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 preparation of ATPase from the membranes of Micrococcus lysodeikticus, solubilized and more than 95% pure, showed two main bands in analytical polyacrylamide gel electrophoresis. They did not correspond to isoenzymes because one band could be converted into the other by exposure to a mildly alkaline pH value. The conversion was paralleled by changes in molecular weight, circular dichroism and catalytic properties. Denaturation by pH at 25 degrees C was followed by means of circular dichroism, ultracentrifugation and polyacrylamide gel electrophoresis. A large conformational transition took place in the acid range with midpoints at about pH = 3.6 (I = 10(-4) M), 4.3 (I = 0.03 M) and 5.3 (I = 0.1 M). The transition was irreversible. Strong aggregation of the protein occurred in this range of pH. The final product was largely random coil, but even at pH 1.5 dissociation into individual subunits was not complete. However, partial dissociation took place at pH 5 (I = 0.028 M). At this pH value the enzyme was inactive, but 20-30% of the activity could be recovered when the pH was returned to 7.5. In the alkaline region the midpoint of the transition occurred near pH = 11 (I = 0.028 M). The pK of most of the tyrosine residues of the protein was about 10.9. The unfolding was irreversible and the protein was soon converted into peptide species with molecular weights lower than those determined for the subunits by gel electrophoresis in the presence of sodium dodecyl sulphate. Conventional proteolysis did not account for the transformation.
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PMID:Conformational and molecular responses to pH variation of the purified membrane adenosine triphosphatase of Micrococcus lysodeikticus. 0 91

Chronic ammonia toxicity in experimental mice was induced by exposing them for 2 and 5 days to 5 % (v/v) ammonia solution. The enzymes concerned with glutamate metabolism (aspartate-, alanine- and tyrosine aminotransferases, glutamate dehydrogenase and glutamine synthetase) and (Na+ + K+)-ATPase were estimated in the three regions of brain (cerebellum, cerebral cortex and brain stem) and in liver. Glutamate, aspartate, alanine, glutamine and GABA, RNA and protein were also estimated in the three regions of brain and liver. A significant rise in the activity of (Na+ + K+)-ATPase in all the three regions of brain along with a fall in the activity of alanine aminotransferase was noticed. Changes in the activities of other enzymes were also observed. A significant increase in alanine and a decrease in glutamic acid was observed while no change was observed in the content of other amino acids belonging to the glutamate family. As a result of this, changes in the ratios of glutamate/glutamine and glutamate + aspartate/GABA was observed. The results indicated that the brain was in a state of more depression and less of excitation. Under these conditions the liver tissue was showing a profound rise in the activity of the enzymes of glutamate metabolism. The results are further discussed.
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PMID:Chronic metabolic effects of ammonia in mouse brain. 9 19

TN-C was purified from bovine cardiac muscle. In the absence of Ca-2+, cardiac TN-C has an intrinsic sedimentation coefficient of 1.93 S and a molecular weight of 18 000 daltons. Cardiac TN-C reverses the inhibitory effect of skeletal TN-I on the Mg-2+-activated ATPase of a skeletal synthetic actomyosin preparation in the presence of skeletal tropomyoson. Circular dichroism (CD) studies indicate that cardiac TN-C undergoes a major conformational change upon binding Ca-2+. A similar response is elicited by Sr-2+, whereas Mg-2+ has a much less pronounced effect. The presence of Mg-2+ does not alter the net effects of either Ca-2+ or Sr-2+. Cardiac TN-C is rich in acidic amino acid residues. UV absorption, near UV CD, and fluorimetric studies show that the protein lacks tryptophan and has a relatively high phenylalanine to tyrosine ratio. The results of this study invite direct comparisons with results reported for the skeletal muscle analogue of cardiac TN-C.
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PMID:Molecular and biological studies on cardiac muscle calcium-binding protein (TN-C). 12 76

G-actin has been nitrated with tetranitromethane in conditions that lead to the modification of one tyrosine residue. The reactive residue was found by earlier workers to be Tyr-69. The nitrated actin is conformationally similar to native G-actin, as judged by sedimentation velocity and circular dichroism analysis. A small proportion only is in the form of covalently linked dimers and trimers. The nitrated G-actin will polymerise to form filaments, indistinguishable in the electron microscope from those of native F-actin, but the polymerisation process is slower. Reduction of the nitrophenol group to the corresponding aminophenol leaves the properties of the protein in respect of polymerisation unchanged. When a dansyl group is introduced at the same point, however, the ability of the actin to polymerise is lost. The nitrated actin and its reduced counterpart will also bind heavy meromyosin, and the characteristic arrowhead formation of the bound molecules along the filaments can be seen in the electron microscope. Neither of the modified F-actins, however, significantly activates or inhibits the myosin ATPase activity. The fluorescence of nitrated actin is strongly quenched through the presence of the nitrophenol chromophore. In soluble complexes with heavy meromyosin the fluorescence is indistinguishable from the sum of the separate contributions of the two protein components. There is thus no measurable excitation transfer between any tryptophan residues on the myosin heads, such as that inferred to be present in the ATPase site, and the nitrotyrosine in position 69 of the actin sequence. Implications of this observation are considered in relation to the different interaction sites in myosin and in actin. The activation of heavy meromyosin ATPase by copolymers containing actin and nitroactin in different proportions has been measured, and is not proportional to the fraction of native actin. The results are consistent with the view that the function of actomyosin depends on the interaction of the myosin heads with more than one actin subunit.
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PMID:Effects of specific chemical modification of actin. 12 59

