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Biomedical subjects

H Katsuki

Publications and source records attributed to H Katsuki.

At least 127 records · Page 7Linked to original sources
PubMed1983

Phosphoenolpyruvate carboxylase of Escherichia coli. Inhibition by various analogs and homologs of phosphoenolpyruvate.

In an attempt to investigate the topography of the catalytic site of phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of Escherichia coli, the inhibitor constants (Ki) for more than 20 compounds were determined with the reaction system containing dioxane, a non-physiological activator of the enzyme. The Ki values for the compounds lacking methylene-, carboxylate-, or phosphate groups were all more than 10-fold larger than the Km value for PEP, indicating the significant contribution of these groups to the binding of PEP with the enzyme. The Ki value for L-phospholactate (0.30 mM) was almost equal to the Km value for PEP (0.25 mM), whereas that for D-phospholactate (0.89 mM) was about 3-fold larger than the Km value. It was presumed that PEP binds with the enzyme on its si-side. Among 6 PEP homologs, the Ki values for phosphoenol alpha-ketobutyrate (0.024 mM) and phosphoenol alpha-ketovalerate (0.034 mM) were about one-tenth the Km value, indicating the presence of a hydrophobic pocket around the binding site of the methylene group of PEP, where the carboxylation reaction is supposed to occur. DL-Phosphomalate, a presumptive carboxylated substrate, was a weak inhibitor with a Ki value of 2.20 mM.

Binding Sites↗
PubMed1982

Formation of dehydrosqualene catalyzed by squalene synthetase in Saccharomyces cerevisiae.

When microsomal fraction of Saccharomyces cerevisiae was incubated with farnesyl pyrophosphate or presqualene pyrophosphate in the presence of Mn2+, 12,13-cis-dehydrosqualene (DeH2Sq) and some related compounds were found to be formed. Incubation in the presence of NADPH gave rise to only squalene. By heat treatment of the microsomal fraction, the DeH2Sq- and squalene-forming activities were inactivated at approximately the same rate. The elution patterns of both activities upon Sephacryl S-200 chromatography of the enzyme solubilized from the microsomal fraction with taurodeoxycholate coincided completely. These results indicate that DeH2Sq formation in yeast is catalyzed by squalene synthetase. Divalent cation was essential for this reaction and Mn2+ was six times more effective than Mg2+. DeH2Sq formation was also observed when microsomes of pig liver were used instead of yeast microsomal fraction, suggesting that this reaction is a ubiquitous one among the eucaryotes which are capable of synthesizing sterols. Based on these observations, the mechanisms of DeH2Sq and squalene formation are discussed.

Animals↗
PubMed1982

Phosphoenolpyruvate carboxylase of Escherichia coli. Hydrophobic chromatography using specific elution with allosteric inhibitor.

The adsorption of Escherichia coli phosphoenolpyruvate carboxylase [EC 4.1.1.31] to butyl-, hexyl-, and octyl-Sepharose gels was investigated. The enzyme was nearly completely adsorbed to the latter two gels both in the absence and presence of high concentrations of ammonium sulfate. At intermediate concentrations--0.1 M in the case of hexyl-Sepharose--virtually no adsorption was observed. Upon application of an increasing or decreasing concentration gradient of the salt, the enzyme was eluted at various concentrations of the salt depending on chain length of the immobilized alkyl groups. The adsorption to hexyl-Sepharose at 0.7 M ammonium sulfate was markedly decreased by L-aspartate, the allosteric inhibitor, whereas it was increased by acetyl-CoA, one of the allosteric activators. Evidence was obtained suggesting that these changes in adsorption were due to conformational alterations of the enzyme elicited by these effectors. The enzyme seemed to have been adsorbed at its hydrophobic regions which were distinct from the allosteric site for long-chain fatty acids. The specific elution with L-aspartate in the presence of 0.82 M ammonium sulfate could successfully be applied to purification of the enzyme. By this hydrophobic interaction chromatography, the enzyme was purified about 55-fold over its partially purified preparation with a recovery of 73%. The obtained enzyme preparation was almost homogeneous as judged from sodium dodecylsulfate-polyacrylamide gel electrophoresis.

Adsorption↗
PubMed1982

Enzymatic formation of nerolidol in cell-free extract of Rhodotorula glutinis.

