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H Katsuki

Publications and source records attributed to H Katsuki.

At least 19 recordsLinked to original sources
PubMed1979

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.

Acyltransferases↗
PubMed1979

Phosphoenolpyruvate carboxylase of Escherichia coli. The role of lysyl residues in the catalytic and regulatory functions.

Phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of E. coli was inactivated by 2,4,6-trinitrobenzene sulfonate (TNBS), a reagent known to attack amino groups in polypeptides. When the modified enzyme was hydrolyzed with acid, epsilon-trinitrophenyl lysine (TNP-lysine) was identified as a product. Close similarity of the absorption spectrum of the modified enzyme to that of TNP-alpha-acetyl lysine and other observations indicated that most of the amino acid residues modified were lysyl residues. Spectrophotometric determination suggested that five lysyl residues out of 37 residues per subunit were modified concomitant with the complete inactivation of the enzyme. DL-Phospholactate (P-lactate), a potent competitive inhibitor of the enzyme, protected the enzyme from TNBS inactivation. The concentration of P-lactate required for half-maximal protection was 3 mM in the presence of Mg2+ and acetyl-CoA (CoASAc), which is one of the allosteric activators of the enzyme. About 1.3 lysyl residues per subunit were protected from modification by 10 mM P-lactate, indicating that one or two lysyl residues are essential for the catalytic activity and are located at or near the active site. The Km values of the partially inactivated enzyme for PEP and Mg2+ were essentially unchanged, though Vmax was decreased. The partially inactivated enzyme showed no sensitivity to the allosteric activators, i.e., fructose 1,6-bisphosphate (Fru-1,6-P2) and GTP, or to the allosteric inhibitor, i.e., L-aspartate (or L-malate), but retained sensitivities to other activators, i.e., CoASAc and long-chain fatty acids. P-lactate, in the presence of Mg2+ and CoASAc, protected the enzyme from inactivation, but did not protect it from desensitization to Fru-1,6-P2, GTP, and L-aspartate. However, when the modification was carried out in the presence of L-malate, the enzyme was protected from desensitization to L-aspartate (or L-malate), but was not protected from desensitization to Fru-1,6-P2 and GTP. These results indicate that the lysyl residues involved in the catalytic and regulatory functions are different from each other, and that lysyl residues involved in the regulation by L-aspartate (or L-malate) are also different from those involved in the regulation by Fru-1,6-P2 and GTP.

Aspartic Acid↗
PubMed1979

Occurrence of thermolabile and regulatory NAD-linked glutamate dehydrogenase in Pseudomonas fluorescens.

NAD-linked glutamate dehydrogeanse [EC 1.4.1.2] was detected together with NADP-linked glutamate dehydrogenase [EC 1.4.1.4] and aspartase [EC 4.3.1.1] in Pseudomonas fluorescens cells. The three enzymes were distinctly separated by DEAE-Sephadex column chromatography. The NAD-linked enzyme was extremely thermolabile and was rapidly inactivated even at temperatures as low as 35--40 degrees C. The combined addition of NAD+ and glutamate, however, effectively stabilized the enzyme. The glutamate saturation profile of the NAD-linked enzyme exhibited cooperativity with a Hill coefficient (n) of 1.4. ATP inhibited the enzyme in an allosteric manner, increasing the n value to 2.2. These results suggest a novel type of metabolic regulation shared by the three enzymes in the biosynthesis and catabolism of amino acids.

Glutamate Dehydrogenase↗
PubMed1979

Studies on delta8-delta7 isomerization and methyl transfer of sterols in ergosterol biosynthesis of yeast.

The formation of cholesta-7,24-dien-3 beta-ol and its activity as a substrate for the sterol side-chain methyltransferase in yeast have not previously been studied. Experiments with acetone-powder extracts of yeast showed that the sterol is formed from zymosterol by delta8-delta7 isomerization. However, direct conversion of cholesta-7,24-dien-3 beta-ol into zymosterol could not be demonstrated. The reversibility of the reaction was proved by the detection of 3H-incorporation into cholesta-8-en-3 beta-ol (with lathosterol as a carrier) from [3H]H2O in the medium. Incubation of cholesta-7,24-dien-3 beta-ol and S-adenosyl-L-[methyl-14C]methionine with the acetone-powder extract resulted in methylation of the sterol to form episterol. Similar incubation of zymosterol gave fecosterol and episterol, suggesting that fecosterol initially formed by the methylation was isomerized to episterol. In intact cells, however, an alternative pathway (zymosterol yields cholesta-7,24-dien-3 beta-ol yields episterol) may also operate. The relative importance of the two pathways is not known.