Ethoxyformylation of sarcoplasmic reticulum vesicles is performed to study a possible role of histidine residues in the calcium translocation process. The influence of the chemical modification is evaluated on the Ca2+-dependent ATPase activity, and on the Ca2+ uptake parameters: VCa (initial rate of calcium uptake) and CCa (amount of cation accumulated at the steady state). The substitution of the amino acids is monitored by three different techniques: (a) by amino acid analysis of the ethoxyformylated material further submitted to modification by diazonium-1-H-tetrazole, or by sulfhydryl titration using 5-5'-dithiobis (2-nitrobenzoic acid); (b) by 14C labeling followed by the removing of labels after NH2OH or imidazole treatment at pH 7; (c) by spectrophotometric measurements at 230 nm. The ethoxyformylation reaction is not specific for histidine at pH 6.1 and 10 degrees. About 1 lysyl group/mol of ATPase is first modified. Then 1 (with a pseudo-first order rate constant of 240 (+/- 20) 10(-3) min-1) or 2 histidines are modified. No substitution of tyrosine or sulfhydryl groups can be detected under our experimental conditions. A decrease of the Ca2+-dependent ATPase activity correlated with the inhibition of both VCa and Cca corresponds to the chemical substitution of the histidine. No direct correlation between the decrease of the activities and the modification of the lysine can be found. After removing the ethoxyformyl group from the histidine, only the Ca2+-dependent ATPase activity is restored to its initial value. No protection is found when the reaction is performed in the presence of ATP or p-nitrophenylphosphate. These results can be explained if one assumes that the ethoxyformylation of the histidine residue(s) induces a conformational change modifying the affinity of the membrane for nucleotides.
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PMID:Mechanism of an active transport of calcium. Ethoxyformylation of sarcoplasmic reticulum vesicles. 13 46

Sodium- and potassium-activated adenosine triphosphatase (NaK-ATPase) was purified from nasal salt glands of the duck (Anas platyrhynchos). Enzyme of specific activity 2,000 to 2,300 mumol of Pi/mg/hour was routinely obtained by sodium dodecyl sulfate treatment of a microsomal fraction of gland homogenate in the presence of 3 mM ATP followed by pelleting of the enzyme through a sucrose density gradient. Purified NaK-ATPase was stable for over 3 months at -20 degree. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography purified NaK-ATPase was shown to contain two polypeptide chains of molecular weight 94,000 and 60,000, the smaller of which was a glycoprotein. Purified enzyme of activity 2,300 mumol of Pi/mg/hour bound 3,600 pmol of ouabain/mg of enzyme protein. Reaction with [gamma-32P]ATP in the presence of Mg2+ and Na+ gave 7,025 pmol of acyl phosphate/mg of enzyme protein. The turnover number calculated from phosphorylation data was 5,460 min-1. Amino acid analysis of the polypeptide components of duck salt gland enzyme after separation by gel filtration chromatography in sodium dodecyl sulfate demonstrated strong compositional homology with highly purified NaK-ATPase preparations from other organs and species. The NH2-terminal amino acid of the 94,000-dalton component was glycine and of the 60,000-dalton component, alanine. With a combination of manual sequencing and automated Edman degradation, the NH2-terminal amino acid sequence of the 94,00-dalton catalytic subunit was found to be Gly-Arg-Asn-Lys-Tyr-Glu-Thr-Thr-Ala-()-Ser-Glu.
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PMID:Sodium- and potassium-activated adenosine triphosphatase of the nasal salt gland of the duck (Anas platyrhynchos). Purification, characterization, and NH2-terminal amino acid sequence of the phosphorylating polypeptide. 13 47

A heat-stable protein has been detected in Saccharomyces cerevisiae which inhibits mitochondrial ATPase activity. The protein inhibitor has been isolated from extracts prepared by brief heat treatment of unbroken cell suspensions. The isolated inhibitor is a small basic protein (molecular weight close to 7000, isoelectric proint 9.05) devoid of tryptophan, tyrosine, and cysteine as well as proline. The NHP2-terminal amino acid is serine. The ultraviolet absorption spectrum shows the vibrational fine structure of the phenyl-alanine band. Like the ATPase inhibitor from bovine heart mitochondria the yeast inhibitor is rapidly destroyed by trypsin. It is also inactivated by the yeast proteinases A and B. Radioimmunological analysis indicates that the inhibitor is synthesized on cytoplasmic ribosomes. Its accumulation seems to be connected to the formation of the mitochondrial ATPase complex, since its specific activity is greatly reduced both in extracts obtained from the F1-ATPase-deficient nuclear mutant pet 936 and from the cytoplasmic petite mutant D 273-10B-1.
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PMID:A protein inhibitor of mitochondrial adenosine triphosphatase (F1) from Saccharomyces cerevisiae. 13 3

