Shigeru Sakajo

Possible involvement of superoxide anion in the induction of cyanide-resistant respiration in Hansenula anomala

A chemiluminescence study showed that Qi site inhibitors such as antimycin A induce O2 − generation in respiring cyanide‐sensitive mitochondria from the yeast, Hansenula anomala. The O2 − generation was suppressed by radical scavengers such as flavone, butylated hydroxyanisole, and Co0. Induction of cyanide‐resistant respiration in H. anomala cells by Qi site inhibitors was also inhibited by these radical scavengers. Furthermore, antimycin A‐induced synthesis of the mitochondrial 36‐kDa protein, which is thought to be the alternative oxidase functional in the cyanide‐resistant respiratory pathway, was abolished by the addition of flavone. These observations suggest that O2 − is somehow involved in the induction of cyanide‐resistant respiration.

Molecular cloning of cDNA for antimycin A-inducible mRNA and its role in cyanide-resistant respiration in Hansenula anomala

A cDNA for mRNA induced by antimycin A in Hansenula anomala was cloned. The mRNA for the cDNA was expressed in the yeast under the conditions expressing the cyanide-resistant respiration activity. The nucleotide sequence revealed a long open reading frame of 342 codons encoding a protein with a molecular weight of 40,282 in the cDNA. An antibody recognizing the protein encoded by the open reading frame was produced. Immunoblotting of H. anomala proteins with this antibody showed that a 36 kDa protein localized in mitochondria was a mature form of the protein encoded by the cDNA. It is suggested that the cloned cDNA encodes a protein involved in the cyanide-resistant respiratory pathway.

An antibiotic, ascofuranone, specifically inhibits respiration and in vitro growth of long slender bloodstream forms of Trypanosoma brucei brucei.

Ascofuranone, a prenylphenol antibiotic isolated from a phytopathogenic fungus, Ascochyta visiae, strongly inhibited both glucose-dependent cellular respiration and glycerol-3-phosphate-dependent mitochondrial O2 consumption of long slender bloodstream forms of Trypanosoma brucei brucei. This inhibition was suggested to be due to inhibition of the mitochondrial electron-transport system, composed of glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) and plant-like alternative oxidase. Ascofuranone noncompetitively inhibited the reduced coenzyme Q1-dependent O2 uptake of the mitochondria with respect to ubiquinol (Ki = 2.38 nM). Therefore, the susceptible site is deduced to be the ubiquinone redox machinery which links the two enzyme activities. Further, ascofuranone in combination with glycerol completely blocked energy production, and potently inhibited the in vitro growth of the parasite. Our findings suggest that ascofuranone might be a promising candidate for the chemotherapeutic agents of African trypanosomiasis.

Erratum to ``An antibiotic, ascofuranone, specifically inhibits respiration and in vitro growth of long slender bloodstream forms of Trypanosoma brucei brucei'': [Mol. Biochem. Parasitol. 81 (1996) 127–136]1

Abstract Ascofuranone, a prenylphenol antibiotic isolated from a phytopathogenic fungus, Ascochyta visiae , strongly inhibited both glucose-dependent cellular respiration and glycerol-3-phosphate-dependent mitochondrial O 2 consumption of long slender bloodstream forms of Trypanosoma brucei brucei . This inhibition was suggested to be due to inhibition of the mitochondria electron-transport system, composed of glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) and plant-like alternative oxidase. Ascofuranone noncompetitively inhibited the reduced coenzyme Q 1 -dependent O 2 uptake of the mitochondria with respect to ubiquinol (K i =2.38 nM). Therefore, the susceptible site is deduced to be the ubiquinone redox machinery which links the two enzyme activities. Further, ascofuranone in combination with glycerol completely blocked energy production, and potently inhibited the in vitro growth of the parasite. Our findings suggest that ascofuranone might be a promising candidate for the chemotherapeutic agents of African trypanosomiasis.

Characterization of the alternative oxidase protein in the yeast Hansenula anomala

The cyanide‐resistant respiratory pathway is induced by respiratory inhibitors in the yeast Hansenula anomala. A monoclonal antibody against the alternative oxidase in the higher plant Sauromatum guttatum cross‐reacted with a 36‐kDa mitochondrial protein induced by antimycin A in H. anomala and with a protein encoded by a cDNA which was previously cloned for an antimycin A‐inducible mRNA in the yeast. There was a similarity in the amino acid sequence between the cDNA‐encoded protein and the plant alternative oxidase protein. We propose that the 36‐kDa mitochondrial protein encoded by the cDNA is a component of alternative oxidase in H. anomala.

