scholarly journals Mechanism of the inhibition of cholesterol biosynthesis by 6-fluoromevalonate

1985 ◽  
Vol 227 (1) ◽  
pp. 247-254 ◽  
Author(s):  
J F Nave ◽  
H d'Orchymont ◽  
J B Ducep ◽  
F Piriou ◽  
M J Jung
Keyword(s):  
Rat Liver ◽  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Competitive Inhibitor ◽  
Isopentenyl Pyrophosphate ◽  
Multienzyme System ◽  
Kinetics Of

6-Fluoromevalonate blocks the incorporation of mevalonic acid, but not that of isopentenyl pyrophosphate, into non-saponifiable lipids in a rat liver multienzyme system. With 3H-labelled 6-fluoromevalonate, it was found that 6-fluoromevalonate is converted to its phospho and pyrophospho derivatives in this system. The kinetics of the two kinases were studied. 6-Fluoromevalonate 5-pyrophosphate is a potent competitive inhibitor of pyrophosphomevalonate decarboxylase (Ki 37 nM). In the multienzyme assay for cholesterol biosynthesis, there is accumulation of mevalonate 5-phosphate and mevalonate 5-pyrophosphate in the presence of 5 microM-6-fluoromevalonate, and 6-fluoromevalonate 5-pyrophosphate is more effective than 6-fluoromevalonate in inhibiting cholesterol biosynthesis. We suggest therefore that 6-fluoromevalonate blocks cholesterol biosynthesis at the level of pyrophosphomevalonate decarboxylase after being pyrophosphorylated.

scholarly journals Incorporation of [2−14C,(5R)-5−3H1]mevalonic acid into cholesterol by a rat liver homogenate and into β-sitosterol and 28-isofucosterol by Larix decidua leaves

1969 ◽  
Vol 114 (4) ◽  
pp. 885-892 ◽  
Author(s):  
L J Goad ◽  
G. F. Gibbons ◽  
Loretta M. Bolger ◽  
H H Rees ◽  
T W Goodwin
Keyword(s):  
Double Bond ◽  
Hydrogen Atom ◽  
Rat Liver ◽  
Cholesterol Biosynthesis ◽  
Liver Homogenate ◽  
Mevalonic Acid ◽  
Atomic Ratio ◽  
Larix Decidua ◽  
Tritium Atom

1. Incubation of a rat liver homogenate with 3R-[2−14C,(5R)-5−3H1]mevalonic acid gave cholesterol with 3H/14C atomic ratio 6:5. 2. Conversion of the labelled cholesterol into 3β-acetoxy-6-nitrocholest-5-ene or cholest-4-ene-3,6-dione resulted in the loss of one tritium atom from C-6. 3. These results show that during cholesterol biosynthesis the 6α-hydrogen atom of a precursor sterol is eliminated during formation of the C-5–C-6 double bond. 4. Incorporation of 3R-[2−14C,(5R)-5−3H1]mevalonic acid into the sterols of larch (Larix decidua) leaves gave labelled cycloartenol and β-sitosterol with 3H/14C atomic ratios 6:6 and 6:5 respectively. 5. One tritium atom was lost from C-6 on conversion of the labelled β-sitosterol into either 3β-acetoxy-6-nitrostigmast-5-ene or stigmast-4-ene-3,6-dione, demonstrating that formation of the C-5–C-6 double bond of phytosterols also involves the elimination of the 6α-hydrogen atom of a precursor sterol. 6. The 3R-[2−14C,(5R)-5−3H1]mevalonic acid was also incorporated by larch (L. decidua) leaves into a sterol that co-chromatographed with 28-isofucosterol. Confirmation that the radioactivity was associated with 28-isofucosterol was obtained by co-crystallization with carrier 28-isofucosterol and ozonolysis of the acetate to give radioactively labelled 24-oxocholesteryl acetate. 7. The significance of these results to phytosterol biosynthesis is discussed.

scholarly journals The kinetics of phenethylhydrazine oxidation by monoamine oxidase