The reaction of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole [NBD-Cl] with purified eel electrophax Na+ and K+ stimulated adenosine triphosphatase [(Na-K)ATPase] has been monitored by changes in the (Na-K)ATPase activity, the K+ stimulated p-nitrophenyl phosphatase [PNPase] activity, and the protein ultraviolet absorption spectrum. The NBD-Cl reacts with two tyrosine residues per mol of enzyme (approximately 6-7 nmol/mg of protein), as judged by changes in protein absorption spectra and incorporation of [14C]NBD-Cl. The modified tyrosine groups are located on the Mr = 95 000 polypeptide chain and react at different rates. Only one tyrosine modification is necessary for complete inhibition of (Na-K)ATPase activity, although both must be modified for complete inhibition of PNPase activity. Reversal of these modifications by 2-mercaptoethanol restores 65% of both activities. Na+ increases the rate of tyrosine modification, K+ decreases the rate, and ATP affords the more reactive tyrosine group complete protection. NBD-Cl modification of approximately 6-7 nmol of tyrosine groups/mg of protein results in a large decrease in ATP affinity as judged by equilibrium binding. These results are compared with similar results obtained from NBD-Cl modification of the coupling factors of oxidative phosphorylation and photophosphorylation. A model is presented suggesting an asymmetric arrangement of two 95 000 polypeptide chains with a single tyrosine residue at the ATP site.
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PMID:Reaction of (Na-K)ATPase with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole: evidence for an essential tyrosine at the active site. 14 73

The mitochondrial F1-ATPase is irreversibly inactivated by the adenine nucleotide analogue, p-fluorosulfonylbenzoyl-5'-adenosine. This inactivation is partly prevented by the presence of bound adenine nucleotides. Inactivations of the ATPase with p-fluorosulfonyl[14C]benzoyl-5'-adenosine were most efficiently accomplished with the nucleotide-free enzyme at pH 7.0, in a buffer containing 20% glycerol. Under these conditions, 4.2 g atoms of 14C are incorporated per 350,000 g of enzyme when the ATPase is inactivated by 90% by its reaction with 2 mM p-fluorosulfonyl[14C]benzoyl-5'-adenosine. Isolation of the component polypeptide chains of the labeled ATPase showed that all of the radioactivity was associated with the two largest subunits. The isolated alpha subunit contained 0.45 g atom of 14C/mol and the isolated beta subunit contained 0.88 g atom of 14C/mol. Hence, the inactivation can be correlated with the incorporation of 14C into the beta subunit. This suggests that the hydrolytic site of the enzyme resides on this subunit. The majority of the radioactivity in a tryptic digest of labeled beta subunit is contained ina tryptic peptide that has the following amino acid sequence: Ile-Met-Asp-Pro-Asn-Ile-Val-Gly-Ser-Glu-His-Tyr-Asp-Val-Ala-Arg, where Tyr is the radioactive derivative of the tyrosine residue that was sulfonylated during the inactivation.
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PMID:Identification of a tyrosine residue at a nucleotide binding site in the beta subunit of the mitochondrial ATPase with p-fluorosulfonyl[14C]-benzoyl-5'-adenosine. 15 Apr 16

Incubation of mitochondria from Neurospora crassa and Saccharomyces cerevisiae with the radioactive ATPase inhibitor [14C]dicyclohexylcarbodiimide results in the irreversible and rather specific labelling of a low-molecular-weight polypeptide. This dicyclohexylcarbodiimide-binding protein is identical with the smallest subunit (Mr 8000) of the mitochondrial ATPase complex, and it occurs as oligomer, probably as hexamer, in the enzyme protein. The dicyclohexylcarbodiimide-binding protein is extracted from whole mitochondria with neutral chloroform/methanol both in the free and in the inhibitor-modified form. In Neurospora and yeast, this extraction is highly selective and the protein is obtained in homogeneous form when the mitochondria have been prewashed with certain organic solvents. The bound dicyclohexylcarbodiimide label is enriched in the purified protein up to 50-fold compared to whole mitochondria. Based on the amino acid analysis, the dicyclohexylcarbodiimide-binding protein from Neurospora and yeast consists of at least 81 and 76 residues, respectively. The content of hydrophobic residues is extremely high. Histidine and tryptophan are absent. The N-terminal amino acid is tyrosine in Neurospora and formylmethionine in yeast.
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PMID:The dicyclohexylcarbodiimide-binding protein of the mitochondrial ATPase complex from Neurospora crassa and Saccharomyces cerevisiae. Identification and isolation. 15 5


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