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:3.6.3.14 (ATP synthase)
7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

(1) The mitochondrial ATPase (EC 3.6.1.3) Ehrlich ascites cell mitochondria, was inhibited by D-glucose under physiological concentrations of ATP. The generation of ADP by the mitochondrial bound hexokinase, seems to be the reason for the D-glucose inhibitory effect. Reversal of the inhibitory effect of ADP on Ehrlich ascites cell mitochondria ATPase by an ATP-regenerating system was achieved. (2) Dissociation of mitochondrial bound hexokinase from the mitochondria eliminated the inhibitory effect of D-glucose. Rebinding of the hexokinase to the mitochondria regenerated the D-glucose inhibitory effect on Ehrlich ascites cell mitochondria ATPase. (3) Bioflavonoids such as quercetin inhibit the mitochondrial hexokinase activity, but do not change the mitochondrial ATPase activity of isolated Ehrlich ascites tumor cell mitochondria. (4) The inhibitory effect of bioflavonoids on mitochondrial bound hexokinase activity is shown to be dissociable from the ascites tumor cell mitochondria and seems to be associated with regulatory rather than catalitic sites of the enzyme.
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PMID:Bioflavonoid regulation of ATPase and hexokinase activity in Ehrlich ascites cell mitochondria. 1 95

Secretory vesicles that accumulate in the temperature-sensitive sec6-4 strain of yeast have been shown to contain a vanadate-sensitive ATPase, presumably en route to the plasma membrane (Walworth, N. C., and Novick, P. J. (1987) J. Cell Biol. 105, 163-174). We have now established this enzyme to be a fully functional form of the PMA1 [H+]ATPase, identical in its catalytic properties to that found in the plasma membrane. In addition, the secretory vesicles are sealed tightly enough to permit the measurement of ATP-dependent proton pumping with fluorescent probes. We have gone on to exploit the vesicles as an expression system for site-directed mutants of the ATPase. For this purpose, a sec6-4 strain has been constructed in which the chromosomal PMA1 gene is under control of the GAL1 promoter; the mutant pma1 allele to be studied is introduced on a centromeric plasmid under the control of a novel heat shock promoter. In galactose medium at 23 degrees C, the wild-type ATPase is produced and supports normal vegetative growth. When the cells are switched to glucose medium at 37 degrees C, however, the wild-type gene turns off, the mutant gene turns on, and secretory vesicles accumulate. The vesicles contain a substantial amount of newly synthesized, plasmid-encoded ATPase (5-10% of total vesicle protein), but only traces of residual wild-type PMA1 ATPase and no detectable mitochondrial ATPase, vacuolar ATPase, or acid or alkaline phosphatase. To test the expression strategy, we have made use of pma1-105 (Ser368----Phe), a vanadate-resistant mutant previously characterized by standard methods (Perlin, D. S., Harris, S. L., Seto-Young, D., and Haber, J. E. (1989) J. Biol. Chem. 264, 21857-21864). In secretory vesicles, as expected, the plasmid-borne pma1-105 allele gives rise to a mutant enzyme with a reduced rate of ATP hydrolysis and a 100-fold increase in Ki for vanadate. Proton pumping is similarly resistant to vanadate. Thus, the vesicles appear well suited for the production and characterization of mutant forms of the PMA1 [H+]ATPase. They should also aid the study of other yeast membrane proteins that are essential for growth as well as heterologous proteins whose appearance in the plasma membrane may be toxic to the cell.
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PMID:Expression of the yeast plasma membrane [H+]ATPase in secretory vesicles. A new strategy for directed mutagenesis. 182 8