Enzymatic formation of nerolidol was demonstrated by incubation of [14C]farnesyl pyrophosphate with the ultracentrifugal supernatant of cell-free extract of Rhodotorula glutinis. Farnesol was also formed concomitantly with the formation of nerolidol and the ratios of formation of both alcohols were from 1:3 to 1:4. Divalent cation was necessary for the reaction and Mn2+ was much more active than Mg2+ for nerolidol formation. No nerolidol was formed when farnesyl monophosphate or farnesol was used instead of farnesyl pyrophosphate as a substrate. Nerolidyl pyrophosphate or nerolidyl monophosphate could not be detected as an intermediate in the reaction. Based on these observations, nerolidol was presumed to be formed not via nerolidyl pyrophosphate or nerolidyl monophosphate but via a carbonium ion intermediate which was formed by cleavage of the carbon-oxygen bond of farnesyl pyrophosphate. This reaction seems to proceed in a similar manner to the acid hydrolysis of farnesyl pyrophosphate to form nerolidol and farnesol.

Farnesol↗
PubMed1982

Effect of detergents on sterol synthesis in a cell-free system of yeast.

In order to obtain information about the reactivity of enzymes in sterol synthesis of yeast, the effects of some detergents were investigated. Among the detergents used, Triton X-100 was found to exert a unique action, and its effect on the incorporation of 14C-labeled acetate, mevalonate, farnesyl pyrophosphate, or S-adenosyl-L-methionine into squalene, 2,3-oxidosqualene, and sterols in a cell-free system was examined. Triton X-100 showed virtually no effect on the enzyme activities in the reactions from acetyl CoA to farnesyl pyrophosphate, but it had a marked effect on reactions from farnesyl pyrophosphate to ergosterol. Evidence was obtained suggesting that Triton X-100 apparently activated squalene synthetase (EC 2.5.1.21) but inhibited squalene epoxidase (EC 1.14.99.7) and delta 24-sterol methyltransferase (EC 2.1.1.41). The activity of epoxidase was protected from the inhibition by increasing the concentration of cell-free extracts or by the prior addition of lecithin liposomes to the reaction mixture. The inhibition of methyltransferase was partially reversed by treatment with Bio-heads SM-2, but that of epoxidase was not reversed by the treatment.

Cell Membrane↗
PubMed1981

Subcellular localization of the enzymes involved in the late stage of ergosterol biosynthesis in yeast.

The subcellular distribution of enzymes involved in the reaction sequence from zymosterol to ergosterol was studied. The spheroplasts obtained from cells aerobically grown on ethanol were gently disrupted and the homogenate was fractionated into the subcellular organelles by differential centrifugation. Inspection of the distribution of several marker enzymes revealed that the fractionation was reasonably effected. As a result of experiments, delta 8-delta 7-sterol isomerase, S-adenosylmethionine: delta 24-sterol methyltransferase and the enzyme involved in the reaction sequence from episterol to ergosterol were localized in microsomes. These results suggest the localization of enzymes involved in the late stage of ergosterol synthesis in microsomes.

Cholestadienols↗
PubMed1981

Purification and properties of sterol-ester hydrolase from Saccharomyces cerevisiae.

Sterol-ester hydrolase [EC 3.1.1.13] from Saccharomyces cerevisiae grown aerobically was solubilized with 1% Tween 20 and purified about 700-fold by the protamine sulfate treatment, DEAE-cellulose-, Sepharose 6B- and DEAE-cellulose column chromatographies. The molecular weight of the enzyme was estimated to be 70,000 by Sepharose 6B gel filtration. The enzyme activity showed two peaks of pH optimum at 4.4 and 6.8. Triton X-100 stimulated the activity as its low concentrations at both pH regions, but decreased the activity at its high concentrations at pH 6.8. The presence of Tween 20 or Tween 80 also stimulated the activity. These results were different from those in the previous report showing no stimulation of the crude enzyme by these detergents. The stimulation of the activity by phosphatidylcholine or low concentrations of lysophosphatidylcholine was similar to that by Triton X-100, and taurocholate was less effective than Triton X-100. The enzyme activity was inhibited by divalent cations such as Hg2+ and Cu2+.

Carboxylic Ester Hydrolases↗
PubMed1981

Regulation of Escherichia coli phosphoenolpyruvate carboxylase by multiple effectors in vivo. II. Kinetic studies with a reaction system containing physiological concentrations of ligands.

