Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.3.14 (ATP synthase)
 7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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 absorbance and fluorescence spectral properties of mitochondrial F1-ATPase confirm that this protein does not contain tryptophan residues and therefore its fluorescence is due to tyrosines. The 36% increase in the fluorescence and the almost 100% increase in quantum yield upon denaturation of the protein suggest that a considerable number of tyrosyl residues have a very low quantum yield in the native enzyme. Quenching experiments using iodide indicate that all of the fluorophores are quenched and also all of them with the same quenching constant. These observations are interpreted as confirmatory of what has been found with several other proteins whose fluorescence originates from tyrosyl residues, where the buried tyrosines fluoresce with a much lower quantum yield than those which are exposed. ATP added to F1 previously depleted of loosely bound nucleotides changes the quenching constant of iodide and the quantum yield and this is interpreted to be due to a conformational change induced by the binding of the nucleotide to the enzyme. Addition of 2-mercaptoethanol decreases, although slightly, the polarization of the fluorescence. However, SDS addition gives a much bigger decrease. Hence disulphide bridges are less important for the tertiary structure of the protein than hydrophobic interactions, hydrogen bonding or other forces. Nevertheless the conformational change induced by reduction of disulphide bridges is detected in iodide quenching experiments and the change of the quantum yield of the enzyme.
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PMID:Structural studies of mitochondrial coupling factor 1 using tyrosine fluorescence. 286 Nov 23

Polar membrane in Campylobacter jejuni has been visualized on membrane vesicles. It was composed of doughnut-shaped particles 5-6 nm in diameter, with stalks, arranged in a hexagonal array. This structure was stabilized on the membrane by a high ionic strength buffer in the presence of 2-mercaptoethanol. Histochemical staining indicated localized ATPase activity at the poles of the cells. An ATPase with distinctive properties has been isolated and purified from this organism; it gives a specific activity of approximately 0.3 units/mg of protein. Electron microscopy showed doughnut-shaped particles 5-6 nm in diameter. Nondissociating and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed, respectively, a single band with ATPase activity and a molecular weight of ca. 75,000 Da. The enzyme was cold labile and activity was abolished by trypsin. Dicyclohexylcarbodiimide inhibited the membrane-bound form of the enzyme, but did not inhibit the soluble form. Oligomycin had no inhibitory activity on either form of the enzyme. The enzyme specifically hydrolysed ATP, but other nucleotide substrates were not degraded. The enzyme was activated by Mg2+ and inhibited by Ca2+, whereas other ions had no effect on activity. Antibodies prepared to this enzyme bound to the polar regions of whole cells as shown by protein A - colloidal gold immunoelectron microscopy. The antibodies to this ATPase cross reacted (shown by Western blotting) with four proteins from a whole-cell extract of this organism, two proteins in Aquaspirillum serpens MW5, and three proteins from Escherichia coli K12. They did not cross-react with any proteins from Spirillum volutans, Methanococcus voltae, Vibrio cholerae, or rat liver mitochondria. Antibodies raised against the F1-ATPase of E. coli K12 cross reacted with six proteins in a whole-cell extract of this organism, and one protein species in each of the whole-cell extracts of V. cholera, A. serpens MW5, S. volutans, and rat liver mitochondria. These antibodies did not recognize any whole cell proteins from either C. jejuni or M. voltae. These results along with the ATPase activity localized by histochemical staining suggest that polar membrane is an assembly of ATPase molecules at the poles of the cell and that the ATPase isolated from C. jejuni is serologically and structurally unusual.
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PMID:The ultrastructure and ATPase nature of polar membrane in Campylobacter jejuni. 297 56

Proteins of similar molecular weights were stripped from submitochondrial particles (A particles) of rat skeletal muscle or bovine heart by treatment with classical chemical uncouplers at 0 degrees C as with Ca2+. Proteins released included two of high molecular weight (about 43 000 and 30 000), an ATPase inhibitor protein (IF1) as well as the Ca2+-binding lipoprotein that has previously been shown to protect the mitochondrial ATPase complex against inhibition by N,N'-dicyclohexylcarbodiimide (DCCD). The latter two proteins were purified to a high degree. The crude fraction obtained by stripping with chemical uncouplers also contained traces of an additional protein (relative mass (Mr) approximately 13 000) which was also found upon aging of the crude fraction stripped by Ca2+. It was not found in aged preparations of either purified IF1 or the lipoprotein, but appeared when IF1 and the lipoprotein were mixed and aged together. Pretreatment of the mixture with 2-mercaptoethanol prior to electrophoresis did not remove the hybrid. More phospholipid was stripped from A particles by chemical uncouplers than by Ca2+ but less protein was stripped. Phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, and cardiolipin were identified in the phospholipid fractions.
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PMID:Stripping of proteins from submitochondrial particles of rat skeletal muscle or bovine heart by chemical uncouplers. 622 47

Biotin-cysteine was used to study protein S-thiolation in isolated rat kidneys subjected to ischemia and reperfusion. After 40 min of ischemia, total protein S-thiolation increased significantly (P < 0.05), by 311%, and remained significantly elevated (P < 0.05), 221% above control, after 5 min of postischemic reperfusion. Treatment of protein samples with 2-mercaptoethanol abolished the S-thiolation signals detected, consistent with the dependence of the signal on the presence of a disulfide bond. With the use of gel filtration chromatography followed by affinity purification with streptavidin-agarose, S-thiolated proteins were purified from CHAPS-soluble kidney homogenate. The proteins were then separated by SDS-PAGE and stained with Coomassie blue. With a combination of matrix-assisted laser desorption ionization time of flight mass spectrometry and LC/MS/MS analysis of protein bands digested with trypsin, a number of S-thiolation substrates were identified. These included the LDL receptor-related protein 2, ATP synthase alpha chain, heat shock protein 90 beta, hydroxyacid oxidase 3, serum albumin precursor, triose phosphate isomerase, and lamin. These represent proteins that may be functionally regulated by S-thiolation and thus could undergo a change in activity or function after renal ischemia and reperfusion.
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PMID:Reversible cysteine-targeted oxidation of proteins during renal oxidative stress. 1287 48

The oxidative phosphorylation inhibitor DBCT (dibutylchloromethyltin chloride) inhibits state 3 respiration at a concentration less than that which stimulates K+ flux into respiring rat liver mitochondria. Inhibition of ADP-stimulated respiration by DBCT can be reversed or blocked by the dithiol 2,3-dimercaptopropanol. The data are consistent with previous suggestions that DBCT may interact with the ATP synthase via reaction with a dithiol group. The stimulation of K+ influx by DBCT is partially reversed by concentrations of 2-mercaptoethanol which fail to affect the inhibition of state 3 respiration by DBCT. The combination of DBCT plus 2,3-dimercaptopropanol inhibits mitochondrial K+ influx. The inhibitory effect of dicyclohexylcarbodiimide on K+ influx is not expressed in the presence of DBCT. Atractyloside has little effect on K+ influx in the presence or absence of DBCT. The combination of DBCT plus uncoupler induces a net loss of endogenous K+. Consideration is given to the alternative hypotheses that the acceleration of K+ influx by DBCT may involve either a direct link to the energy transduction apparatus, or may occur via separate activation of a passive transport mechanism.
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PMID:Interacting effects of dibutylchloromethyltin chloride, 2,3-dimercaptopropanol, and other reagents on mitochondrial respiration and K+ flux. 1825 Nov 11