1971 ◽  
Vol 125 (2) ◽  
pp. 521-524 ◽  
Author(s):  
K. F. Tipton ◽  
I. P. C. Spires
Keyword(s):  
Monoamine Oxidase ◽  
Rat Liver ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Parallel Lines ◽  
Hydrazine Oxidation ◽  
Pig Brain ◽  
Kinetics Of

1. In the presence of the substrate benzylamine, phenethylhydrazine has been shown to be a competitive inhibitor of monoamine oxidase from rat liver and pig brain. 2. Phenethylhydrazine is also a substrate for monoamine oxidase. Reciprocal plots for hydrazine oxidation give families of intersecting lines in contrast with the parallel lines previously reported for tyramine oxidation. 3. Two possible modifications of the mechanism obeyed by tyramine oxidation are suggested, but the product inhibition results are insufficient to distinguish between these two mechanisms.

scholarly journals Some aspects of the kinetics of rat liver pyruvate carboxylase

1970 ◽  
Vol 120 (1) ◽  
pp. 79-93 ◽  
Author(s):  
Janet M. Wimhurst ◽  
K. L. Manchester
Keyword(s):  
Rat Liver ◽  
Binding Sites ◽  
Pyruvate Carboxylase ◽  
Competitive Inhibitor ◽  
Mitochondrial Fraction ◽  
Spectrophotometric Assay ◽  
Time Dependent ◽  
Malonyl Coa ◽  
Kinetics Of ◽  
Hill Plots

1. The kinetics of rat liver pyruvate carboxylase were examined and the effect of various agents as activators or inhibitors determined. 2. Essentially similar results were obtained in comparisons of kinetics determined by a radioactivity method involving extracts of acetone-dried powders from whole livers and with a spectrophotometric assay using partially purified enzyme from the mitochondrial fraction. Activity per g of liver from fed or starved rats assayed under optimum substrate and activator conditions was 3 or 6 μmol of oxaloacetate formed/min at 30°C, respectively. 3. The enzyme exhibited cold-lability and lost activity on standing, even in 1.5m-sucrose. 4. The Km towards pyruvate was about 0.33mm and towards bicarbonate 4.2mm. Km towards MgATP2− was 0.14mm. Mg2+ ions activated the enzyme, in addition to their role in MgATP2− formation. From calculations of likely concentrations of free Mg2+ in the assay medium a Ka towards Mg2+ of about 0.25mm was deduced. Mn2+ also activated the enzyme as well as Mg2+, but at much lower concentrations. It appeared to be inhibitory when concentrations of free Mn2+ as low as 0.1mm were present. 5. Excess of ATP is inhibitory, and this appears at least in part independent of the trapping of Mg2+. 6. Both Co2+ and Zn2+ were inhibitory; 2mol of the latter appeared to be bound even in the presence of excess of Mg2+ and the inhibition was time-dependent. 7. Ca2+ inhibited by competition with Mg2+ (Ki about 0.38mm). The inhibition due to Ca2+ was less pronounced when activation was with Mn2+. Inhibition by Ca2+ and ATP appeared to be additive. 8. Hill plots suggested that no interactions occurred between ATP-binding sites. Although similar plots for total Mg2+ gave n=3.6, no conclusions could be drawn due to the chelation of the cation with other components of the assay. Similar difficulties arose in assessing the values for Ca2+. 9. The enzyme was inactive in the absence of acetyl-CoA and showed a sigmoidal response in its presence. Ka was about 0.1mm with possibly up to four binding sites. Malonyl-CoA was a competitive inhibitor, with Ki 0.01mm. 10. There was no apparent inhibition by glucose, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, acetoacetate, β-hydroxybutyrate, malate, aspartate, pyruvate, palmitoylcarnitine, octanoate, glutathione, butacaine, triethyltin or potassium chloride under the conditions used. Inhibition was found with citrate (possibly by chelation) and adenosine, and also by phosphoenolpyruvate, AMP, ADP and cyclic AMP, Ki towards the last four being 0.55, 0.76, 0.25 and 1.4mm respectively.

scholarly journals Studies of the specificity and kinetics of rat liver spermidine/spermine N1-acetyltransferase