In an attempt to clarify the kinetic properties of Escherichia coli phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] in vivo and to evaluate the physiological significance of the individual effectors, saturation curves were obtained for each ligand with reaction mixtures (pH 7.3) containing "physiological concentrations" of the other ligands in various combinations. As the "physiological concentrations" of ligands, which are defined as the concentrations of ligands found in the glucose-grown cells, the following values were employed: PEP, 0.2 mM; acetyl-CoA(CoA-SAc), 0.4 mM; fructose 1,6-bisphosphate(Fru-1,6-P2), 2.0 mM; GTP, 1.0 mM; L-aspartate, 1.0 mM; L-malate, 1.0 mM (Morikawa, M., Izui, K., Taguchi, M., & Katsuki, H. (1980) J. Biochem. 87, 441--449). In the absence of any activator the enzyme activity was very low. CoASAc was the most powerful activator. The other two activators (Fru-1,6-P2 and GTP) exhibited essentially no activation alone, but produced a strong synergistic activation with CoASAc. The severe inhibition by L-aspartate or L-malate was effectively alleviated only through this synergistic action of the activators. The presence of all three activators decreased the half-saturation concentration (S0.5) of PEP from 15 mM to 0.35 mM and increased the maximal velocity attainable at infinite concentration of PEP about 15-fold. In the system containing all five effectors, which is close to the in vivo condition, the saturation curve of PEP was sigmoidal with a Hill coefficient of 1.6 and with an S0.5 value of 3.0 mM, which is about 15-fold larger than its "physiological concentration." On the basis of the rate-concentration curve for each effector obtained with the reaction mixture containing PEP and the other effectors at "physiological concentrations," it was suggested that all five effectors significantly contribute to the enzyme activity in vivo. Palmitoleate, another activator of the enzyme, showed no activation in such a reaction mixture. The sensitivity of the enzyme to the "physiological concentration" of each effector was also observed in an in situ system using permeabilized E. coli cells, where the enzyme concentration was as high as in vivo.

Carboxy-Lyases↗
PubMed1981

Stringent control of intermediary metabolism in Escherichia coli: pyruvate excretion by cells grown on succinate.

A large amount of pyruvate was excreted into the medium by CP78 (rel+) cells grown on succinate when they were starved for amino acids. In contrast, no such excretion was observed with CP79 (rel-) cells. This phenomenon was also seen with two other isogenic pairs of strains: NF161 (rel+) and NF162 (rel-), and 10B601 (rel+) and 10B602 (rel-). Besides succinate, L-malate, and fumarate were effective carbon sources for the excretion, but glucose, glycerol, and acetate were not. When DL-lactate was used, not only CP78 but also CP79 cells excreted pyruvate. Experiments using [1,4-14C]succinate as a carbon source revealed that pyruvate was formed by decarboxylation of one carboxyl group of succinate and that the pyruvate excretion amounted to about 40% of the total succinate degraded. Experiments designed to elucidate the mechanism of the excretion yielded the following observations. (i) The concentration of pyruvate in CP78 cells grown on the C4-dicarboxylic acids mentioned above was not significantly changed upon amino acid starvation. (ii) Guanosine 5'-diphosphate-3'-diphosphate exerted no effect on the activities of several enzymes thought to be involved in pyruvate-related metabolism. It is suggested firstly that the excretion was not due to some impairment in the biosynthetic pathway of a particular amino acid, but was due to the stringent control of central amphibolic metabolism, and secondly that no de novo protein synthesis was involved in the excretion.

Amino Acids↗
PubMed1980

Regulation of Escherichia coli phosphoenolpyruvate carboxylase by multiple effectors in vivo. Estimation of the activities in the cells grown on various compounds.

Intracellular concentrations of phosphoenolpyruvate (PEP) and five kinds of allosteric effectors (acetyl-CoA, fructose 1,6-bisphosphate, GTP, L-aspartate, and L-malate) of PEP carboxylase were measured in E. coli cells grown on various compounds as a carbon source. Based on the data obtained, reaction systems which contained a definite concentration of the enzyme and the ligands at the concentrations found in vivo were constructed and the enzyme activities were measured. The ratio of each activity thus obtained to the maximal activity attainable with the same concentration of enzyme and saturating concentrations of the activators was estimated. For the cells grown on glucose, glycerol, or lactate, the extent of exhibition of the enzyme activity was 2-15% of the maximal activity. For the cells grown on acetate or oleate, the extent was 1-3%. For the cells grown on succinate, L-aspartate, L-malate, or glucose plus L-aspartate, the extent was less than 0.4%. Consideration of the data obtained in the present studies, together with those obtained in our previous studies on the enzyme level (Teraoka, H. et al. (1970) J. Biochem. 67, 567-575), showed that the control of the enzyme reaction in vivo is considerably different from that expected from the in vitro experiments, and that deficiencies of "coarse control" are covered by a "fine control."

