The arginase pathway in Rhodobacter: Metabolism of L-ornithine

Abstract Fungi of the genus Pleurotus were shown to produce the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor mevinolin. Crude methanol extracts and purified inhibitor from three different species, P. sapidus, P. saca and P. ostreatus , were tested using the solubilized microsomal HMGCoA reductase from Chinese hamster ovary cells. The identity of the inhibitor was also confirmed by thin layer chromatography, high pressure liquid chromatography and mass spectroscopy.     

Isolation and characterization of mevinolin resistant mutants of Saccharomyces cerevisiae

Mutant of Saccharomyces cerevisiae resistant to mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme-A (HMGCoA) reductase (EC1.1.1.34) were isolated and one mutant (MV71) was extensively characterized. While growth of resistant strains in the presence of mevinolin was growth. Diploids produced by mutant/wild-type matings showed levels of mevinolin resistance which indicated incomplete dominance. Sterol synthesis in the presence of mevinolin was inhibited in strain MV71 but to a lesser degree than seen in the wild-type strain. All mevinolin resistant mutants also demonstrated a slight resistance to the antibiotic nystatin. The subcellular location of HMGCoA reductase activity in MV71 and the wild-type strain were determined and it was shown that yeast HMGCoA reductase is not regulated by a dephosphorylation mechanism as has been shown for mammalian reductases. In vivo and in vitro studies of strain MV71 and the wild-type indicated that mevinolin resistance did not result in changes in HNGCoA reductase activity as has been demonstrated in mammalian systems. Based on growth data, sterol analysis, and the lack of detection of HMGCoA reductase activity differences between strain MV71 and the wild-type, mevinolin resistance is concluded to result possibly from a mutation in HMG2, one of the two functional yeast HMGCoA reductase genes, which accounts for a minor (up to 17%) amount of total cellular reductase activity.     

HMGCoA reductase and cholesterol 7 alpha-hydroxylase in human liver

The activities of HMGCoA reductase and cholesterol 7 alpha-hydroxylase were assayed in liver biopsies of patients with or without cholestyramine treatment. The active dephosphorylated form of HMGCoA reductase and the activity of cholesterol 7 alpha-hydroxylase were under the detection limits in untreated subjects. After cholestyramine treatment activities of both enzymes were stimulated and the active form of HMGCoA reductase became detectable in four out of the five tested patients. In two subjects who received cholestyramine, the effect of exogenously added [4-14C] cholesterol on cholesterol 7 alpha-hydroxylase was tested. In the presence of Tween 80, the detergent by which [14C]cholesterol was suspended, the enzyme activity was profoundly inhibited and synthesis of 7 alpha-hydroxycholesterol was extremely low.     

Synthesis and biological testings as inhibitors of HMGCoA reductase of the seco-acid of tuckolide and its C-7 epimer.

The seco-acid of the natural macrolactone, tuckolide (decarestrictin D) and the C-7 epimer have been prepared in enantiomerically pure form from D-gluconolactone and poly(3-hydroxy butyric acid). The key steps are Horner Emmons olefination and stereoselective reduction of the resulting enone to provide both epimers at C-7. None of the seco-acids inhibit microsomal HMGCoA reductase of pea or rat liver. It may be concluded that the cholesterol biosynthesis inhibiting effect of tuckolide is unlikely to proceed via HMGCoA reductase inhibition.     

HMGCoA reductase inhibition partially mediates tumor necrosis factor alpha-induced apoptosis in human U-937 and HL-60 cells

We have examined the effects of tumor necrosis factor alpha (TNF alpha) and its second messenger, ceramide, on HMGCoA reductase, the rate-limiting enzyme in the mevalonate pathway. Treatment of human U-937 and HL-60 cells with TNF alpha or C2-ceramide inhibited both expression and activity of HMGCoA reductase in a time-dependent manner. Maturation of p21(ras) was also inhibited in a mevalonate-dependent fashion. The addition of mevalonate to both U-937 and HL-60 cells could also partially prevent TNF alpha and ceramide-induced apoptosis. These results support the hypothesis that the inhibition of HMGCoA reductase expression and the subsequent decrease in prenylation of proteins such as p21(ras) are part of the mechanism by which TNF alpha induces apoptosis in these cells.     

