the modification. Adenylyl imidodiphosphate and ADP accelerated Na+,K+-ATPase activity in the presence of 2.7 microM ATP by decreasing the extent of the fluorescence without affecting the amount of phosphoenzyme, irrespective of the modification. These data suggest that Na+,K+-ATPase activity was accelerated due to the acceleration of the breakdown of K+-bound enzyme by high concentrations of ATP and ATP analogues.
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PMID:The acceleration of Na+,K+-ATPase activity by ATP and ATP analogues. 304 Jul 15

Frozen aqueous suspensions of partially purified membrane-bound renal (Na+ + K+)-ATPase have been irradiated at -135 degrees C with high-energy electrons. (Na+ + K+)-ATPase and K+-phosphatase activities are inactivated exponentially with apparent target sizes of 184 +/- 4 kDa and 125 +/- 3 kDa, respectively. These values are significantly lower then found previously from irradiation of lyophilized membranes. After reconstitution of irradiated (Na+ + K+)-ATPase into phospholipid vesicles the following transport functions have been measured and target sizes calculated from the exponential inactivation curves: ATP-dependent Na+-K+ exchange, 201 +/- 4 kDa; (ATP + Pi)-activated Rb+-Rb+ exchange, 206 +/- 7 kDa and ATP-independent Rb+-Rb+ exchange, 117 +/- 4 kDa. The apparent size of the alpha-chain, judged by disappearance of Coomassie stain on SDS-gels, lies between 115 and 141 kDa. That for the beta-glycoprotein, though clearly smaller, could not be estimated. We draw the following conclusions: (1) The simplest interpretation of the results is that the minimal functional unit for (Na+ + K+)-ATPase is alpha beta. (2) The inactivation target size for (Na+ + K+)-dependent ATP hydrolysis is the same as for ATP-dependent pumping of Na+ and K+. (3) The target sizes, for K+-phosphatase (125 kDa) and ATP-independent Rb+-Rb+ exchange (117 kDa) are indistinguishable from that of the alpha-chain itself, suggesting that cation binding sites and transport pathways, and the p-nitrophenyl phosphate binding site are located exclusively on the alpha-chain. (4) ATP-dependent activities appear to depend on the integrity of an alpha beta complex.
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PMID:Minimal functional unit for transport and enzyme activities of (Na+ + K+)-ATPase as determined by radiation inactivation. 608 87

Membrane-bound (Na+, K+)ATPase from avian nasal salt glands was exposed to limited papain digestion. Such treatment results in the selective removal of the beta-subunit rendering the alpha-subunit still membrane-bound and expressing full enzymic activity. With further exposure to papain the alpha-chain becomes fragmented into two major polypeptide components. The fragmented membrane-bound catalytic chain is extremely sensitive to detergent treatment and cannot be solubilized in an active state.
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PMID:The (Na+, K+)ATPase exhibits enzymic activity in the absence of the glycoprotein subunit. 630 51

A ouabain p-aminobenzenediazonium derivative with a high specific radioactivity has been synthesized from ouabain and used as a photolabel for the (sodium plus potassium)-activated adenosinetriphosphatase from Electrophorus electricus electric organ and from dog kidney. In the dark it binds reversibly to the digitalis receptor site, with binding characteristics comparable to those of ouabain. The photoactivation of the ouabain derivative to produced covalent labeling of the receptor was obtained by energy transfer from a tryptophan residue in the (Na+,K+)ATPase to the ouabain p-aminobenzenediazonium molecule bound at the active site. The great advantage of this procedure compared to previous methods is that free molecules of the photoactivatable derivative are not photodecomposed. Analysis of the photolabeled polypeptides on sodium dodecyl sulfate gel electrophoresis showed that over 90% of the total radioactivity incorporated was found in the large molecular weight alpha-chain of the kidney enzyme (Mr 93 000). The same specific labeling of the alpha-subunit was obtained with a crude microsomal fraction from Electrophorus electricus. A mild tryptic fragmentation of the subunit into two peptide fragments of Mr 58 000 and 41 000, respectively, shows that the digitalis receptor is located in the N-terminal 41 000 fragment.
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PMID:Specific photoaffinity labeling of the digitalis binding site of the sodium and potassium ion activated adenosinetriphosphatase induced by energy transfer. 631 46

Gel filtration of (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.8) solubilized in octaethyleneglycol dodecylmonother ( C12E8 ) has been performed under conditions where active (alpha beta)2 dimers (Mr 265000) are obtained, and under conditions where dissociation into alpha beta monomers occurs without appreciable loss of activity. It is shown that the alpha beta monomers aggregate with time to form (alpha beta)2 dimers at low detergent concentrations with no change in enzymatic activity. At high detergent concentrations the aggregation is much slower, but the enzymatic activity is lost rapidly. Polyacrylamide gel electrophoresis in the presence of C12E8 also suggest that high concentrations of detergent dissociate the (alpha beta)2 dimer into smaller particles, and conditions for gel electrophoresis are described. The inactivating effect of C12E8 at high C12E8 /protein ratios can be related to a delipidation of the enzyme, with about 0.19 mg phospholipid required per mg protein for optimal activity. The experiments suggest that the solubilized (Na+ + K+)-ATPase can be disrupted into particles containing only one alpha-chain and one or two beta-chains without irreversible loss of activity, and that the stable form of the enzyme is an (alpha beta)2 dimer.
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PMID:The distribution of C12E8-solubilized oligomers of the (Na+ + K+)-ATPase. 632 42

