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Antibodies against isolated beef-heart ubiquinol- cytochrome c reductase (complex III) have been characterized. Antibodies to complex III react strongly with isolated beef heart complex III and intact beef heart mitochondria, as shown by immunodiffusion and rocket electrophoresis experiments. The complex III content of intact mitochondria can be quantitated with rocket electrophoresis using isolated complex III as a standard. Antibodies to complex III also react with beef liver mitochondria and with both heart and liver mitochondria from rats. The latter are very weak antigens compared to beef heart material. Antibodies to complex III do not react with respiratory chain complexes I and IV, or F1-ATPase from beef heart mitochondria, but gives a slight, but variable, reaction with complex II and the membrane fraction isolated from complex V (oligomycin-sensitive ATPase). Antigenic sites are located on at least five of the seven peptides of complex III. These peptides are presumably lacking in respiratory chain complexes which do not react with antibodies to complex III, and are assumed to be uniquely located in complex HI. Antiserum against complex III inhibits duroquinol-cytochrome c reductase activity in isolated complex III and in complex III incorporated into phospholipid vesicles. Oxidation of NADH and succinate is not affected in submitochondrial particles treated with 6-times more antibody than required for complete inhibition of enzyme activity in free complex III or in complex III-phospholipid vesicles. [ABSTRACT FROM AUTHOR]

The arrangement of the peptides of free complex III and complex III incorporated into phospholipid vesicles was investigated by labeling with 125I + lactoperoxidase and with p-diazoniumbenzene [35S]sulfonate (N=N-PhSO3H). The peptides were separated by sodium dodecylsulfate acrylamide gel electrophoresis and scanned for radioactivity. In free complex III, the most extensive iodination occurred in peptides II and IV, whereas all polypeptides (I -VI), except peptide VII, could be coupled to N=N-PhSO3H. Peptide VII was, however, labeled in the sodium-dodecylsulfate- dissociated enzyme. These results show that, with the possible exception of peptide VII, all peptides are at least partially exposed on the surface of the isolated complex. Complex III incorporated into phospholipid vesicles showed a labeling pattern which differed from the free enzyme, both with respect to the total incorporation and to the relative incorporation between the peptides. Total incorporation of N=N-Ph35SO3H and 125I was decreased by 60 - 70% in complex III=vesicles, suggesting that a large number of the binding sites on the free enzyme become buried within the membrane. In addition, peptides I, II, IV and V were relatively less labeled in complex III-vesicles than were peptides III and VI, suggesting a deeper location of the former peptides within the membrane. This is supported by the finding that labeling of deoxycholate-treated complex III-vesicles with N = N - Ph35SO3H results in an incorporation pattern similar to that in free complex III. Binding of N = N -PhSO3H and its effects on enzymatic activity was also studied in free and membrane-bound complex III. Both the binding of N = N -Ph35SO3H and inactivation of duroquinol -cytochrome c reductase were significantly slower in complex Ill-vesicles. These results indicate that many of the N = N -PhCO3H-binding sites on the free complex are inaccessible when the enzyme is incorporated into a vesicle. This approach was also used to ascertain intactness of the vesicles, and to show that N = N -PhSO3H labeled only those peptides exposed on the outer surface. [ABSTRACT FROM AUTHOR]

The peptides of purified complex III from beef heart mitochondria have been studied by electrophoresis on dodecylsulfate gels. Of the 12 peptides consistently observed, only eight appear to be integral peptides of the functional complex. Attempts to identify these peptides have been made through co-electrophoresis of complex [I and fractions in which the individual peptides were either purified or greatly enriched. Electrophoresis of complex III preparations which were not reduced by mercaptoethanol indicates that intermolecular disulfide bonds play no significant role in stabilizing the complex. [ABSTRACT FROM AUTHOR]

The import and processing of cytochrome c1 and the iron sulfur protein of the cytochrome b-c1 complex were studied in Zajdela hepatoma ascites cells. Both peptides were synthesized as larger percursor molecules which were approximately 2–3 kDa and 5–6kDa larger than the mature forms of apocytochrome c1 and apo-iron sulfur protein, respectively. Comparison of these precursors to those reported for functionally homologous peptides in yeast and Neurospora indicate significant size changes have occurred in mammals. Rhodamine 6G, a specific vital stain for mitochondria, is a potent inhibitor of precursor processing in isolated hepatoma cells. Both precursor to cytochrome c1 and precursor to FeS accumulate in the soluble and particulate fractions obtained by digitonin treatment of tumor cells treated with Rhodamine 6G. Appearance of the mature peptides was abolished. The precursors are unstable, however, and disappear from the cytosolic and membrane fractions during a 10min chase. Comparison of the effects of Rhodamine 6G and carbonylcyanide m-chloro- phenylhydrazone on precursor processing shows that: (a) Rhodamine 6G is a more effective inhibitor of processing. (b) it has less of an inhibitory effect on cellular protein synthesis, and (c) it inhibits processing trader conditions in which it appears to have little influence on coupled respiration in whole cells. The data suggest that the most likely mode of action of Rhodamine 6G is on the matrix processing step. [ABSTRACT FROM AUTHOR]