Hormonal control of cholesterogenesis and related enzymes in isolated rat hepatocytes during pre- and postnatal development

The effect of glucagon and insulin on the incorporation of 1-14C-acetate into cholesterol and fatty acids and on the enzymes involved in the first steps of cholesterol synthesis (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, 3-hydroxy-3-methyl-glutaryl-coenzyme A synthase, and acetoacetyl-coenzyme A thiolase) was investigated. Isolated rat hepatocytes at different stages of fetal and postnatal development were employed. Data obtained show the appearance of hormonal control on the 18th day of fetal life, indicating the same pattern, as regards acetate incorporation and HMGCoA reductase prepared and assayed in the presence of NaF. On the contrary, HMGCoA reductase, prepared without NaF, HMGCoA synthase, and acetoacetyl CoA thiolase, does not appear to respond to hormonal stimulation. In the perinatal period, the hormonal effect is no longer detectable, probably because of a hormone resistance of this metabolic pathway.     

双歧醋对高脂饮食大鼠血脂影响的机制探讨

目的 探讨双歧醋对高脂饮食大鼠血脂的影响机制.方法 48只SD大鼠被随机分成双歧醋低剂量组[1.8 mL/(kg·BW)]、中剂量组[3.4 mL/(kg· BW)]、高剂量组[6.8 mL/(kg·BW)]和市售醋组[3.4 mL/(kg·BW)]、高脂模型组以及基础对照组,观察双歧醋对高脂饮食大鼠血脂的影响作用机制,测定指标包括大鼠肝脏脂质、大鼠LDLR的免疫组化分析,HMGCoA还原酶和LDLR的RT-PCR分析.结果 双歧醋各组对于HMGCoA还原酶表达有抑制作用,但可以上调LDLR的基因和蛋白质表达.结论 双歧醋可以通过调节HMGCoA还原酶和LDLR的表达预防高脂饮食大鼠高血脂的发生.     

Cholesterogenesis and related enzymes in isolated rat hepatocytes during pre- and postnatal life

Cholesterogenesis pathway during pre- and postnatal development was studied in isolated rat hepatocytes. No modified activity of cytosol acetoacetyl coenzyme A (CoA), thiolase, or 3-hydroxy-3-methylglutaryl CoA (HMGCoA) synthase was detectable at the different stages examined. Minimal levels of 1(14)C-acetate incorporation into cholesterol and HMGCoA reductase activity were present at 16 days of fetal development in newborn and suckling rats, whereas both parameters increased rapidly before birth. The pattern of NaF nonsuppressible reductase activity showed a different activation state of the enzyme, suggesting the appearance of a modulation state, probably related to the development of some short-term regulatory mechanisms.     

Development of a large-scale continuous substrate feed process for the biotransformation of simvastatin by Nocardia s.p

This article describes a process for microbial hydroxylation of simvastatin by a Nocardia sp. Simvastatin (Zocor) belongs to the family of HMGCoA reductase inhibitors used as cholesterol-lowering drugs. Studies at 14 L scale showed that high substrate (simvastatin) concentrations inhibited product formation; consequently, continuous slow feeding of the substrate was introduced to maintain low residual simvastatin concentrations. Dissolved oxygen levels above 50% air saturation were desirable for the biotransformation. The process was scaled up to 19,000-L fermentors using an on-line filter sterilization system for substrate feeding. The feed rate was regulated by off-line high-pressure liquid chromatography (HPLC) assays to keep the substrate concentration below 20 mg/L. Intermittent addition of nutrients helped to boost the bioconversion rate to give final titers of 400 mg/L 6-beta-hydroxymethyl simvastatin. Enrichment of the nutrient medium led to bioconversion titers of 800 mg/L 6-beta-hydroxymethyl simvastatin. Bioconversion efficiencies (desired product/substrate) of 22-25% with a ratio of desired product/side products of 0.7 were obtained by this process.     

Sterol and Fatty Acid Metabolism in Fusarium oxysporum

The effects of temperatures ranging from 10°C to 35°C on sterol and fatty acid production and hydroxymethylglutaryl CoA reductase (EC 1.1,1.34, HMGCoA reductase) activity have been examined. Growth, based on dry weight, was maximal at 25°C to 30°C. Sterol production and the reductase activity were highest at 15°C after 28~32 hr incubation when the total fatty acids were minimal. Fatty acid unsaturation generally increased with decrease in temperature.     

