Studies on Cyanide-Resistant Respiration in yeast, Hansenula anomala

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(1) Mitochondria exihibiting cyanide-resistant respiration were isolated from Hansenula anomala incubated in the presence of antimycin A or a certain sulfur-compounds. Analysis of mitochondrial proteins by gel electrophoresis indicates that a 36 kDa protein was induced. When the cells were labeled with ^<35>S-methionine in the presence of both antimycin A and CCCP, the radioactivity was incorporated into a 39 kDa mitochondrial protein. Upon removal of CCCP, this protein was processed into a 36 kDa form, suggesting that the 39 kDa protein is the precursor of 36 kDa protein.(2) The antimycin A-dependent induction of cyanide-resistant respiration was completely repressed by specific chelators for Fe^<2+>. The addition of Fe^<2+> to the cells resulted in a rapid increase in cyanide-resistant respiration activity, suggesting that Fe^<2+> is involved in the reaction mechanism of this respiration.(3) Since a plant-like alternative oxidase was reported to serve as a sole terminal oxidase of Tr … More ypanosoma brucei brucei, the studies on the specific inhibitors of cyanide-resistant respiration have taken on a great sibnificance for the chemotherapy of trypanosome infections. We found specific inhibitory effects on cyanide-resistant respiration of some fatty acids with nonconjugated duoble bonds, radical scavengers such as CoQ derivatives and flavonoids, and cation ionophores.(4) Q_i site inhibitors such as antimycin A induce O_2 generation in respiring cyanide-sensitive mitochondria of H. anomala. Radical scavengers suppressed O_2 generation, and inhibited both the induction of cyanide-resistant respiration and the appearance of the mitochondrial 36 kDa protein. These results suggest that O_2 is involved in the induction of cyanide-resistant respiration.(5) A cDNA for mRNA induced by antimycin A was cloned. The nucleotide sequence revealed a long open reading frame of 342 codons encoding a protein. Immunoblotting showed that the 36 kDa protein localized in mitochondria was a mature form of the protein encoded by the cDNA. Less