Several inner membrane proteins from rat liver mitochondria have been translated for the first time in rabbit reticulocyte lysates. These include the Rieske iron-sulfur protein, cytochrome c1 and core protein I of the cytochrome bc1 complex, the α and β subunits of F1 ATPase, and subunit IV of cytochrome oxidase. All were translated from free polysomes as larger-molecular-mass precursors, and were processed to their mature forms by isolated liver mitochondria or by the isolated mitochondrial matrix fraction. In vitro processing, catalyzed by the isolated matrix fraction, is inhibited by rhodamine 6G. The latter is a fluorescent probe, which accumulates specifically in mitochondria of whole cells and which is used extensively to visualize mitochondrial morphology. The concentration of rhodamine 6G required for inhibition in vitro is similar to that of o-phenanthroline. Rhodamine 6G inhibits matrix-catalyzed processing of all precursors tested, indicating that the mechanism of inhibition is common for a variety of functionally unrelated precursors. The novel action of rhodamine 6G reported here can form the basis for its inhibition of precursor processing in intact hepatoma cells.

Focuses on the isolation of the cytochrome-bc1 complex from rat-liver mitochondria. Information about various methods of isolation of the cytochrome-bc1 complex from rat-liver mitochondria; Purpose of electrophoresis. [ABSTRACT FROM AUTHOR]

Purified complex III (ubiquinol-cytochrome c reductase) from beef heart mitochondria was alkylated with iodo[1-14C]acetamide. After 6–8 h of incubation with iodo[1-14C]acetamide, duroquinol and ubiquinol-2-cytochrome c reductase activities were inhibited approximately 50%. During this time 4.5 ± 1.6 nmol of iodo[1-14C]acetamide reacted per mg of complex III protein. Experiments carried out over 24 h indicated that enzyme activity could be inhibited to 70% and that alkylation of complex III was proportional to inhibition. The rates of cytochrome b and c1 reduction by duroquinol are also decreased upon treatment of complex III with iodoacetamide. Separation of the peptides of complex III by electrophoresis in sodium dodecylsulfate shows that all of the radioactivity is located in a single peptide of 50000 molecular weight, which has been identified as one of the two core proteins. The possible functions of core protein are discussed. [ABSTRACT FROM AUTHOR]

1. The incorporation of small amounts of ubiquinone (Q) into pentane-extracted submitochondrial particles has been studied. A procedure is described which allows nearly quantitative incorporation of as little as 0.2 to 0.3 nmol Q/mg protein, i.e: less than 10 % of the Q present in the lyophilized particles prior to extraction. 2. It is shown that both NADH oxidase and succinate oxidase activities can be restored to 100 % of that in lyophilized particles by incorporation of 6-8 nmol Q/mg protein. The relative activities of both oxidases show a parallel response to an increase of the Q content of the particles between 0.2 and 22 nmol Q/mg protein, 50% reactivation being obtained at about 2 nmol Q/mg protein. 3. 50 to 60% of the incorporated Q can be reduced by NADH or succinate, independent of the total concentration of Q in the membrane. Similar reduction values were obtained for lyophilized particles prior to extraction, suggesting that incorporated Q functions similarly to that originally present in the particles. 4. Succinate dehydrogenase activity, which is decreased % by extraction of Q. can be completely restored upon incorporation of only 1.5 nmol Q/mg protein. 5. Extraction of lyophilized particles with pentane + 10% acetone results in a more effective removal of Q, but also in a differential Q requirement for NADH oxidase and succinate oxidase. Thin-layer chromatography shows that extraction with pentane + acetone removes an additional, unidentified, nonpolar lipid. 6. The present data do not support the belief that succinate oxidase and NADH oxidase communicate with separate pools of Q. [ABSTRACT FROM AUTHOR]

The effects of thyroid hormone on nuclear-encoded mitochondrial inner membrane proteins were investigated by in vitro translation of the endogenous mRNA present in a postmitochondrial fraction from the livers of rats treated in vivo with hormone. The levels of the mRNAs were estimated by quantitative immunoabsorption of the translation mixture. Total protein synthesis was increased 2.6-fold after 4 days of in vivo hormone treatment, but only 10-15% of the polypeptides were dramatically altered (> 5-fold). Among the most highly elevated were cytochrome c1 (> 10-fold increase) and the Rieske iron-sulfur protein of the cytochrome bc1 complex. Other inner membrane proteins (core protein 1, β subunit of F1 ATPase, subunit IV of cytochrome oxidase, 3-hydroxybutyrate dehydrogenase) and non-mitochondrial proteins (rat serum albumin, β2-microglobulin) were not altered significantly by hormone treatment. Cytochrome c1 and the Rieske protein increased after 12 h of hormone treatment, a relatively early response in mammalian mitochondrial biogenesis. The possible significance of this response for the regulation of mitochondrial synthesis and assembly is discussed. [ABSTRACT FROM AUTHOR]

Thyroid hormone regulation of rat liver mitochondrial transcription was investigated. Steady-state levels of mitochondrial transcripts were measured by Northern blot analysis using cloned fragments of rat mtDNA. Thyroid hormone increased the steady-state concentrations of all mitochondrial mRNAs by 2–8 fold after 1–3 days of hormone treatment, whereas no significant change in the mitochondrial rRNA was observed. Analysis of transcript synthesis in isolated mitochondria shows that part or all of this increase is accounted for by elevated synthesis. Mechanisms by which thyroid hormone regulates transcription of the mitochondrial genome are discussed. [ABSTRACT FROM AUTHOR]