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H Katsuki

Publications and source records attributed to H Katsuki.

At least 109 records · Page 6Linked to original sources
PubMed1985

Cloning of phosphoenolpyruvate carboxylase gene from a cyanobacterium, Anacystis nidulans, in Escherichia coli.

The phosphoenolpyruvate carboxylase gene (ppc) from Anacystis nidulans, a cyanobacterium (blue-green alga), was cloned in Escherichia coli. Chromosomal DNA of A. nidulans was partially digested with Sau3AI, and the obtained DNA fragments were ligated in the BamHI site of pBR322. The hybrid plasmids were first transformed into E. coli K802 (hsdR-, hsdM+) to obtain the gene bank of A. nidulans. The bank consisted of about 12,000 clones. These hybrid plasmids were then transformed into E. coli PCR1 (ppc2-, recA1-, hsdR+, hsdM+), and the transformants were selected by complementation of the ppc mutation (phenotype of glutamate requirement). In the cell-free extracts of E. coli strains having the cloned ppc gene, PEPCase activities were detected, but their properties were different from those of the E. coli enzyme. Analysis by subcloning showed that the ppc gene was included in a DNA fragment 3,500 base pairs long and the maxicell method revealed that the molecular weight of the gene product was about 108,000. It is suggested that the ppc gene is expressed in E. coli mainly by read-through transcription, being initiated by the promoter of tetracycline-resistance gene of pBR322, but the significant expression in reversed orientation of the cloned ppc gene indicates that the gene includes a promoter capable of functioning in E. coli cells.

Carboxy-Lyases↗
PubMed1985

Effect of photooxidation on catalytic and regulatory properties of NAD-linked malic enzyme from Escherichia coli.

In an aim to elucidate the structure-function relationship of NAD-linked malic enzyme [EC 1.1.1.38] from Escherichia coli W, the effect of chemical modification on the catalytic and regulatory properties of the enzyme was studied. Upon photooxidation of the enzyme in the presence of methylene blue, a time-dependent inactivation occurred following pseudo-first order kinetics. The pH-dependence of the inactivation rate exhibited a pK value of 6.1. L-Malate, NAD+, and Mn2+ markedly protected the enzyme against the inactivation. Prior masking of the catalytically essential sulfhydryl groups with p-mercuribenzoate did not result in a retardation of the rate of photoinactivation. This excluded the possibility of an involvement of sulfhydryl group modification in the photoinactivation. Although the Km values for L-malate and NAD+ were not affected by photooxidation, the S0.5 value and the Hill coefficient for Mn2+ were considerably altered, and the cooperative nature of the saturation profile for Mn2+ in the native enzyme was completely abolished. The activating effect of L-aspartate on the native enzyme was completely abolished upon photooxidation, and the inhibitory effect of CoA was also diminished to a marked extent upon the treatment. The oxaloacetate decarboxylating activity of the enzyme was lost in parallel with the loss of the activity for oxidative decarboxylation of L-malate. These results suggest a possible involvement of histidyl residue(s) in the catalytic and regulatory functions of the enzyme.

Escherichia coli↗
PubMed1985

Distinct effects of pyridoxal phosphate on NAD- and NADP- linked malic enzymes of Escherichia coli.

NADP-linked malic enzyme from Escherichia coli W was inactivated by pyridoxal 5'-phosphate (PLP) following pseudo-first order kinetics. The inactivation was, however, reversed upon addition of an aminothiol, such as penicillamine and cysteamine, whereas the activity was not restored, when the PLP-inactivated enzyme was treated with NaBH4 prior to the addition of aminothiol. The inactivating effect was specific to PLP and no other structural analogs of PLP tested inactivated the enzyme, except that pyridoxal exhibited a similar effect, though to a lesser extent. In contrast, NAD-linked malic enzyme from the same micro-organism was insensitive to PLP, even in the presence of 0.8 M guanidine hydrochloride.

Escherichia coli↗
PubMed1984

Reaction mechanism of phosphoenolpyruvate carboxylase. Bicarbonate-dependent dephosphorylation of phosphoenol-alpha-ketobutyrate.