Cholestadienols↗
PubMed1979

Studies on regulatory functions of malic enzymes. VII. Structural and functional characteristics of sulfhydryl groups in NADP-linked malic enzyme from Escherichia coli W.

NADP-linked malic enzyme from Escherichia coli W contains 7 cysteinyl residues per enzyme subunit. The reactivity of sulfhydryl (SH) groups of the enzyme was examined using several SH reagents, including 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM). 1. Two SH groups in the native enzyme subunit reacted with DTNB (or NEM) with different reaction rates, accompanied by a complete loss of the enzyme activity. The second-order modification rate constant of the "fast SH group" with DTNB coincided with the second-order inactivation rate constant of the enzyme by the reagent, suggesting that modification of the "fast SH group" is responsible for the inactivation. When the enzyme was denatured in 4 M guanidine HCl, all the SH groups reacted with the two reagents. 2. Althoug the inactivation rate constant was increased by the addition of Mg2+, an essential cofactor in the enzyme reaction, the modification rate constant of the "fast SH group" was unaffected. The relationship between the number of SH groups modified with DTNB or NEM and the residual enzyme activity in the absence of Mg2+ was linear, whereas that in the presence of Mg2+ was concave-upwards. These results suggest that the Mg2+-dependent increase in the inactivation rate constant is not the result of an increase in the rate constant of the "fast FH group" modification. 3. The absorption spectrum of the enzyme in the ultraviolet region was changed by addition of Mg2+. The dissociation constant of the Mg2+-enzyme complex obtained from the Mg2+- dependent increment of the difference absorption coincided with that obtained from the Mg2+- dependent enhancement of NEM inactivation. 4. Both the inactivation rate constant and the modification rate constant of the "fast SH group" were decreased by the addition of NADP+. The protective effect of NADP+ was increased by the addition of Mg2+. Based on the above results, the effects of Mg2+ on the SH-group modification are discussed from the viewpoint of conformational alteration of the enzyme.

Cysteine↗
PubMed1979

Phosphoenolpyruvate carboxylase of Escherichia coli. Affinity labeling with bromopyruvate.

Phosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli W was alkylated by incubation with bromopyruvate, substrate analog, leading to irreversible inactivation. The reaction followed pseudo-first-order kinetics. Mg2+, an essential cofactor for catalysis, enhanced the inactivation, and the enhancing effect increased as the pH increased. The inactivation rate showed a tendency to saturate with increasing concentrations of bromopyruvate, indicating that an enzyme-bromopyruvate complex was formed prior to the alkylation. DL-Phospholactate, a potent competitive inhibitor with respect to phosphoenolpyruvate, protected the enzyme from inactivation in a competitive manner. Examination of the acid hydrolysate of the enzyme modified with [14C]bromopyruvate by paper chromatography showed that radioactivity was solely incorporated into carboxyhydroxyethyl cysteine. In addition, determination of sulfhydryl groups of the native and modified enzymes with 5,5'-dithiobis(2-nitrobenzoate) showed that inactivation occurred concomitant with the modification of one cysteinyl residue per subunit. The results indicate that bromopyruvate reacted with the enzyme as an active-site-directed reagent.

Affinity Labels↗
PubMed1979

Studies on the delta 5-desaturation in ergosterol biosynthesis in yeast.

Studies were carried out on the delta 5-desaturation reaction in ergosterol biosynthesis with a particulate fraction of cell-free extract of yeast. A reduced pyridine nucleotide coenzyme and molecular oxygen were required for the reaction. It was shown that the enzyme activity is located in a fraction corresponding to microsomes. The reaction was inhibited by KCN, but not by CO. Menadione and potassium ferricyanide inhibited the NADPH- and NADH-dependent reactions, respectively, and cytochrome c inhibited both of them. These results suggested an involvement in delta 5-desaturation of a mixed function oxidase system resembling that for the fatty acyl-CoA desaturation reaction.

Ascorbic Acid↗
PubMed1979

Studies on regulatory functions of malic enzymes. VI. Purification and molecular properties of NADP-linked malic enzyme from Escherichia coli W.