Preparation of a soluble 58kDa-3-hydroxy-3-methylglutaryl CoA reductase from liver microsomes and its inhibition by ethoxysilatrane,a hypocholesterolemic compound

On repeated thawing at room temperature of frozen preparations of heavy microsomes from rat livers, HMGCoA reductase activity was solubilized due to limited proteolysis. This soluble enzyme was partially purified by fractionation with ammonium sulfate and filtration on Sephacryl S-200 column. The active enzyme was coeluted with a major 92 kDa-protein and was identified as a 58kDa-protein after separation by SDS-PAGE and immunoblotting. Ethoxysilatrane, a hypocholesterolemic compound, which decreased the liver-microsomal activity of HMGCoA reductase on intra-peritonial treatment of animals, showed little effect on the enzyme activity with isolated microsomes or the 50kDa-soluble enzyme when added in the assay. But it was able to inhibit the activity of the soluble 58kDa-enzyme in a concentration-dependent, reversible manner. Cholesterol and an oxycholesterol were without effect whereas chlorophenoxyisobutyrate and ubiquinone showed small inhibition under these conditions. The extra region that links the active site domain (50kDa protein) to the membrane, present in the 58kDa-protein appears to be involved in mediating the inhibition by silatrane.     

The inhibitory effect of ATP on HMGCoA reductase

The inhibitory effect of ATP on HMGCoA reductase activity from rat liver microsomes in the system described by Beg $\text{et al.}$ was examined. The inhibition by magnesium ATP is confirmed but varies widely from zero to complete. A requirement for a cytosolic fraction to enhance the inhibition could not be established. ATP labeled uniformly with ¹⁴C in the adenine portion and ³²P in the terminal phosphate was incubated with the enzyme in a situation where strong inhibition was observed. The enzyme protein was precipitated with trichloroacetic acid, or subjected to column fractionation. No evidence of labeling was found in the protein. Finally, the enzyme protein was specifically isolated by immunoprecipitation with a specific antibody to the HMGCoA reductase. In no instance could labeling of the enzyme protein be detected. These results show that the mechanism of the inhibition does not involve phosphorylation or adenylation of the enzyme protein.     

Circannual variations in mevalonate utilization in frog (Rana esculenta)

1. The fate of mevalonate, the product of HMGCoA reductase, was studied in male and female frogs (Rana esculenta) in order to explain the circannual variations of enzyme activity. 2. The incorporation of 2-14C MVA into unsaponifiable lipids, cholesterol and dolichol in liver, plasma and eggs was followed. 3. Labeled MVA shows a different utilization depending on season and sex. In spring and summer cholesterol synthesis is related to hepatic reserve storage in both sexes, while the peak of enzyme activity, present only in females in fall, seems committed to cholesterol export into the blood and uptake by the oocytes. 4. The presence of a MVA-derived protein identifiable with vitellogenin and labeled on the lipid moiety, suggests that HMGCoA reductase activity in fall is committed to the lipidation of this protein essential for oocyte maturation.     

Desensitization to ATP-Mg inhibition of 3-hydroxy-3-methylglutarylCoA reductase by heat treatment of microsomes

HMGCoA reductase is found to be inhibited by palmitylCoA and free CoA. The inhibition of this enzyme by ATP-Mg, but not by palmityl CoA, is lost on preincubation of microsomes at 50°C for 15 min.     

Investigations on the mechanism of the hypocholesterolemic action of 1-ethoxysilatrane

Summary Intraperitoneal administration of the nontoxic silicon compound, 1-ethoxysilatrane, to the rat did not cause proliferation of hepatic mitochondria or of endoplasmic reticulum, nor did it affect mitochondrial oxidative phosphorylation. The activities of cholesterol 7 -hydroxylase in hepatic microsomes and of cholesterol oxidase in mitochondria respectively were unaffected by silatrane treatment. The rate of release of bile, whose composition remained unchanged, also was not increased in silatrane-treated animals. The results indicated that the compound did not affect the pathway of cholesterol degradation.A progressive decrease in the activity of hepatic microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase was observed on administration of the compound over a period of three weeks. Consistent with this, cholesterol biosynthesis in liver as measured by incorporation of radioactive precursors, acetate and water but not mevalonate, was significantly decreased in silatrane-treated animals. However, enzyme-linked immunosorbant assay revealed that the concentration of HMGCoA reductase protein was not decreased by the treatment indicating that inactivated enzyme was also present in such microsomes. Addition of silatrane to microsomes in the assay system did not cause inhibition indicating that the inactivation is by an indirect mechanism. It is concluded that the hypocholesterolemic action of the compound rested entirely on the inhibition of cholesterol biosynthesis in vivo by inactivation of the rate-limiting enzyme HMGCoA reductase.     

