Hirohiko Katsuki

Improved direct method for determination of keto acids by 2,4-dinitrophenylhydrazine.

An improved method was devised for the determination of keto acid by 2,4-dinitrophenylhydrazine with the omission of the extraction procedure with organic solvent. Measurement of the coloration after addition of alkali was conducted at a particular wavelength not affected by the proportion of cis and trans isomers of the hydrazone. This method was applied to the analysis of some aliphatic keto acids; almost complete recoveries were obtained.

Further analysis of cDNA clones for maize phosphoenolpyruvate carboxylase involved in C4 photosynthesis Nucleotide sequence of entire open reading frame and evidence for polyadenylation of mRNA at multiple sites in vivo

Four clones of cDNA for phosphoenolpyruvate carboxylase [EC 4.1.1.31] were obtained from a maize green leaf cDNA library by colony hybridization. The largest cDNA was of full‐length (3335 nucleotides), being 243 nucleotides longer than the cDNA cloned previously [(1986) Nucleic Acids Res. 14, 1615–1628]. Alignment of the sequence for the N‐terminal coding region found in two of the four clones with the sequence reported previously, established the sequence of the entire coding region for the enzyme. The sequencing of 3′‐untranslated region of the clones revealed that the poly(A) tract is attached at multiple sites in vivo.

Involvement of cytochrome P-450 in Δ22-desaturation in ergosterol biosynthesis of yeast

Abstract Δ 22 -Desaturation of ergosta-5,7-dien-3β-ol to form ergosterol was studied with the microsomal fraction of Saccharomyces , cerevisiae . NADPH and molecular oxygen were necessary for the reaction. The reaction was inhibited by carbon monoxide, metyrapone, cytochrome c , ferricyanide, menadione or p -hydroxymercuribenzoate but not inhibited by cyanide or azide. These results strongly suggested the involvement of cytochrome P-450-containing monooxygenase system in the reaction.

Physiological functions of NAD- and NADP-linked malic enzymes in Escherichia coli

Abstract Regulatory mechanisms for biosyntheses of NAD- and NADP-linked malic enzymes in E. coli cells were studied. The NAD-enzyme was repressed by glucose and induced by malate. The repression by glucose was overcome by the addition of malate. In contrast, the NADP-enzyme was repressed by glucose, glycerol, lactate or acetate in decreasing order of magnitude, in the absence and presence of malate which gave a high level of the enzyme. These results, together with 14C-incorporation experiments into fatty acids from 1,4- or 2,3-14C-labeled succinate by using resting cells and the results so far reported, suggest that the NAD-enzyme takes a role in the catabolism of malate, while the NADP-enzyme, in the supply of acetyl-CoA from malate via pyruvate.

Characterization of sterol-ester synthetase in saccharomyces cerevisiae

Cell-free extracts of Saccharomyces cerevisiae grown under aerobic as well as semi-anaerobic conditions were found to catalyze the synthesis of fatty acid ester of sterol from cholesterol, fatty acid, ATP and CoA, or from cholesterol and fatty acyl-CoA. This result indicates that the enzyme involved in the formation of the ester is acyl-CoA:sterol O-acyltransferase (EC 2.3.1.26). The enzyme had a broad substrate specificity for sterols and acyl-CoAs. The enzyme levels in the cells grown under aerobic and semi-anaerobic conditions were almost equal. The enzyme was located in the microsomal fraction of the aerobically grown cells.

Involvement of cytochrome P-450 and a cyanide-sensitive enzyme in different steps of lanosterol demethylation by yeast microsomes

Summary In the presence of NADPH, NAD+, and molecular oxygen, microsomes prepared from Saccharomyces cerevisiae converted [1,7,15,22,26,30-14C]lanosterol to 4,4-dimethylzymosterol, 4-methylzymosterol, and zymosterol, and this conversion was accompanied by the liberation of 14CO2 derived from the methyl group (C-30) at the 4-position. The 14CO2 formation was inhibited by antibodies to yeast cytochrome P-450 and by cyanide. Gas chromatographic evidence indicated that the antibodies inhibited the conversion of lanosterol to 4,4-dimethylzymosterol, whereas the demethylation of the latter to 4-methylzymosterol was sensitive to cyanide. It is concluded that cytochrome P-450 and a cyanide-sensitive enzyme are involved in the 14α- and 4-demethylations of lanosterol, respectively, by yeast microsomes.