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) of Escherichia coli was found to catalyze the cleavage reaction of phosphoenol-alpha-ketobutyrate, a potent competitive inhibitor with the substrate, to yield inorganic phosphate and alpha-ketobutyrate. The rate of phosphate liberation was about 1/20 th of that in the normal reaction with phosphoenolpyruvate. Although HCO3- and Mg2+ were the necessary components in this reaction as in the normal reaction, no CO2 fixation could be detected. When the reaction was carried out in the presence of [18O]HCO3-, multiple incorporations of 18O atoms into the liberated phosphate molecule were observed. The molar proportions of phosphate having one, two, and three 18O atoms were 70, 25, and 5%, respectively. No multiple but only one 18O atom incorporation was observed when phosphoenolpyruvate was used as a substrate. These results suggest that the liberation of phosphate can proceed without CO2 fixation, being not consistent with the concerted mechanism [ Maruyama , H., Easterday , R. L., Chang, H. C., & Lane, M. D. (1966) J. Biol. Chem. 241, 2405-2412] but essentially consistent with the current stepwise mechanism [O'Leary, M. H., Rife , J. E., & Slater , J. D. (1981) Biochemistry 20, 7308-7314].

Bicarbonates↗
PubMed1984

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.

Bacteria↗
PubMed1984

Phosphoenolpyruvate carboxylase of Escherichia coli. Specificity of some compounds as activators at the site for fructose 1,6-bisphosphate, one of the allosteric effectors.

An investigation was performed to elucidate some unusual phenomena which had been observed with phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of Escherichia coli. (i) Fructose 1,6-bisphosphate (Fru-1,6-P2) and GTP--the allosteric activators--were competitive with each other in the activation. (ii) Some analogs of PEP such as DL-2-phospholactate and 2-phosphoglycolate, which behaved as inhibitors in the presence of the activator (acetyl-CoA or dioxane), activated the enzyme to some extent in the absence of the activator. (iii) Ammonium sulfate deprived the enzyme of sensitivity to Fru-1,6-P2 or GTP but had no effect on the sensitivity to other effectors. It was found that the activation by the analogs was lost upon desensitization of the enzyme to Fru-1,6-P2 by reaction with 2,4,6-trinitrobenzene sulfonate. The activation by the analogs was not observed in the presence of 200 mM ammonium sulfate. In the presence of lower concentrations (0.1 mM) of PEP, ammonium sulfate activated the enzyme at concentrations less than 700 mM but had an inhibitory effect on the desensitized enzyme. These findings suggest that the unusual phenomena described above are a result of binding of the phosphate esters and sulfate ions with the Fru-1,6-P2 site of the enzyme or the active site depending on the reaction conditions.

Allosteric Site↗
PubMed1984

The primary structure of phosphoenolpyruvate carboxylase of Escherichia coli. Nucleotide sequence of the ppc gene and deduced amino acid sequence.

The nucleotide sequence of the ppc gene, the structural gene for phosphoenolpyruvate carboxylase [EC 4.1.1.31], of Escherichia coli K-12 was determined. The gene codes for a polypeptide comprising 883 amino acid residues with a calculated molecular weight of 99,061. The amino acid sequence deduced from the nucleotide sequence was entirely consistent with the protein chemical data obtained with the purified enzyme, including the NH2- and COOH-terminal sequences and amino acid composition. The coding region is preceded by two putative ribosome binding sites, and is followed closely by a good representative of rho-independent terminator. The codon usage in the ppc gene suggests a moderate expression of the gene. The secondary structure of the enzyme was predicted from the deduced amino acid sequence.

Amino Acid Sequence↗
PubMed1984

Purification, crystallization, and molecular properties of aspartase from Pseudomonas fluorescens.

Aspartase [L-aspartate ammonia-lyase, EC 4.3.1.1] of Pseudomonas fluorescens was highly purified to homogeneity and crystallized. The purified enzyme sedimented as a monodisperse entity upon ultracentrifugation with a s0(20),w value of 8.6S. Upon polyacrylamide gel electrophoresis (PAGE), the enzyme migrated as a single band. The molecular weight of the native enzyme was 173,000 +/- 3,000, as determined by sedimentation equilibrium analysis, and that of the enzyme subunit was determined to be 50,000 +/- 1,500 by sodium dodecyl sulfate (SDS)-PAGE. Cross-linking experiments using dimethyl suberimidate followed by SDS-PAGE indicated that the native enzyme was composed of four subunits with identical molecular weight. The amino acid composition of the enzyme was determined.

Amino Acids↗
PubMed1984

Preparation of 14C-labeled and unlabeled sterol intermediates from yeast using metabolic inhibitors.