This study concerns the assembly into a multisubunit enzyme complex of a small hydrophobic protein imported into isolated mitochondria. Subunit 8 of yeast mitochondrial ATPase (normally a mitochondrial gene product) was expressed in vitro as a chimaeric precursor N9L/Y8-1, which includes an N-terminal-cleavable transit peptide to direct its import into mitochondria. Assembly into the enzyme complex of the imported subunit 8 was monitored by immunoadsorption using an immobilized anti-F1-beta monoclonal antibody. Preliminary experiments showed that N9L/Y8-1 imported into normal rho+ mitochondria, with its complement of fully assembled ATPase, did not lead to an appreciable assembly of the exogenous subunit 8. With the expectation that mitochondria previously depleted of subunit 8 could allow such assembly in vitro, target mitochondria were prepared from genetically modified yeast cells in which synthesis of subunit 8 was specifically blocked. Initially, mitochondria were prepared from strain M31, a mit- mutant completely incapable of intramitochondrial biosynthesis of subunit 8. These mit- mitochondria however were unsuitable for assembly studies because they could not import protein in vitro. A controlled depletion strategy was then evolved. An artificial nuclear gene encoding N9L/Y8-1 was brought under the control of a inducible promoter GAL1. This regulated gene construct, in a low copy number yeast expression vector, was introduced into strain M31 to generate strain YGL-1. Galactose control of the expression of N9L/Y8-1 was demonstrated by the ability of strain YGL-1 to grow vigorously on galactose as a carbon source, and by the inability to utilize ethanol alone for prolonged periods of growth. The measurement of bioenergetic parameters in mitochondria from YGL-1 cells experimentally depleted of subunit 8, by transferring growing cells from galactose to ethanol, was consistent with the presence in mitochondria of a mosaic of ATPase, namely fully assembled functional ATPase complexes and partially assembled complexes with defective F0 sectors. These mitochondria demonstrated very efficient import of N9L/Y8-1 and readily incorporated the imported processed subunit 8 protein into ATPase. Comparison of the kinetics of import and assembly of subunit 8 showed that assembly was noticeably delayed with respect to import. These findings open the way to a new systematic analysis of the assembly of imported proteins into multisubunit mitochondrial enzyme complexes.
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PMID:Assembly of imported subunit 8 into the ATP synthase complex of isolated yeast mitochondria. 213 40

An ATPase from anaerobic Lactobacillus casei has been isolated and 100-times purified. The 400 kDa enzyme molecule was found to have a hexagonal structure 10 nm in diameter composed of at least six protein masses. SDS-electrophoresis reveals four or, under certain conditions, five types of subunit, of apparent molecular masses 57 (alpha), 55 (beta), 40 (gamma), 22 (delta) and 14 (epsilon) kDa with stoichiometry of 3 alpha, 3 beta, gamma, delta, epsilon. The following features resembling F1-ATPases from other sources were found to be inherent in the solubilized L. casei ATPase. (i) Detachment from the membrane desensitizes ATPase to low DCCD concentrations and sensitizes it to water-soluble carbodiimide. (ii) Soluble ATPase is inhibited by Nbf chloride and azide, is resistant to SH-modifiers and is activated by sulfite and octyl glucoside, the activating effect being much stronger than in the case of the membrane-bound ATPase. Substrate specificity of the enzyme is also similar to that of other factors F1. Divalent cations strongly activate the soluble enzyme when added at a concentration equal to that of ATP. An excess of Mn2+, Mg2+ or Co2+ inhibits ATPase activity of F1, whereas that of Ca2+ induces its further activation. No other F1-like ATPases are found in L. casei. It is concluded that this anaerobic bacterium possesses a typical F1-ATPase similar to those in mitochondria, chloroplasts, aerobic and photosynthetic eubacteria.
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PMID:The F1-type ATPase in anaerobic Lactobacillus casei. 213 82

The gene coding for the yeast Saccharomyces cerevisiae mitochondrial oligomycin sensitivity-conferring protein (OSCP) has been sequenced, and the gene products have been characterized. The OSCP is subunit 5 of the mitochondrial ATP synthase, a multimeric protein complex. As such, the gene coding for the yeast OSCP is referred to here as the ATP5 gene. From the predicted primary sequence, the calculated molecular weight of the immature yeast OSCP is 22,813 and the amino acid sequence is 35% identical and 65% homologous to bovine OSCP. A null mutant has been constructed. This mutant strain is unable to grow on glycerol medium, has no detectable oligomycin-sensitive ATPase activity, and has no detectable immune reactive proteins with the corresponding molecular weight of the OSCP (using antibodies reactive to the yeast OSCP). The transcription products of the yeast gene have been characterized. There is a single major transcript from the ATP5 gene of 1.05 kilobases. The level of the transcription product is increased from 3-5-fold after growth in galactose medium as compared to cells grown in glucose medium. The transcriptional initiation sites were determined to occur at +68(G) and +69(T) at comparable frequency and were not dependent on the growth medium. These results suggest that transcription of the ATP5 gene is catabolite-repressed.
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PMID:The gene coding for the yeast oligomycin sensitivity-conferring protein. 214 69