Two species of cytochrome P-450 involved in ergosterol biosynthesis of yeast

Discrimination of cytochrome P-450 involved in delta 22-desaturation of ergosta-5,7-dien-3 beta-o1 (P-450(22)-DS) from that involved in lanosterol 14 alpha-demethylation (P-450(14)-DM) in ergosterol biosynthesis was investigated with microsomes of several strains of Saccharomyces cerevisiae. In mutant N22 which is partially defective in the delta 22-desaturation, the 14 alpha-demethylation was not blocked. In contrast, mutant SG1 which is known to lack the 14 alpha-demethylation showed a significant activity of the delta 22-desaturation. The delta 22-desaturation activity was markedly increased upon aerobic adaptation of yeast cells but the 14 alpha-demethylation was not affected. Buthiobate, a specific inhibitor of P-450(14)-DM, and rabbit antibodies against P-450(14)-DM did not inhibit the delta 22-desaturation activity at all. It is evident from the obtained observations that these phenomena are not explainable in terms of NADPH-cytochrome P-450 reductase. These results indicate that P-450(22)-DS is different from P-450(14)-DM in molecular species.

Molecular cloning of the phosphoenolpyruvate carboxylase gene, ppc, of Escherichia coli

The ColE1 hybrid plasmid, pLC20-10, carrying the ppc gene and the argECBH gene cluster of Escherichia coli K-12, was characterized. The ppc gene coding for phosphoenolpyruvate carboxylase (EC 4.1.1.31), was subcloned into the plasmid pBR322 to give the plasmids pS2 and pS3. These plasmids carried a 4.4-kb SalI segment containing the ppc gene, in both orientations. The specific activity of the enzyme was increased approx. 20-fold by these plasmids. Experiments with maxicells harboring pS2 showed that the 90-kDal enzyme subunit was encoded by the plasmid. The location of the ppc gene in pS2 and the direction of transcription of the gene were determined. In DNA-DNA hybridization experiments using pS2 as a probe, significant hybridizations were observed with DNAs from E. coli strains K-12 and W, and from Salmonella typhimurium, but not with those from Chlorella regularis, Anacystis nidulans, Rhodospirillum rubrum, and Pseudomonas AM-1.

Activation of phosphoenolpyruvate carboxylase of Escherichia coli by free fatty acids or their coenzyme a derivatives

Abstract Phosphoenolpyruvate carboxylase of Escherichia coli was found to be remarkably activated by free fatty acid, such as laurate and oleate, and by its CoA derivative. The regulatory site of the enzyme for binding with these compounds was discriminated from the site for acetyl-CoA (one of the allosteric activators, on the basis of the isolation of the mutant having the altered phosphoenolpyruvate carboxylase which is almost insensitive to these compounds but still sensitive to acetyl-CoA. Physiological significance of this activation was also discussed.

The determination of pyruvic acid by 2,4-dinitrophenylhydrazine method

Abstract A modified method for pyruvic acid (PA) determination was devised based on the following two facts: (a) cis and trans isomers of 2,4-dinitrophenylhydrazones (DNPH) of PA, by adding an equal volume of petroleum ether to an ethyl acetate layer, could be completely extracted from the organic solvent layer with 0.1 N sodium carbonate. (b) PA-DNPH could be determined by measuring either the absorbance of their sodium carbonate solution at 355 mμ or that of their sodium hydroxide solution at 417 mμ, irrespective of the proportion of both isomers. PA, in the presence of α-ketoglutaric acid (KGA), was found to be determined by combining the present method with the salting-out extraction method for KGA determination.