The method for the preparation of zymosterol was improved (13 mg of zymosterol/g dry cells) by the aerobic adaptation of the cells in the presence of 1 mM DL-ethionine. Lanosterol was also found to accumulate (5.0 mg/g dry cells) when the cells were adapted aerobically in the presence of 10(-4) M buthiobate. Pure lanosterol could be obtained by separation of the unsaponifiable lipids on TLC. Pure [14C]lanosterol with a high specific radioactivity (56 Ci/mol) could be prepared by incubation of the desiccated cells with [14C]isopentenyl pyrophosphate, cofactors such as ATP and NADPH-generating system, and buthiobate in phosphate buffer. The method using desiccated cells may also be applicable to the preparation of other radioactive sterol intermediates.

Anaerobiosis↗
PubMed1984

Specific 14C-labeling of isoprenoids of intact E. coli cells.

Upon rehydration of lyophilized E. coli cells with phosphate buffer containing [14C]isopentenyl pyrophosphate, 14C was incorporated into the cells. Radioactivity was found in prenylquinones and some polyprenyl phosphate derivatives. Evidence was obtained suggesting that the latter compounds were interconverted to each other and that the "lipid cycle" operated in these cells.

Carbon Radioisotopes↗
PubMed1984

[Surgery in medical education].

What is expected from medical education is not always one to both sides -the side expecting, the side expected. People expect it to give training of medical care for their own lives, in short, the art and morals in medicine. Medical students expect to learn medical science and the medical art. Medical educators expect medical science and to bring up researchers to advance it, namely in the present medical education we see mingled expectations of medicine, which to be a science and the right way of medical care in social life. Medicine is indispensable to medical care, while the former can be apart by itself. What men should be to keep social life is not fundamentally different, irrespective of the presence of intervention of medicine, but the reason why morals in medicine is especially emphasized is that consideration of life is covered in the name of medicine, namely science. Surgery, as it is generally called, is a general term for a field where operations are jointly used as a means of treating diseases. Operation is not fundamentally different from medication or radio therapy in point of being artificial, but its artificial participation as a remedy is remarkable and complete. In surgical treatment destruction and mending are a means and object as well. In life-saving, urgent surgery the means after comes close to the object, sometimes the order is inverted. Training for meeting active actions has an important mission.(ABSTRACT TRUNCATED AT 250 WORDS)

Education, Medical↗
PubMed1983

Cell volume change of Escherichia coli under stringent control. Apparent increase of K+ in rel- cells.

With several pairs of rel+ and rel- strains of Escherichia coli, the effects of amino acid starvation on the intracellular concentration of K+ and the rate of uptake of 42K+ were investigated. In the early phase of the experiments, the intracellular concentration of K+ was estimated by the conventional method in which the cell volume per A660 value of the culture was assumed to be constant, being not influenced by the variation of growth condition and strain. Apparently, the K+ concentration of rel+ cells was kept almost constant, while that of rel- cells increased about 1.5-fold 2 h after the exposure to amino acid starvation. Unexpectedly, however, the above assumption was found not to be valid in the present study. The cell volume per A660 changed only slightly in CP78 (rel+) cells, while it increased markedly in CP79 (rel-) cells after the exposure to amino acid starvation. Reestimation of the K+ concentrations based on the estimated respective values of cell volumes per A660 revealed no significant difference between both strains. After all, the above apparent phenomenon was found to be due to the fact that the increase in cell volume of the rel+ cells was arrested upon amino acid starvation whereas that in the rel- cells was not. The 42K+ uptake by the rel+ cells was depressed upon amino acid starvation, whereas that by the rel- cells increased. Some regulatory mechanism was suggested to operate in both strains to keep their K+ concentrations constant. When intracellular concentration of a metabolite is to be determined, importance of measurement of cell volume under the respective conditions, without assuming the constancy of the cell volume per A660 of the culture, was pointed out.

Amino Acids↗
PubMed1983

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.

Cytochrome P-450 Enzyme System↗
PubMed1983

Buthiobate: a potent inhibitor for yeast cytochrome P-450 catalyzing 14 alpha-demethylation of lanosterol.