1983 ◽  
Vol 213 (3) ◽  
pp. 701-706 ◽  
Author(s):  
F Della Ragione ◽  
A E Pegg
Keyword(s):  
Rat Liver ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Aliphatic Chain ◽  
Amino Group ◽  
Acetyl Coa ◽  
Competitive Kinetics ◽  
Primary Amino ◽  
Secondary Amino ◽  
Kinetics Of

The substrate specificity and kinetic mechanism of spermidine N1-acetyltransferase from rat liver was investigated using a highly purified (18 000-fold) preparation from the livers of rats in which the enzyme was induced by treatment with carbon tetrachloride (1.5 ml/kg body wt. 6h before death). The enzyme catalysed the acetylation of spermidine, spermine, sym-norspermidine, sym-norspermine, N-(3-aminopropyl)-cadaverine, N1-acetylspermine, 3,3′-diamino-N-methyldipropylamine and 1,3-diaminopropane, but was inactive with putrescine, cadaverine, sym-homospermidine and N1-acetylspermidine. These results suggest that the enzyme is highly specific for the acetylation of a primary amino group that is separated by a three-carbon aliphatic chain from another nitrogen atom (i.e. the substrates are of the type H2N[CH2]3NHR). The maximal rates of acetylation of 1,3-diaminopropane and 3,3′-diamino-N-methyldipropylamine were much lower than the maximal rates with spermidine or sym-norspermidine as substrates, suggesting a preference for a secondary amino group bearing the aminopropyl group that is acetylated. The best substrates for acetylation were sym-norspermidine and sym-norspermine, which had Km values of about 10 micrograms and Vmax. values of about 2 mumol of product/min per mg of enzyme compared with Km of 130 microM and Vmax. of 1.3 mumol/min per mg for spermidine. N1-Acetylspermidine (the product of the reaction) and N8-acetylspermidine were weak inhibitors and were competitive with spermidine, having Ki values of about 6.6 mM and 0.4 mM respectively. N1-Acetylspermidine was a non-competitive inhibitor with respect to acetyl-CoA. CoA was also inhibitory to the reaction, showing non-competitive kinetics when either [acetyl-CoA] or [spermidine] was varied. These results suggest that the reaction occurs via an ordered Bi Bi mechanism in which spermidine binds first and N1-acetyl-spermidine is the final product to be released.

Acetyl-CoA hydrolase: relation between activity and cholesterol metabolism in rat

1988 ◽  
Vol 255 (5) ◽  
pp. R724-R730
Author(s):  
S. Ebisuno ◽  
F. Isohashi ◽  
Y. Nakanishi ◽  
Y. Sakamoto
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Cholesterol Metabolism ◽  
Cholesterol Biosynthesis ◽  
Physiological Role ◽  
Competitive Inhibitor ◽  
Isobutyric Acid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Acetyl Coa ◽  
Coa Hydrolase

To examine whether cytosolic acetyl-CoA hydrolase in rat liver is involved in regulation of cholesterol biosynthesis, we investigated the alteration of the enzyme activity under conditions of stimulation (cholestyramine treatment) and suppression [cholesterol feeding, a potent competitive inhibitor of microsomal 3-hydroxy-3-methylglutaryl-CoA reductase (CS 514) treatment, and a hypolipidemic drug [alpha-(p-chlorophenoxy)isobutyric acid, CPIB] injection) of cholesterol biosynthesis. The enzyme activity in rat liver increased significantly in the early diabetic, cholesterol-fed, CS 514-, and CPIB-treated groups, but no change in its activity was observed in chronic diabetic groups. Cholestyramine treatment to cholesterol-fed rats made the enzyme activity return to the initial level. When chronic diabetic rats were given a cholesterol diet or treated with CS 514 or CPIB, the activity increased significantly. Inhibition of cholesterol biosynthesis caused by these treatments induced increase in the enzyme activity with increase in the enzyme protein, judging from results obtained by enzyme-linked immunosorbent assay. These results suggest that this enzyme has a physiological role in maintenance of the equilibrium between the cytosolic acetyl-CoA concentration and CoA-SH pool for cholesterol metabolism.

