Nobuo Miki

Transcriptional activation of the alternative oxidase gene of the fungus Magnaporthe grisea by a respiratory-inhibiting fungicide and hydrogen peroxide

Alternative oxidase (AOX) is dramatically induced when the fungus Magnaporthe grisea is incubated with the fungicide SSF-126, which interacts with the cytochrome bc1 complex in the electron transport system of mitochondria. A full-length cDNA for the alternative oxidase gene (AOX) was obtained, and the deduced amino acid sequence revealed marked similarity to other AOXs, but lacks two cysteine residues at corresponding sites which are conserved in plant AOXs and play essential roles in the post-translational regulation. Northern blot experiments showed that treatment of M. grisea cells with SSF-126 induces accumulation of AOX mRNA in a dose-dependent manner, and the level was correlated with the activity of alternative respiration. H2O2 also induced the accumulation of the transcript with a short half-life (<15 min). Nuclear run-on experiments showed that the AOX gene was transcribed constitutively in unstimulated cells. Cycloheximide did not change the basal level of transcription, but induced the accumulation of the transcript, indicating that active degradation of the transcript occurs by factor(s) sensitive to cycloheximide. On the other hand, SSF-126 enhanced the transcriptional activity of AOX gene threefold compared to that of control cells, and H2O2 was also potent for enhancement of the transcription. From these results, it is concluded that the respiratory inhibitor-dependent activation of the transcription is a primary determinant for the induction of alternative respiration in M. grisea. Because we have previously shown that SSF-126 treatment of M. grisea mitochondria induced the generation of superoxide, active oxygen species are thought to be signal mediators to activate AOX gene transcription in M. grisea.

The obligatory requirement of cytochrome b5 in the p-nitroanisole O-demethylation reaction catalyzed by cytochrome P-450 with a high affinity for cytochrome b5

The role of cytochrome b5 in the p-nitroanisole O-demethylation was studied with a reconstituted system containing a unique cytochrome P-450, isolated from rabbit liver microsomes as a species with a high affinity for cytochrome b5. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome b5 reductase, and Triton X-100 in addition to cytochrome b5. The omission of cytochrome b5 from the complete system entirely abolished the activity. These results clearly show that cytochrome b5 is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome b5 in such a way that the second electron is transferred from cytochrome b5 and thus exhibits the demethylase activity.

Effect of the methoxyiminoacetamide fungicide, SSF129, on respiratory activity in Botrytis cinerea

(E)-2-Methoxyimino-N-methyl-2-[2-(2,5-dimethylphenoxymethyl)phenyl]acetamide (SSF129) has been developed as a broad-spectrum systemic fungicide for control of cereal and fruit diseases. This compound inhibited NADH-oxidation by submitochondrial particles from mycelial cells of Botrytis cinerea, with an EC 50 value of 14.5 nM, due to blockage of electron transport through the cytochrome bc 1 complex in the mitochondrial respiratory chain. However, SSF129 did not suppress, but rather increased, oxygen consumption by mycelial cells of the fungus. This was because mycelial cells contain an alternative oxidase protein and the cells have the ability to rapidly switch electron flux from the main cytochrome pathway to the alternative pathway on blockage of the former by SSF129. The alternative pathway of the mycelia seems not to be operative when the cytochrome pathway is functional. Naturally occurring flavonoids inhibited the alternative oxidase of the mycelial cells in a dose-dependent manner, with EC 50 values of 68.4 μM for flavone and 63.7 μM for flavanone. These observations suggested that plant components play an important role in control of gray mould by SSF129.

Anaerobic dehalogenation of halothane by reconstituted liver microsomal cytochrome P-450 enzyme system☆

Abstract Cytochrome P-450 from liver microsomes of phenobarbital-treated rabbits catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) when combined with NADPH and NADPH-cytochrome P-450 reductase. Cytochromes P-450B1 and P-448 from liver microsomes of untreated rabbits were less active. Triton X-100 accelerated the reaction. Unlike anaerobic dehalogenation of halothane in microsomes, the major product was 2-chloro-1,1,1-trifluoroethane and 2-chloro-1,1-difluoroethylene was negligible. These products were not detected under aerobic conditions, and dehalogenation activity was inhibited by carbon monoxide, phenyl isocyanide and metyrapone.

Thermally induced conformational and spin-state changes of cytochrome b5

Reversible changes of the conformation and spin-state of cytochrome b5 with varying temperature were studied by absorption spectroscopy. Above 45°C, the Soret absorption peak due to the low-spin state decreased in intensity with a blue-shift of 25 nm. This spectral transition from the low- to the high-spin state was found to take place in a relatively limited temperature range from 45 to 65°C, and furthermore, this transition was found to be reversible at temperatures below 55°C. Thermodynamic parameters were obtained for the spin-state change: ΔH value of 32.3 kcal/mol, ΔS, 97.5 e.u. and ΔG°, 3.25 kcal/mol. Above 55°C, the irreversible denaturation of cytochrome b5 started to occur besides the equilibrium between the low- and high-spin forms of native cytochrome b5. The small decreases in the electron-transfer activity from NADH-cytochrome b5 reductase to cytochrome c and in the reconstitution capability of p-nitroanisole O-demethylase were observed during the denaturation process in parallel with a small decrease in absorbance at 413 nm. These results indicate that the conformational and spin-state changes in the cytochrome b5 molecule are completely reversible in the temperature range below the transition temperature and that the cytochrome b5 molecule does not lose the physical and enzymatic properties of the native cytochrome b5 as long as the treatment is done below the transition temperature.

A prominent feature of the conversion of P-450 to P-420 of cytochrome P-450B1 among the cytochrome P-450 isozymes

Conversion of cytochrome P-450 to P-420 was observed with the use of three isozymes of P-450, P-450PB, P-450MC and P-450B1. The last one, which was isolated and characterized in our laboratories, is the cytochrome P-450 with high affinity for cytochrome b5. Of these isozymes, cytochrome P-450B1 is predominantly fast in the rate of conversion from P-450 to P-420 in the reduced state under carbon monoxide. p-Nitroanisole, which is the substrate of P-450B1 for demethylation, accelerated the conversion, whereas the effects of the compound on the rate of conversion of the other P-450S were small. The effect of cholate on the conversion was distinct and rapid but not very selective among the isozymes. Stabilization with glycerol for prevention of the conversion was found to be effective, for any of these isozymes. No remarkable difference was observed in the stability of the oxidized state among these isozymes when detected in the CO-reduced form. The rates of the reaction from the oxidized to the CO-reduced form were measured with these isozymes. The rate of P-450B1 was the highest in both the medium with glycerol and that without glycerol. Circular dichroism was measured with respect to conversion of P-450 to P-420. The absorption at 450 nm was related to the significant circular dichroism, while the increased absorption at 420 nm due to the conversion was not accompanied by distinct circular dichroism. These data support the concept that the heme vicinity of cytochrome P-450B1 is more labile in the structure of the reduced form under carbon monoxide than those of the other isozymes.

PURIFICATION AND SOME PROPERTIES OF CYTOCHROME P-450 WITH HIGH AFFINITY FOR CYTOCHROME b5

Cytochrome P-450, which shows high affinity for cytochrome b5, has been purified from rabbit liver microsomes using immobilized cytochrome b5 as an affinity adsorbent to a gel-electrophoretically homogeneous state. The purified cytochrome has a minimum molecular weight of 52,000 and shows a different electrophoretic mobility from that of the phenobarbital-inducible or 3-methylcholanthrene-inducible form on polyacrylamide gel in the presence of sodium dodecyl sulfate.