The F1F0-ATP synthase from the alkaliphilic Bacillus firmus OF4 was purified in a reconstitutively active form, in good yield and with a high specific ATPase activity when appropriately activated. The purification procedure involved octyl glucoside extraction of washed membrane vesicles in the presence of 20% glycerol and asolectin followed by ammonium sulfate fractionation and sucrose density gradient centrifugation. The purified preparation was resolved into seven bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to the five F1 subunits, alpha, beta, gamma, delta, and epsilon, and to the b and c subunits of the F0. Two-dimensional sodium dodecyl sulfate-poly-acrylamide gel analysis revealed a candidate for the alpha subunit of F0. The MgATPase activity of B. firmus OF4 F1F0 was barely detectable but could be stimulated, optimally more than 100-fold, by sulfite, methanol, and octyl thioglucoside. The enzyme was inhibited by N,N'-dicyclohexylcarbodiimide and sodium azide, but not by aurovertin, an inhibitor of the F1 from Escherichia coli. The F1F0 reconstituted into proteoliposomes catalyzed ATPase activity, ATP-Pi exchange, and ATP-dependent delta pH and delta psi formation. ATP hydrolysis was stimulated by protonophores while the other activities were abolished by protonophores. These activities were neither dependent on added sodium ions nor significantly affected by them. F1F0 proteoliposomes made from crude octyl glucoside extracts that also contained the Na+/H+ antiporter were shown to catalyze ATP-dependent Na+ uptake that was completely sensitive to carbonyl cyanide m-chlorophenyl-hydrazone; Na+ uptake activity was absent in proteoliposomes containing more purified F1F0 but lacking the Na+/H+ antiporter. These data show that the F1F0 translocates protons and does not substitute Na+ for H+ in energy coupling.
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PMID:Purification and reconstitution of the F1F0-ATP synthase from alkaliphilic Bacillus firmus OF4. Evidence that the enzyme translocates H+ but not Na+. 217 11

Three mutants of Saccharomyces cerevisiae resistant to triethyltin (an inhibitor of mitochondrial ATPase) on non-fermentative media, and non-resistant to this drug on fermentative media, were isolated and named TTR1, TTR2 and TTR3. Apart from triethyltin resistance, these mutants show the following common characteristics: (1) Increased intracellular cytochrome c concentration. (2) Increased respiration rate. (3) Decreased growth yield. (4) Increased growth sensitivity to several drugs inhibiting oxidative phosphorylation: namely, CCCP (permeabilizing inner mitochondrial membrane to protons), valinomycin (permeabilizing inner mitochondrial membrane to potassium) and oligomycin (inhibitor of mitochondrial ATPase). (5) Increased sensitivity to carbon source starvation. For each mutant, these characteristics appeared to be due to a single pleiotropic nuclear mutation. Mutation TTR1 causes additional phenotypic characteristics which do not appear in mutants TTR2 and TTR3: (1) Pinkish coloration of colonies which is more pronounced after a long growth period. (2) Inability of the cells to store glycogen. (3) Growth defect of the cells on a galactose-containing medium. (4) Inability of a diploid homozygote mutant strain to sporulate. All these phenotypic characteristics have already been described in yeast mutants deregulated in cAMP-dependent protein phosphorylation. Crossing of a strain bearing the TTR1 mutation with a strain mutated in the adenylate cyclase structural gene suggested that the TTR1 phenotype is due to a modification in regulation of cAPK by cAMP, making cell multiplication possible without intracellular cAMP.
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PMID:Isolation and genetic study of triethyltin-resistant mutants of Saccharomyces cerevisiae. 220 22