Hb Hirosaki is a new unstable variant in which CE 1 phenylalanine of alpha-chain is substituted by leucine. Seven patients with hemolytic anemia were found in one family and Hb Hirosaki was detected in four of them. In clinical feature some differences of severity were observed between child and adult patients. The child cases showed relatively severe symptoms with repeated hemolytic crises and drug sensitivity. Otherwise most of adult cases had only mild hemolytic process throughout their past life. The variety of clinical severity suggests that the expression of abnormal gene may be various in this disorder. The mode of inheritance seemed to be autosomal dominant and all the cases in this report were heterozygous state. Macrocytic and hypochromic anemia was characteristic in most cases and red blood cells containing Heinz bodies were dominant in peripheral blood from the splenectomized patients. Accelerated glycolysis and enhanced ATPase activity were outstanding features in the metabolism of erythrocyte in this disorder. The instability of reduced glutathione was also found in three of five cases.
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PMID:Clinical features and biochemical aspects of red blood cells in Hb Hirosaki. 645 79

Tubulin tyrosine ligase catalyzes the reversible addition of tyrosine to the C-terminus of tubulin alpha chains. By using ligase and carboxypeptidase A in conjunction, we have previously shown that brain cytoplasmic tubulin exists in three forms: 15-40% already has C-terminal tyrosine, another 10-30% can accept additional tyrosine, and about one-half is an uncharacterized species which is not a ligase substrate. A membrane-bound fraction of brain tubulin, purified by vinblastine precipitation from a detergent extract, has been found to differ by the complete absence of preexisting tyrosine. The membrane fraction from which tubulin was extracted also contained masked forms of both ligase and a distinct detyrosylating enzyme, which can be released by detergent extraction. The turnover of alpha-chain C-terminal tyrosine in vivo was studied by incubating brain mince with labeled tyrosine, or injecting it intracerebrally, under conditions where protein synthesis was inhibited. Tyrosine appeared to turn over to about the same extent in membrane-bound, as in soluble, tubulin. This apparently paradoxical result was not due to ATPase in the membrane fraction, which might have allowed ligase-catalyzed exchange between free and fixed tyrosine. Authentic [14C]tyrosylated tubulin added to the brain membrane fraction was not detyrosylated or subject to endoprotease digestion during subsequent procedures to isolate tubulin. The unexpected finding that tubulin tyrosylated at the C-terminal in vivo appears to be in the "non-substrate" fraction points toward a possible resolution of the paradox.
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PMID:An apparent paradox in the occurrence, and the in vivo turnover, of C-terminal tyrosine in membrane-bound tubulin of brain. 745 80

Multilamellar 3-dimensional (Type I) microcrystals of detergent-solubilized crude microsomes or purified protein preparations of membrane-bound gastric (H+, K+)-ATPase from rabbit or hog stomachs develop in media consisting of 0.1 M KCl, 20 mM imidazole, 5 mM MgCl2, 3 mM NaN3, 5 mM DTT, 25 IU/ml Trasylol, 2 micrograms/ml DTBpC and 20-40% glycerol, using nonionic detergent of C12E8 or BRIJ 36 for solubilization. Crystals developed in a pH-range of 6.0-7.25, during 3-10 days of incubation, at 2 degrees C. For C12E8, the most effective detergent:protein ratio for crystallization varied between (1.8-2.0):1 for the microsomes and between (0.25-0.75):1 for the purified preparations. The results of biochemical and structural analysis of the (H+, K+)-ATPase crystals showed close resemblance to those of the sarcoplasmic reticulum Ca(2+)-ATPase from skeletal muscle and plasmamembrane (Na+, K+)-ATPase from kidney (J. Biol. Chem., 269, 10107-111, 1994). Based on these identities and the high (62%) overall sequential homology to the (Na+, K+)-ATPase, we conclude that the new crystals of the (H+, K+)-ATPase could also contain only the alpha-chain of the alpha beta heterodimers found in the native membrane. High-resolution electron microscopy of frozen-hydrated crystalline (H+, K+)-ATPase samples are in progress to give unit cell dimensions and molecular packing of the new crystals.
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PMID:Three-dimensional (type I) microcrystals of detergent-solubilized membrane-bound gastric (H+, K+)-ATPase enzyme from hog and rabbit stomachs. 778 47

When pig stomach membrane H+,K(+)-ATPase preparations were incubated with [gamma-32P]ATP and Mg2+ with vanadate, 32P was incorporated into the alpha-chain of H+,K(+)-ATPase to a steady-state level of approximately 0.7 mol of phosphotyrosine (Tyr(P))/mol of phosphoenzyme intermediates. The addition of a membrane H+,K(+)-ATPase preparation with Mg2+ accelerated the liberation of 32P from Tyr(P) residues in the alpha-chain. Mild tosylphenylalanyl chloromethyl ketone-trypsin treatment solubilized 32P-containing peptides from the alpha-chain almost completely. A reverse-phase column chromatography of the supernatant gave two peaks of 32P-peptide with similar total radioactivities. The amino acid sequence of both peaks was shown to be Gly-Lys-Ala-Glu-Asn-Tyr-Glu-Leu-Tyr-Gln--, which is consistent with the amino-terminal sequence of the alpha-chain of H+,K(+)-ATPase deduced from cDNA from pig stomach except that the initial Met was absent. The comparison of the recovery of amino acid from each Edman cycle showed that the phosphorylation of Tyr10 occurred preceding the phosphorylation of Tyr7. These data and others suggested the presence of a novel membrane-bound enzyme system to participate in reversible phosphorylation of both Tyr residues in the alpha-chain of H+,K(+)-ATPase.
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PMID:Reversible phosphorylation of both Tyr7 and Tyr10 in the alpha-chain of pig stomach H+,K(+)-ATPase by a membrane-bound kinase and a phosphatase. 779 39


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