Buthiobate (S-n-butyl S'-p-tert-butylbenzyl N-3-pyridyldithiocarbon-imidate), a fungicide, inhibited 14 alpha-demethylation of lanosterol catalyzed by a reconstituted enzyme system consisting of cytochrome P-450 (P-450(14)-DM) and NADPH-cytochrome P-450 reductase both purified from Saccharomyces cerevisiae. Concentration of buthiobate necessary for the 50% inhibition was 0.3 microM and this value was markedly lower than those of metyrapone and SKF-525A. Buthiobate bound stoichiometrically to P-450(14)-DM and induced Type II spectral change of the cytochrome. Buthiobate inhibited lanosterol-dependent enzymatic reduction of the cytochrome. These facts indicate that buthiobate binds to P-450(14)-DM with high affinity and acts as a potent inhibitor on the cytochrome.

Cytochrome P-450 Enzyme Inhibitors↗
PubMed1983

Altered cytochrome P-450 in a yeast mutant blocked in demethylating C-32 of lanosterol.

Spectroscopic and enzymatic analysis of a Saccharomyces cerevisiae mutant in sterol biosynthesis (SG1 (erg 11); Trocha, P. J., Jasne, S. J., and Sprinson, D. B. (1977) Biochemistry 16, 4721-4726) that was blocked in demethylating C-32 of lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol) showed that it contained low levels of cytochromes P-450 and b5 compared to those present in the parent strain D-587, while NADPH-cytochrome c reductase activity was elevated. The fungicide buthiobate (S-n-butyl S'-p-tert-butylbenzyl N-3-pyridyldithiocarbonimidate), which bound specifically to yeast cytochrome P450 responsible for demethylating C-32 of lanosterol and effected a Type II spectral change, did not react with SG1 cytochrome P-450. On the other hand, cholate-solubilized microsomes from SG1 formed a single precipitin line on Ouchterlony plates with antibodies raised against purified (Yoshida, Y., Aoyama, Y., Kumaoka, H., and Kubota, S. (1977) Biochem. Biophys. Res. Commun. 78, 1005-1010) yeast cytochrome P-450 specific for demethylating C-32 of lanosterol. Hence, mutant SG1 contained an altered protein which retained the antigenicity of cytochrome P-450 responsible for demethylating C-32 of lanosterol but lost its catalytic activity.

Cytochrome P-450 Enzyme System↗
PubMed1983

Studies on isoprenoid biosynthesis with bacterial intact cells.

For the study on the regulation of isoprenoid biosynthesis with intact cells, some strains of bacteria capable of growing on mevalonate as a sole carbon source were isolated from soil. Many of them incorporated [14C]-mevalonate, [14C]isopentenyl- and [14C]farnesyl pyrophosphates into the cells. However, radioactivity was found in their degradation products but not in isoprenoids. Addition of [14C]isopentenyl pyrophosphate, farnesyl pyrophosphate and Mg2+ ions in combination to the culture of a strain of Arthrobacter gave rise to 14C-incorporation into isoprenoids. Radioactivity was found in polyprenol, its pyrophosphate, monophosphate and fatty acid esters. The reactions of isopentenyl- and farnesyl pyrophosphates syntheses seemed to be rate-limiting steps.

Bacteria↗
PubMed1983

Characterization of a Saccharomyces cerevisiae mutant, N22, defective in ergosterol synthesis and preparation of [28-14C]ergosta-5,7-dien-3 beta-ol with the mutant.

Analysis of sterols was made with a Saccharomyces cerevisiae mutant, N22, which was resistant to nystatin and defective in ergosterol synthesis, and with its parent strain, M10 (haploid type, methionineless, petite). The main sterol of M10 was ergosterol, whereas that of N22 was ergosta-5,7-dien-3 beta-ol. A small amount of ergosterol was found also in N22. This indicates that the delta 22-desaturation reaction in N22 is blocked, though not completely. Ergosta-5,8-dien-3 beta-ol, an unusual sterol, was detected in N22 but not in M10. Variations in the amounts and compositions of free and esterified sterols of both strains were examined during cultivation and the subsequent aerobic adaptation. The contents of free sterols, which were mostly composed of the respective main sterols of both strains, did not change markedly. In contrast, the contents and compositions of esterified sterols of both strains varied depending on the growth conditions. When the cells of both strains were aerobically adapted, the total contents of esterified sterols increase, as did, as the intermediate sterols. Upon aerobic adaptation in the presence of [methyl-14C]methionine, [28-14C]ergosta-5,7-dien-3 beta-ol accumulated markedly in N22 cells. Using this mutant, we devised a convenient method for preparation of the radioactive sterol in high yield.

Chemical Phenomena↗