Acetyl Coenzyme A↗
PubMed1980

Augmentation of glycogen synthesis under stringent control in Escherichia coli.

When Escherichia coli strain CP78 (rel+) was starved for isoleucine by the addition of valine, the amount of glucose in polymeric form in the cells increased markedly compared to that of the control cells. In contrast, this phenomenon was not seen in strain CP79 (rel-). The increase in CP78 was shown to be due to the increase of glycogen. These results indicate that glycogen synthesis was augmented under stringent control. This was confirmed using other isogenic pairs of rel+ and rel- strains starved for other amino acids. When the cultivation temperature of strains 10B601 (rel+) and 10B602 (rel-) possessing temperature-sensitive valyl-tRNA synthetase was shifted from 30 degrees C to 40 degrees C, no difference was observed in the response of glycogen synthesis between the two strains. These results indicate that protein synthesis was necessary for the augmentation of glycogen synthesis and that guanosine 5'-diphosphate 3'-diphosphate did not exert its effect through stimulation of the activity of pre-existing enzyme(s) involved in glycogen synthesis. These conclusions were supported by the results of experiments using chloramphenicol and rifampicin. The rates of glucose utilization of CP78 and CP79 were decreased to nearly the same extent by valine addition. This suggests that the regulation site of glycogen synthesis under stringent control resides in a step after the transport of glucose by the phosphotransferase system.

Arginine↗
PubMed1980

Sterol-content lowering action of o-chlorobenzylchloride in yeast.

o-Chlorobenzylchloride, a simple aromatic halogen compound, was found to inhibit the growth of Saccharomyces cerevisiae and to lower the contents of sterols and fatty acids. The growth inhibition was considerably alleviated by the presence of sterols such as ergosterol and cholesterol and of unsaturated fatty acids such as oleate and linolenate. Inspection of effect of the inhibitor on the electron transport system related to the biosyntheses of these compounds revealed that the cytochrome contents and some enzyme activities in the system of the inhibited cells were much lower than those of the control cells. The features of the inhibition were similar to those of inhibition for other organisms by the hypocholesterolemic compounds such as triparanol and benzmalecene.

Benzyl Compounds↗
PubMed1980

Biosynthesis of ergosterol in cell-free system of yeast.

We previously proposed the occurrence of multiple pathways in the ergosterol biosynthesis of yeast, based on the results of examination of 14C-incorporation into sterols from L-[methyl-14C]methionine which was given to the intact cells of yeast. This led us to investigate the validity of the pathways by experiments with the cell-free system. Highly active cell-free extracts could be prepared by disruption of yeast cells with a Vibrogen Cell Mill in the presence of 0.1 mM dithiothreitol. This preparation catalyzed 14C-incorporation from [14C]methionine into ergosterol with a high yield. This preparation was found to be favorable for elucidation of ergosterol synthesis, since only a small amount of radioactivity was incorporated into fatty acid ester form of sterols which were reported to be inactive as a substrate for sterol synthesis reaction. Time course experiment of 14C-incorporation from [14C]methionine into various sterols under aerobic conditions showed that ergosta-8,24(28)-dien-3 beta-ol was a precursor for ergosta-7,24(28)-dien-3 beta-ol and that radioactivities were converted through ergosta-5,7,24(28)-trien-3 beta-ol and ergosta-5,7,22,24(28)-tetraen-3 beta-ol into ergosterol with time. In contrast, similar experiments under anaerobic conditions showed that ergosta-7,24(28)-dien-3 beta-ol accumulated and very little conversion of radioactivity into ergosterol occurred. In addition, the results indicated that oxygen was required for the introduction of double bond into 22 position as well as into 5 position. The results obtained with the cell-free system supported the validity of the proposal of multiple pathways of ergosterol synthesis in the intact cells.

Anaerobiosis↗
PubMed1980

Augmentation of cyclopropane fatty acid synthesis under stringent control in Escherichia coli.

An abrupt increase of cyclopropane fatty acid (CFA) occurred concomitant with a decrease of the corresponding unsaturated fatty acids in CP78 (rel+) of Escherichia coli at the onset of the stationary growth phase, whereas such variations were slight in CP79 (rel-). When the cells were starved for isoleucine, the CFA content increased in CP78 but not in CP79. The rate of 14C-incorporation from [methyl-14C]methionine into CFAs increased in CP78 abut two-fold due to the starvation. The apparent level of CFA synthase also increased due to the starvation. These results that the CFA formation is augmented under stringent control.

Cell Cycle↗
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↗