Akio Yoshimoto

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.

Studies on yeast sulfite reductase I. Purification and characterization

1. NADPH-sulfite reductase, catalyzing the reduction of sulfite to sulfide by NADPH, was purified from bakers yeast to an almost homogeneous state. The purified enzyme showed a sedimentation coefficient of 14.8 S. A minimal molecular weight of 350 000 was estimated from its flavin content. 2. The oxidized enzyme was greenish yellow in color, showing absorption peaks at 386, 455 and 587 mμ. The intensity of these peaks was lowered considerably by NADPH or dithionite. 3. The enzyme contained 1 mole each of FMN and FAD per 350 000 g of protein. About 5 atoms of non-heme iron were detected per mole of FMN or FAD. The enzyme also contained a non-flavin chromophore (or chromophores) absorbing at 587 mμ and at about 386 mμ. The 587-mμ chromophore was unstable to treatment causing alterations in protein conformation. Five to six moles of p-chloromercuribenzoate combined with 350 000 g of the enzyme protein. 4. The enzyme catalyzed, besides the NADPH-sulfite reductase activity, the reduction of sulfite by reduced viologen dyes, the reductions by NADPH of nitrite, hydroxylamine, ferricyanide, cytochrome c, quinones, and 2,6-dichlorophenolindophenol, and the reduction of NADP+ by reduced methyl viologen. 5. All the NADPH-linked activities were inhibited by NADP+, 2′-AMP and p-chloromercuribenzoate. NADP+ and 2′-AMP, however, did not affect the reduction of sulfite by reduced viologen dyes. 6. Cyanide strongly inhibited the NADPH-sulfite reductase activity only when the reaction was started by adding sulfite to the enzyme which had been incubated with NADPH and cyanide. The addition of cyanide to the NADPH-reduced (but not the oxidized) enzyme caused an irreversible conversion to a reddish violet form. This conversion was prevented by the presence of sulfite.

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.

Studies on yeast sulfite reductase: III. Further characterization

Abstract 1. 1. Acid ammonium sulfate treatment of purified yeast NADPH-sulfite reductase caused selective detachment of FMN from the protein, leaving FAD still attached to the precipitated enzyme. The NADPH-linked activities of the treated enzyme could be partly restored by the addition of FMN or FAD. 2. 2. The NADPH-linked activities, but not the reduced methyl viologen (MVH)-sulfite reductase activity, of the enzyme were inactivated on exposure to low ionic strength. This inactivation was accompanied by a decrease in sedimentation coefficient for the enzyme protein. The NADPH-sulfite reductase activity thus inactivated could be restored, though to very limited extents, by the addition of a protein fraction from yeast mutants incapable of reducing sulfite. 3. 3. The presence in yeast crude extracts of at least two types of NADPH-cytochrome c reductase activity was demonstrated; one type of activity was associated with the sulfite reductase and sensitive to low salt concentrations, whereas the other was stable to low ionic strength. The sensitive reductase activity was absent in all the mutant strains which are genetically blocked in the sulfite reduction step. 4. 4. Heat treatment of the purified enzyme decreased the heights of absorption peaks at 587 and 386 mμ, and the loss of the MVH-sulfite reductase activity paralleled the decrease in the intensity of the 587-mμ peak. 5. 5. The results of ultraviolet irradiation of the enzyme provided evidence that the 587-mμ chromophore also absorbs in the 386-mμ region. 6. 6. The enzyme possessed two different fluorescent species; one seemed to be flavin(s) and the other was probably the 587-mμ chromophore. 7. 7. The ESR spectrum of the reduced enzyme, but not of the oxidized enzyme, showed a sharp signal at g = 2.

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.

Studies on yeast sulfite reductase II. Partial purification and properties of genetically incomplete sulfite reductases

Abstract Enzymes catalyzing the reduction of sulfite by reduced methyl viologen (MVH) were partially purified from four mutants of Saccharomyces cerevisiae, strains 6, 11, 20 and 21, which are genetically blocked in the sulfite reduction step in the sulfate assimilation pathway. Unlike NADPH-sulfite reductase from the wild-type strain, the enzymes from the mutants showed no activities coupled to NADPH oxidation. Sedimentation coefficients of these mutant enzymes ranged from 5.1 S to 6.6 S, values which are much smaller than the value of 14.8 S determined for yeast NADPH-sulfite reductase. All the mutant enzymes contained a chromophore or chromophores absorbing at 386 and 587 mμ. In contrast to the wild-type enzyme possessing both FMN and FAD, the enzymes from strains 6, 11 and 20 contained only FMN, and that from strain 21 lacked both flavins. Iron and acid-labile sulfide were detected in these mutant enzymes as well as in the wild-type enzyme.

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.