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The significance of the incorporation of [14C] leucine into different protein fractions by isolated ox heart mitochondria.

ABSTRACT: The problem of whether isolated mitochondria are able to synthesize specific proteins was investigated, particular consideration being paid to the possible contribution of micro-organisms to this activity. With ox heart mitochondria it was shown that: (1) The medium used for the incubations inhibits the exponential phase of bacterial growth for at least 8h either in the absence or the presence of fresh mitochondria, but the inhibition disappears after 4h when mitochondria damaged by freezing and thawing are used. (2) The incorporation of [(14)C]leucine into total proteins is linear up to at least 8h, although part of the radioactivity at the later periods might be due to some incorporation by resting-phase bacteria. (3) A contamination by as little as 800 cells/mg of mitochondrial protein is enough to contribute substantially to the total radioactivity incorporated by the mitochondrial preparations. (4) Purified cytochrome b and cytochrome oxidase are labelled even under conditions of minimal contamination by micro-organisms (less than 60 cells/mg of mitochondrial protein) and the contribution of bacterial proteins to the radioactivity found in cytochromes is negligible, as shown by double-labelling experiments. (5) At 4h the specific radioactivities of cytochrome b and cytochrome oxidase are seven- and 16-fold lower respectively than that of a structural protein-rich fraction, suggesting that the labelling of cytochromes is due to a residual contamination by these proteins.

SUBMITTER: Krymkiewicz N

PROVIDER: S-EPMC1185355 | biostudies-other | 1970 Jan

REPOSITORIES: biostudies-other

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Project description:We report the identification of an NADH-dependent haem-degrading system in ox heart mitochondria. The activity was localized to the mitochondrial inner membrane, specifically associated with complex I (NADH:ubiquinone oxidoreductase). The mitochondrial NADH-dependent haem-degradation activity was highly effective and displayed a rate nearly 60% higher than that of the microsomal activity. The following observations suggested the enzymic nature of the activity: (i) haem degradation by complex I did not proceed upon exposure to elevated temperature and extremes of pH; (ii) it displayed substrate specificity; (iii) it was inhibited by a substrate analogue; and (iv) it showed a cofactor requirement. Moreover, the activity was distinctly different from the ascorbate-mediated haem-degradation activity. Also, complex I differed from the microsomal NADPH:cytochrome c (P-450) reductase inasmuch as the formation of an effective interaction with the microsomal haem oxygenase could not be detected. Addition of purified haem oxygenase to complex I neither influenced the rate of haem degradation nor resulted in the formation of biliverdin IX alpha. In contrast, addition of haem oxygenase to NADPH:cytochrome c (P-450) reductase enhanced the rate of haem degradation by nearly 8-fold, and more than 60% of the degraded haem could be accounted for as biliverdin IX alpha. The haem-degrading activity of complex I appeared to involve the activity of H2O2, as the reaction was inhibited by nearly 90% by catalase, and propentdyopents were detected as reaction products. Intact haemoproteins such as cytochrome c and myoglobin were not effective substrates. However, the haem undecapeptide of cytochrome c was degraded at a rate equal to that observed for haem. Haematohaem was degraded at a rate 50% lower than that observed for haem. It is suggested that the NADH-dependent haem-degradation system may have a biological role in the regulation of the concentration of respiratory haemoproteins and the disposition of the aberrant forms of the mitochondrial haemoproteins.

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The significance of the incorporation of [14C] leucine into different protein fractions by isolated ox heart mitochondria.

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