Oral and Intraperitoneal treatment of Trypanosoma brucei brucei with a combination of ascofuranone and glycerol in mice

Abstract On the basis of our previous report of ascofuranone, an antibiotic isolated from Ascochyta visiae , which strongly inhibited both the mitochondrial O 2 consumption in mitochondrial preparation and growth of in vitro cultured bloodstream forms of Trypanosoma brucei brucei in combination with glycerol, we investigated the chemotherapeutic efficacy of ascofuranone on experimental African trypanosomiasis in mice. A suspension of ascofuranone (6–200 mg/kg) was given and then glycerol (1 g/kg) was administered orally or intraperitoneally at 30-min intervals to heavily parasitemic mice. Both orally (100 mg/kg) and intraperitoneally (25 mg/kg) administered ascofuranone combined with a total dose of 3 g/kg glycerol showed potent antitrypanosomal activity in infected mice. The trypanocidal activity of ascofuranone was very powerful and all trypanosomes disappeared within 30 and 180 min after final intraperitoneal and oral treatment, respectively. This combination treatment showed high efficacy and low toxicity. Our results strongly suggest that ascofuranone in combination with glycerol may be an effective tool in chemotherapy for African trypanosomiasis.

A 36-kDa mitochondrial protein is responsible for cyanide-resistant respiration in Hansenula anomala

Antimycin A‐dependent induction of cyanide‐resistant respiration in Hansenula anomala was reversibly blocked by carbonylcyanide‐m‐chlorophenylhydrazone (CCCP). When the cells were pulse‐labeled with [35S]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. The increase in the 36 kDa protein completely paralleled that in cyanide‐resistant respiration activity, suggesting that the 39 kDa protein is the precursor of the 36 kDa protein, which is responsible for cyanide‐resistant respiration.

Cell-free synthesis of succinate dehydrogenase and mitochondrial adenosine triphosphatase of sweet potato

Polyadenylated mRNA was isolated from aged slices of sweet potato root tissue and translated in a wheat germ cell-free system. The synthesis of apoprotein of the flavoprotein subunit of succinate dehydrogenase and two of the subunits of mitochondrial adenosine triphosphatase were detected by indirect immunoprecipitation. The molecular weights of the immunologically identified products were 3,000 and 8,000-9,000 daltons larger than the mature flavoprotein subunit of succinate dehydrogenase and the mature subunits of adenosine triphosphatase, respectively.

In vitro synthesis of catalase protein in sweet potato root microbodies

Poly(A)+ RNA prepared from sweet potato root tissue was translated in a wheat germ in vitro translation system. A translation product was immunoprecipitated with anti‐sweet potato catalase immunoglobulin G. The product was identical to the subunit of the catalase with respect to the mobility on an SDS‐polyacryl‐amide gel and the pattern of the peptide map, indicating that the catalase protein is synthesised in vitro in the same size as the mature subunit. No amino acids were released from the purified enzyme protein by Edman degradation, suggesting the occurrence of a minor modification in the N‐terminal part of the protein during the enzyme formation.

Functional expression of the ascofuranone-sensitive Trypanosoma brucei brucei alternative oxidase in the cytoplasmic membrane of Escherichia coli.

Trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms (LS forms) of African trypanosoma, which causes sleeping sickness in human and nagana in cattle. TAO is a cytochrome-independent, cyanide-insensitive quinol oxidase and these properties are quite different from those of the bacterial quinol oxidase which belongs to the heme-copper terminal oxidase superfamily. Only little information concerning the molecular structure and enzymatic features of TAO have been available, whereas the bacterial enzyme has been well characterized. In this study, a cDNA encoding TAO from Trypanosoma brucei brucei was cloned into the expression vector pET15b (pTAO) and recombinant TAO was expressed in Escherichia coli. The growth of the transformant carrying pTAO was cyanide-resistant. A peptide with a molecular mass of 37 kDa was found in the cytoplasmic membrane of E. coli, and was recognized by antibodies against plant-type alternative oxidases from Sauromatum guttatum and Hansenula anomala. Both the ubiquinol oxidase and succinate oxidase activities found in the membrane of the transformant were insensitive to cyanide, while those of the control strain, which contained vector alone, were inhibited. This cyanide-insensitive growth of the E. coli carrying pTAO was inhibited by the addition of ascofuranone, a potent and specific inhibitor of TAO ubiquinol oxidase. The ubiquinol oxidase activity of the membrane from the transformant was sensitive to ascofuranone. These results clearly show the functional expression of TAO in E. coli and indicate that ubiquinol-8 in the E. coli membrane is able to serve as an electron donor to the recombinant enzyme and confer cyanide-resistant and ascofuranone-sensitive growth to E. coli. This system will facilitate the biochemical characterization of the novel terminal oxidase, TAO, and the understanding on the mechanism of the trypanocidal effect of ascofuranone.