NADP-linked malic enzyme [EC 1.1.1.40] was highly purified from Escherichia coli W cells. The purified enzyme was homogeneous as judged by ultracentrifugation and gel electrophoresis. The apparent molecular weights obtained by sedimentation equilibrium analysis, from diffusion and sedimentation constants, and by disc electrophoresis at various gel concentrations were 471,000, 438,000, and 495,000, respectively. The subunit molecular weights obtained by sedimentation equilibrium analysis in the presence of 6 M guanidine hydrochloride and gel electrophoresis in the presence of sodium dodecyl sulfate were 76,000 and 82,000, respectively. The sedimentation coefficient (S(0)20, W) was 13.8S, and the molecular activity was 44,700 min-1 at 30 degrees C. The amino acid composition of the enzyme was determined, and the results were compared with those of NAD-linked malic enzyme from the same organism and those of pigeon liver NADP-linked malic enzyme. The partial specific volume was calculated to be 0.738 ml/g. The Km value for L-malate was 2.3 mM at pH 7.4. Malonate, tartronate, glutarate, and DL-tartrate competitively inhibited the activity. The saturation profile for L-malate exhibited a marked cooperativity in the presence of both chloride ions and acetyl-CoA. However, acetyl-CoA alone did not show cooperativity or produce inhibition in the absence of chloride ions. Vmax and Km were determined as a function of pH. The optimum pH for the reaction was 7.8. Inspection of the Dixon plots suggested that three ionizable groups of the enzyme are essential for the enzyme activity. In addition to the oxidative decarboxylase activity, the enzyme preparation exhibited divalent metal ion-dependent oxaloacetate decarboxylase and alpha-keto acid reductase activities. Based on the above results, the molecular properties of the enzymatic reaction are discussed.

Acetyl Coenzyme A↗
PubMed1979

Phosphoenolpyruvate carboxylase of Escherichia coli. Effect of proteolytic modification on the catalytic and regulatory propties.

Phosphoenolpyruvate carboxylase from Escherichia coli W was treated with ten proteases, and the effects of the treatments on the enzyme activity and sensitivity to effectors were investigated. Proteases such as trypsin, alpha-chymotrypsin, papain, and subtilisin inactivated the enzyme, whereas elastase, carboxypeptidase Y and leucine aminopeptidase had no effect on the enzyme activity. Elastase and carboxypeptidase Y, however, inactivated the enzyme in the presence of 1 m urea. Subtilisin and alpha-chymotrypsin caused not only inactivation of the enzyme but also a significant desensitization to the effectors. DL-Phospholactate, a potent competitive inhibitor, markedly protected the enzyme from inactivation by subtilisin but did not protect it from desensitization to the effectors. Acetyl-CoA, fructose 1, 6-bisphosphate, and GTP-the allosteric activators--protected the enzyme from subtilisin inactivation, while laurate, the other allosteric activator, accelerated the inactivation. These activators did not protect the enzyme from desensitization to themselves. In contrast, modification with subtilisin in the present of l-aspartate, the allosteric inhibitor, caused an apparent transient activation of the enzyme. The enzyme modified in the presence of L-aspartate retained its sensitivity to L-aspartate, but the sensitivities to the other effectors were reduced to about one-half their initial values. Based on these results, a possible mode of desensitization of the enzyme by subtilisin modification and the possible existence of a multiplicity of conformational states of the enzyme, induced upon binding with the various effectors, are discussed.

Carboxy-Lyases↗
PubMed1978

Characterization of sterol-ester hydrolase in Saccharomyces cerevisiae.

A homgenate of Saccharomyces cerevisiae grown under semi-anaerobic as well as aerobic conditions was found to catalyze the hydrolysis of fatty acid esters of sterols in the presence of Triton X-100. The enzyme levels in cells grown under various conditions were similar and the enzyme had a broad substrate specificity for sterol esters. The enzyme was localized in the mitochondrial fraction for the aerobically grown cells and in the mitochondrial and cytosolic fractions for the semi-anaerobically grown cells.

Carboxylic Ester Hydrolases↗
PubMed1978

Quantitative aspects of free and esterified sterols in Saccharomyces cerevisiae under various conditions.

