ATP Exchange Activity Research Articles

Reconstitution of beef heart mitochondrial F 0F 1 in reverse phase evaporation vesicles

Beef heart mitochondrial F 0F 1 was reconstituted in proteoliposomes by a new procedure. MF 0F 1 was inserted in preformed reverse phase evaporation vesicles of large diameters prepared from asolectin (MF 0F 1–REV). Reconstitution was mediated by Triton X-100, which was subsequently removed by treatment with Bio-Beads. Parameters which resulted in optimal reconstitution were described. The MF 0F 1–REV proteoliposomes catalyzed an exchange between P i and ATP and were capable of proton pumping. Both reactions were inhibited by oligomycin and uncoupler of oxidative phosphorylation. The range of P i–ATP exchange activity of the proteoliposomes (70–110 nmol min −1 mg −1) compared favorably with activities obtained in vesicles reconstituted by cholate dialysis or cholate dilution. The most important aspect of this method is that, unlike other reconstitution methods, exogenous F 1 and other coupling factors are not required to obtain high P i–ATP exchange activity by MF 0F 1–REV. This simple and rapid reconstitution procedure should be useful for future studies dealing with functional analysis of MF 0F 1.

Mitochondrial H +-ATPase in mutants of saccharomyces cerevisiae with defective subunit 8 of the enzyme complex

Mutants of Saccharomyces cerevisiae carrying defined lesions in the mitochondrial aap1 gene, coding for membrane subunit 8 of the H+-ATPase, have been investigated to examine the consequence of the mutations on the function and assembly of the enzyme complex. These include three mit- mutants, which cannot grow by oxidative metabolism due to their inability to synthesize full-length subunit 8, and three partial revertants of one of the mutants. The mutations in these strains have been previously characterized by DNA sequencing. The use of a monoclonal antibody to the beta subunit of the H+-ATPase as a probe of assembly defect revealed that the presence of subunit 8 is essential for the assembly of subunit 6 to the enzyme complex. Mitochondria isolated from the mit- mutants have negligible [32Pi]ATP exchange activity and they exhibited ATPase activity which is not sensitive to inhibition by oligomycin, indicating a defective membrane F0 sector. Normal assembly of subunit 8 (and subunit 6) was observed in the revertant strains, despite 8-9 amino-acid substitutions in the membrane-spanning region of the H+-ATPase subunit 8 in two of the strains. The assembled complex, however, exhibited reduced [32Pi]ATP exchange activity and low sensitivity to oligomycin, indicating that the product of the aap1 gene is a functional subunit of the mitochondrial H+-ATPase.

Coupling factor B involvement in the inhibition of P i—ATP exchange activity by N-ethylmaleimide

Coupling factor B involvement in the inhibition of P i—ATP exchange activity by N-ethylmaleimide

Enhancement of reconstituted ADP,ATP exchange activity by phosphatidylethanolamine and by anionic phospholipids

FEBS LettersVolume 119, Issue 2 p. 257-260 Full-length articleFree Access Enhancement of reconstituted ADP,ATP exchange activity by phosphatidylethanolamine and by anionic phospholipids R. Krämer, R. Krämer Institut für Physikalische Biochemie der Universität München Goethestrasse 33, 8000 München 2, FRGSearch for more papers by this authorM. Klingenberg, M. Klingenberg Institut für Physikalische Biochemie der Universität München Goethestrasse 33, 8000 München 2, FRGSearch for more papers by this author R. Krämer, R. Krämer Institut für Physikalische Biochemie der Universität München Goethestrasse 33, 8000 München 2, FRGSearch for more papers by this authorM. Klingenberg, M. Klingenberg Institut für Physikalische Biochemie der Universität München Goethestrasse 33, 8000 München 2, FRGSearch for more papers by this author First published: October 06, 1980 https://doi.org/10.1016/0014-5793(80)80266-3Citations: 72AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume119, Issue2October 06, 1980Pages 257-260 ReferencesRelatedInformation