The isolation,characterisation, and chromosomal assignment of the gene for human 3-hydroxy-3-methylglutaryl coenzyme A reductase, (HMG-CoA reductase)

Summary We have used a cDNA clone for Chinese hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase to isolate a genomic recombinant for human HMG-CoA reductase. The identity of the gene was confirmed by partial sequence analysis. Several unique fragments that will be useful for restriction fragment length polymorphism (RFLP) studies were identified. In situ hybridisation of a 2.6kb unique fragment of the gene to human metaphase chromosomes localised the human HMGCoA reductase gene to human chromosome 5q12.     

Inhibition of 3-hydroxy-3-methylglutaryl coenzyme a reductase by cytosolic proteins - real or artifact

The inhibitory effect of FeSO₄-dependent cytosolic protein on microsomal HMGCoA reductase is on the enzyme activity and not an artifact of loss of the product, mevalonate, through phosphorylation, unlike that of ATP.Mg effect.     

HMG-CoA reductase kinase: measurement of activity by methods that preclude interference by inhibitors of HMG-CoA reductase activity or by mevalonate kinase

Assay of HMG-CoA reductase kinase activity requires HMG-CoA reductase (reductase, E.C. 1.1.1.34) free of associated reductase kinase. Microsomal reductase insensitive to inactivation by Mg-nucleotides alone may be prepared by heating microsomes at 50 degrees C for 15 min. The reductase in these microsomes may subsequently be inactivated by Mg-nucleotides only after addition of reductase kinase. Inactivation is a linear function of time and of cytosol protein concentration and may be reversed by treatment with a phosphoprotein phosphatase. The extent of inactivation observed under standard conditions provides an assay for reductase kinase activity. Factors present in cytosol that hinder measurement of either reductase or reductase kinase activity must be removed or inhibited. Reductase phosphatase is inhibited by 50 mM NaF. Reductase kinase kinase activity is not expressed under the assay conditions used. Mg-Nucleotide-independent inhibitors of reductase activity are removed by chromatography on DEAE-Sephacel or Blue Sepharose. Mevalonate kinase and reductase kinase are separable by chromatography on DEAE-Sephacel or Sephadex G-200. We describe a rapid chromatographic procedure for separating reductase kinase of crude fractions from mevalonate kinase and from Mg-nucleotide-independent inhibitors of reductase activity. The 1.0 M KCl eluate from DEAE-Sephacel contains all of the cytosol reductase kinase activity. This method is applicable to measurement of reductase kinase activity in cytosol or more purified fractions.     

Isoflavones found in Korean soybean paste as 3-hydroxy-3-methylglutaryl Coenzyme A reductase inhibitors

3-Hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase is the rate-limiting enzyme in the biosynthesis of cholesterol in mammals. Some microbial metabolites have been found to be HMG-CoA reductase inhibitors. Korean soybean paste is a unique food fermented by many microorganisms. The enzymatic method using the catalytic domain of Syrian hamster HMG-CoA reductase was employed for the screening of HMG-CoA reductase inhibitors. Soybean paste extract was fractionated by vacuum liquid chromatography. Fractions showing relatively high HMG-CoA reductase inhibition were further purified through Sephadex LH-20 column chromatography and C18 preparative HPLC, and the inhibitory compounds were identified as genistein, daidzein, and glycitein.     

Characterization of active and inactive forms of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase

Rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was purified to homogeneity using agarose-HMG-CoA affinity chromatography. Additional protein was isolated from the affinity column with 0.5 M KCl that demonstrated no HMG-CoA reductase activity, yet comigrated with purified HMG-CoA reductase on sodium dodecyl sulfate-polyacrylamide gels. This protein was determined to be an inactive form of HMG-CoA reductase by tryptic peptide mapping, reaction with anti-HMG-CoA reductase antibody, and coelution with purified HMG-CoA reductase from a molecular-sieving high-performance liquid chromatography column. This inactive protein was present in at least fourfold greater concentration than active HMG-CoA reductase, and could not be activated by rat liver cytosolic phosphoprotein phosphatases. Immunotitration studies with microsomal and solubilized HMG-CoA reductase isolated in the presence and absence of proteinase inhibitors suggested that the inactive protein was not generated from active enzyme during isolation of microsomes or freeze-thaw solubilization of HMG CoA reductase.