For extraction of free and esterified sterols from yeast cells, a method was devised in which both forms of sterols were extracted with light petroleum after the treatment of the cells with acetone, and then with dimethylsulfoxide. The content of sterol esters in the cells under aerobic conditions markedly increased with time, amounting to 95% of the total sterols under some conditions. However, the formed sterol esters were decreased, accompanied with an increase of free sterols, when the cells were put under anaerobic conditions. Variations of radioactivities of both sterols which had been labeled in the side chain by incubation of the cells with [Me[-14C]methionine were examined on the cells grown under various conditions. No variation was observed on the cells under aerobic conditions. On the other hand, the labeled esters were hydrolyzed to yield free sterols in the cells under anaerobic conditions. In the cells under aerobic conditions, the free sterols were found to consist mainly of ergosterol, whereas the esterified sterols contained considerable amounts of zymosterol, lanosterol, and other intermediate sterols besides ergosterol.

Aerobiosis↗
PubMed1978

Ergosterol biosynthesis in yeast. Pathways in the late stages and their variation under various conditions.

[Methyl-14C]methionine was supplied to yeast cells under aerobic and anaerobic conditions for the investigation of the pathway for ergosterol biosynthesis after the methylation of the side-chain. Under aerobic conditions, the incorporation of radioactivity into ergosterol was high. With a limited oxygen supply, in contrast, the radioactivity was first accumulated in ergosta-7,24(28)-dien-3beta-ol and ergosta-8,24(28)-dien-3beta-ol, and then transferred to ergost-7-en-3beta-ol, ergost-8-en-3beta-ol and ergosta-7,22-dien-3beta-ol with time. Under strictly anaerobic conditions, a double bond was introduced neither to delta5 nor to delta22. The results of the tracer experiments suggested the operation of several pathways in the late stages of ergosterol biosynthesis. It was also suggested that the main pathways varied depending on the conditions such as oxygen supply and other factors. The above conclusion was supported by the results of the analyses of the sterol compositions of the cells grown under various conditions.

Aerobiosis↗
PubMed1978

Accumulation of zymosterol in yeast grown in the presence of ethionine.

In order to identify the methyl acceptor for the methylation of sterol side-chains in ergosterol biosynthesis, Saccharomyces cerevisiae (wild type) was grown in the presence and absence of ethionine which was expected to be an inhibitor of the methylation. Gas-liquid chromatographic analyses of the sterols in the cells grown in the absence of ethionine showed that ergosterol was the most abundant sterol. On the other hand, a sterol, named sterol Z, accounted for more than 50% of the total sterols in the cells grown in the presence of ethionine. As a result of experiments to raise the yield of sterol Z, the best concentration of DL-ethionine for the production was found to be 1.0 mM. The use of the methionine-less mutant was less effective for the production of sterol Z. Sterol Z was isolated by repeated TLC and was identified as zymosterol from its melting point, GLC and mass spectrometry. The role of zymosterol and other sterols as the methyl-acceptor sterol in ergosterol biosynthesis is also discussed.

Cholestadienols↗
PubMed1978

Studies on regulatory functions of malic enzymes. V. Comparative studies of malic enzymes in bacteria.

Screening of four malic enzymes--NAD-linked enzyme [EC 1.1.1.38], NAD, NADP-linked enzyme [EC 1.1.1.39], NADP-linked enzyme [EC 1.1.1.40], and D-malic enzyme--was carried out with cell-free extracts of the following 16 strains of bacteria by the aid of Sepharose 6B column chromatography: 9 strains of enteric bacteria, 3 strains of Pseudomonas, Alcaligenes faecalis, Agrobacterium tumefaciens, Rhodospirillum rubrum, and Clostridium tetanomorphum. All the strains tested contained at least one malic enzyme. The NADP-linked enzyme activity was found in all the strains except C. tetanomorphum, the NAD-linked enzyme activity in 12 strains--8 strains of enteric bacteria, 2 strains of Pseudomonas, Ag. tumefaciens, and C. tetanomorphum--and D-malic enzyme activity in 4 strains--A, aerogenes (IFO 3319 and 12059), Ps. fluorescens, and R. rubrum. The NADP-linked and NAD-linked enzyme activities of two strains of Pseudomonas were not separated by the chromatography. The available evidence suggested that the NAD, NADP-linked enzyme was not present in these 16 strains. The comparative studies of molecular, enzymatic, and serological properties of the malic enzymes in these 16 strains revealed a close similarity of the same types of malic enzymes among enteric bacteria.

Alcaligenes↗