Reconstituted mitochondrial oligomycin-sensitive ATPase (F 0F 1) with intermediate P i ⇌ HOH exchange but no P i ⇌ ATP exchange activity

Reconstituted mitochondrial oligomycin-sensitive ATPase (F 0F 1) with intermediate P i ⇌ HOH exchange but no P i ⇌ ATP exchange activity

Effects of ethacrynic acid and furosemide on phosphory-lation reactions of kidney mitochondria. Inhibition of the adenine nucleotide translocase

Previous reports that ethacrynic acid and furosemide diminish mitochondrial P : O ratios and reduce ( Na + + K +)-ATPase activity suggested that these diuretics may inhibit mitochondrial phosphorylation reactions. This possibility was initially studied by determining the effects of ethacrynic acid and furosemide on [ 32P]ATP exchange activity of rat kidney mitochondria. Concentrations of both drugs at 10 −4 M or greater, significantly inhibited [ 32P]ATP exchange. To investigate the mechanism of this inhibition, the effects of ethacrynic acid and furosemide on the ATPase activity of intact mitochondria and sonicated submitochondrial particles were determined. Both diuretics inhibited ATPase activity of intact mitochondria at 10 −4 M. In contrast, ATPase of submitochondrial particles was significantly less susceptible to inhibition by the diuretics. These results suggested that ethacrynic acid and furosemide inhibit adenine nucleotide transport across the mitochondrial membrane. This was directly tested by determining the effects of the diuretics on the mitochondrial adenine nucleotide translocase. At 5 · 10 −4 M, both ethacrynic acid and furosemide significantly inhibited adenine nucleotide transport. These findings suggest that ethacrynic acid and furosemide may diminish renal tubular solute reabsorption by direct inhibition of adenine nucleotide transport across the mitochondrial inner membrane.

Restoration of P i-ATP exchange in the oligomycin-sensitive ATPase: Effect of a coupling factor

Summary P i -ATP exchange activity has been restored to the bovine heart mitochondrial ATPase. The exchange rates can be doubled by addition of coupling factor 1 (F 1 and the oligomycin-sensitivity conferring protein (OSCP), and further stimulated 2·5-fold by the addition of a coupling factor which has Factor B as a major component. The activity is sensitive to oligomycin, uncoupler or valinomycin + K + .

Modification of phenylalanyl-tRNA synthetase from Escherichia coli by histidine-specific reagents. Effects on structure and function.

Phenylalanyl‐tRNA synthetase from Escherichia coli was subjected to chemical modification with diethylpyrocarbonate at pH 6.0 and to photochemical oxidation in the presence of rose Bengal, both methods giving rise to loss of enzymic activity. The highly sensitive reaction of the enzyme with either reagent is pseudo‐first order. The specificity of the modification methods employed has been investigated.All substrates were studied for their ability to protect the enzyme against inactivation. Presence of phenylalanine, ATP and Mg2+ provides the most pronounced effect. Under this condition the tRNA‐aminoacylation activity may be completely abolished, whereas the pyrophosphate–ATP exchange activity is only partly affected. However, such enzyme is still able to form a specific complex with tRNAPhe. The results presented therefore suggest that histidine residues of phenylalanyl‐tRNA synthetase accessible to modification by diethylpyrocarbonate or to photooxidation do not seem to be involved in the binding of tRNA but might possibly take part in the esterification reaction.Quantitative analysis showed that a total of 50 histidine residues are accessible to diethylpyrocarbonate in the unprotected enzyme under non‐denaturing conditions. In the presence of phenylalanine, ATP and Mg2+ the loss of aminoacylation activity is correlated with the modification of 2–4 residues.During extensive modification of the enzyme a dissociation process takes place. It was found that the observed loss of quaternary structure is not correlated with the decrease of the aminoacylation activity of the enzyme upon modification.