Kinetic Aspects of the Interaction of Blood Clotting Enzymes

1968 ◽  
Vol 19 (03/04) ◽  
pp. 364-367 ◽  
Author(s):  
H. C Hemker ◽  
P. W Hemker
Keyword(s):  
Enzyme Kinetics ◽  
Blood Clotting ◽  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Kinetics Of

SummaryThe enzyme kinetics of competitive inhibition under conditions prevailing in clotting tests are developed and a method is given to measure relative amounts of a competitive inhibitor by means of the t — D plot.

VANILLINSÄURE ALS ENDPRODUKT DES STOFFWECHSELS VON ADRENALIN UND NORADRENALIN. II.

1963 ◽  
Vol 44 (1) ◽  
pp. 101-106 ◽  
Author(s):  
Wilhelm Dirscherl ◽  
Helmut Thomas
Keyword(s):  
Rat Liver ◽  
Paper Chromatography ◽  
Column Chromatography ◽  
Vanillic Acid ◽  
Hippuric Acid ◽  
Single Spot ◽  
Solvent Systems ◽  
Perfusion Experiment ◽  
Kinetics Of

ABSTRACT Perfusion of rat liver with vanillic acid yielded only one metabolite. In paper chromatography with three different solvent systems, the substance showed the same RF-values as vanillyolglycine (3-methoxy-4-hydroxyhippuric acid) and in mixed chromatograms there was only one single spot. After separation by column chromatography, the UV- and IRspectra of the reaction product were identical with those of 3-methoxy4-hydroxy-hippuric acid. During the perfusion experiment, the kinetics of the conjugation were investigated.

Kinetics of oestradiol-dependent induction of the prolactin receptor in rat liver

1988 ◽  
Vol 117 (4_Suppl) ◽  
pp. S88
Author(s):  
R. GÜNTHER ◽  
H. C. BLOSSEY
Keyword(s):  
Rat Liver ◽  
Prolactin Receptor ◽  
Kinetics Of

scholarly journals Characterizing the Role of HMG-CoA Reductase in Aryl Hydrocarbon Receptor-Mediated Liver Injury in C57BL/6 Mice

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Peter Dornbos ◽  
Amanda Jurgelewicz ◽  
Kelly A. Fader ◽  
Kurt Williams ◽  
Timothy R. Zacharewski ◽  
...  
Keyword(s):  
Liver Injury ◽  
Aryl Hydrocarbon Receptor ◽  
Cholesterol Biosynthesis ◽  
Competitive Inhibitor ◽  
Cholesterol Homeostasis ◽  
Hepatic Glycogen ◽  
Alcoholic Fatty Liver ◽  
Female Mice ◽  
Aryl Hydrocarbon ◽  
The Impact

Abstract The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor. The prototypical ligand of the AHR is an environmental contaminant called 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD exposure is associated with many adverse health outcomes in humans including non-alcoholic fatty liver disease (NAFLD). Previous studies suggest that AHR ligands alter cholesterol homeostasis in mice through repression of genes involved in cholesterol biosynthesis, such as Hmgcr, which encodes the rate-limiting enzyme of cholesterol biosynthesis called 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR). In this study, we sought to characterize the impact of HMGCR repression in TCDD-induced liver injury. C57BL/6 mice were exposed to TCDD in the presence or absence of simvastatin, a competitive inhibitor of HMGCR. Simvastatin exposure decreased TCDD-induced hepatic lipid accumulation in both sexes, but was most prominent in females. Simvastatin and TCDD (S + T) co-treatment increased hepatic AHR-battery gene expression and liver injury in male, but not female, mice. In addition, the S + T co-treatment led to an increase in hepatic glycogen content that coincides with heavier liver in female mice. Results from this study suggest that statins, which are amongst the most prescribed pharmaceuticals, may protect from AHR-mediated steatosis, but alter glycogen metabolism and increase the risk of TCDD-elicited liver damage in a sex-specific manner.

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