The effect of rhein, 4,5-dihydroxyanthraquinone-2-carboxylic acid, on oxygen consumption and the rate of aerobic and anaerobic lactate production by Ehrlich ascites tumor cells has been investigated. The rate of oxygen uptake decreases with the increase of rhein concentration. Rhein also inhibits aerobic and anaerobic glycolysis. The rate of aerobic lactate production decreases with the drug concentration and the maximal effect was observed at 0.100 mM. Anaerobic lactate production is also inhibited and the maximum effect is reached at 0.220 mM. The possibility that the lactate production decrease was secondary to an effect on mitochondrial ATPase was excluded on the basis of the data with DNP and oligomycin. Rhein reduces the intracellular level of lactate, pyruvate and glucose-6-phosphate. Glucose utilization and 2-deoxy-D-glucose uptake are decreased to the same extent as the inhibition of aerobic lactate production, whereas glucose phosphorylation is unaffected. It is, therefore, concluded that the inhibition of glycolysis of Ehrlich ascites tumor cells by rhein is caused by an impairment of glucose uptake.
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PMID:Effect of rhein on the glucose metabolism of Ehrlich ascites tumor cells. 237 63

The mannose receptor mediates the transport of high-mannose glycoproteins from the cell surface to lysosomes in macrophages. The binding of ligand to the receptor is dependent on both pH and Ca2+. Upon internalization, ligands enter an acidic pre-lysosomal compartment where receptor-ligand dissociation takes place. Acidification is driven by an endosomal proton pump and anion transport is coupled to this acidification step. A permeabilized-cell assay has been designed to characterize the ionic requirements for receptor-ligand dissociation in endosomes. The plasma membrane of macrophages has been permeabilized selectively with digitonin without affecting endosomal membranes. Receptor-ligand dissociation in permeabilized cells required ATP and was blocked by proton ionophores. Di-isothiocyanostilbene-disulphonic acid and N-ethylmaleimide also blocked dissociation, but mitochondrial ATPase inhibitors and vanadate were ineffective. To explore the nature of the anion requirement for acidification, the ability of different anions to compensate for Cl- was tested. For the halide series, Br- was as equally effective as Cl- in supporting receptor-ligand dissociation, but I- was inhibitory. Citrate and gluconate were only partially effective, while SO4(2-), NO3- and PO4(2-) blocked dissociation. Addition of Ca2+ to permeabilized-cell preparations impaired ATP-dependent dissociation without affecting endosome acidification. These results suggest that the endosomal membrane has a Ca2+ conductance that would permit the rapid efflux of Ca2+ from endosomes during acidification, and this would appear to be a necessary step for efficient sorting of Ca2+-dependent receptors from their ligands.
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PMID:The use of permeabilized cells to study the ion requirements of receptor-ligand dissociation in endosomes. 247 13

At the optimal pH for growth (pH 10.5), alkalophilic Bacillus firmus RAB, an obligate aerobe, exhibits normal rates of oxidative phosphorylation despite the low transmembrane proton electrochemical gradient, about -60 mV (delta psi = -180 mV and delta pH = +120 mV). This bioenergetic problem might be resolved by use of an Na+ coupled ATP synthase; otherwise an F1F0-ATPase must be able to utilize low driving forces in this organism. The ATPase activity was extracted from everted membrane vesicles by low ionic strength treatment and purified to homogeneity by hydrophobic interaction chromatography and sucrose density gradient centrifugation. The ATPase preparation had the characteristic F1-ATPase subunit structure, with Mr values of 51,500 (alpha), 48,900 (beta), 34,400 (gamma), 23,300 (delta), and 14,500 (epsilon); the identity of the alpha and beta subunits was confirmed by immunoblotting with anti-beta of Escherichia coli and anti-B. firmus RAB F1. Methanol and octyl glucoside, agents that stimulated the low basal membrane ATPase activity 10- to 12-fold, dramatically elevated the MgATPase activity of the purified F1, more than 150-fold, to 50 mumol min-1 mg protein-1. Anti-F1 inhibited membrane ATPase activity greater than or equal to 80%. The membranes exhibited no Na+-stimulated or vanadate-sensitive ATPase activity when prepared in the absence or presence of Na+ or ATP. These findings, which are consistent with previous studies, establish that in alkalophilic bacteria, ATP hydrolysis, and presumably ATP synthesis is catalyzed by an F1F0-ATPase rather than a Na+ ATPase.
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PMID:The membrane ATPase of alkalophilic Bacillus firmus RAB is an F1-type ATPase. 287 76


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