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G C Ness

Publications and source records attributed to G C Ness.

At least 73 records · Page 4Linked to original sources
PubMed1987

Development of a noncompetitive, solid phase, bridged biotin-avidin enzyme immunoassay for measurement of human leukocyte microsomal HMG-CoA reductase protein concentration.

Methods were developed for determination of human mononuclear leukocyte HMG-CoA reductase protein concentration by a noncompetitive, solid phase, bridged biotin-avidin enzyme immunoassay procedure. Leukocyte microsomal HMG-CoA reductase, first immobilized onto a nitrocellulose filter, is sequentially reacted with 1) monospecific, polyclonal rabbit anti-rat liver HMG-CoA reductase antiserum, which crossreacts with the human liver and leukocyte enzymes; 2) biotinylated donkey anti-rabbit immunoglobulin; 3) a streptavidin-horseradish peroxidase conjugate; and 4) 4-chloro-1-naphthol and H2O2 to visualize the quantity of horseradish peroxidase bound to the immunocomplex. Color development was proportional to the quantity of either purified liver or leukocyte microsomal HMG-CoA reductase applied to the nitrocellulose. Color development was not observed, however, when HMG-CoA reductase was omitted from the nitrocellulose, when one of the reactant species was omitted from the incubation reactions, or when anti-rat liver HMG-CoA reductase antiserum was pre-absorbed with either rat liver or human leukocyte HMG-CoA reductase. Immunoreactivity of microsomal HMG-CoA reductase was independent of the phosphorylation state of the enzyme, but was inversely related to the concentration of thiol-reducing agents present in the microsomal preparation up to 4 mM. Further increases in thiol-reductant failed to produce changes in immunoreactivity. Freshly isolated mononuclear leukocyte microsomal HMG-CoA reductase protein concentration in leukocytes from 31 healthy, normocholesterolemic subjects was a linear function of HMG-CoA reductase activity (R = 0.65; P less than 0.001). The catalytic efficiency of the freshly isolated mononuclear leukocyte enzyme was 313 +/- 34 pmol of mevalonate formed per min of incubation at 37 degrees C per mg immunoreactive protein. This methodology, in conjunction with that recently developed to measure human leukocyte HMG-CoA reductase activity (1984. J. Lipid Res. 25: 967-978), should prove useful in discriminating between HMG-CoA reductase regulatory mechanisms involving changes in enzyme protein concentration and those resulting from changes in enzyme catalytic efficiency.

Animals↗
PubMed1986

Regulation of the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin.

In hypophysectomized--diabetic rats full restoration of hepatic HMG-CoA reductase activity required administration of both triiodothyronine and insulin. Giving triiodothyronine alone resulted in full restoration of reductase mRNA and immunoreactive protein but not of enzyme activity. Increasing the ratio of dithiothreitol to microsomal protein resulted in full HMG-CoA reductase activity. With 5mM glutathione, HMG-CoA reductase activity was 5 to 10 fold higher in non-diabetic animals. The data suggest that insulin acts by increasing the portion of HMG-CoA reductase present in its active free sulfhydryl form.

Animals↗
PubMed1986

Characteristics of rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

A procedure for the preparation of rat liver microsomal fractions essentially devoid of contaminating lysosomes is described. When this preparation was examined by immunoblotting with a rabbit antiserum to rat 3-hydroxy-3-methylglutaryl-CoA reductase, a single band corresponding to an Mr of 100000 was observed. No evidence was found for glycosylation of rat liver-3-hydroxy-3-methylglutaryl-CoA reductase. Native rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase differs from the purified proteolytically modified species in that it displays allosteric kinetics towards NADPH.

Acetylglucosaminidase↗
PubMed1985

Sulfhydryl/disulfide forms of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Using radiation inactivation and immunoblotting techniques, evidence for functionally active forms of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase with molecular weights of about 100,000 and 200,000 was obtained. In liver microsomes isolated from rats fed both mevinolin and colestipol, the Mr 100,000 form was the predominant species, whereas in microsomes from animals fed only colestipol, the Mr 200,000 species was the major form. This Mr 200,000 form could be converted to the Mr 100,000 form by addition of dithiothreitol or beta-mercaptoethanol. Although both forms appear to possess catalytic activity, the Mr 200,000 species displays sigmoidal kinetics with respect to the concentration of NADPH, whereas the Mr 100,000 form exhibits typical hyperbolic kinetics.

Animals↗
PubMed1985

Activation of rat liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase by NADPH. Effects of dietary treatments.

The sigmoidal curves observed for rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase with NADPH as the varied substrate were markedly affected by feeding the animals diets containing colestipol, mevinolin and colestipol or cholesterol. Feeding of mevinolin and colestipol decreased the S0.5 for NADPH from 270 to 40 microM, while cholesterol feeding increased the value to 1.3 mM. Immuno-blotting analysis revealed that the Mr 100,000 form of HMG-CoA reductase predominated in cases where the S0.5 value was lowest, and the Mr 200,000 species was the major form where the S0.5 values were highest. Activation of HMG-CoA reductase by NADPH was not due to conversion of the Mr 200,000 form to the 100,000 form.

Allosteric Regulation↗
PubMed1984

Topography of rat liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The orientation of 3-hydroxy-3-methylglutaryl coenzyme A reductase within the endoplasmic reticulum was investigated. Microsomal reductase activity was not latent, as addition of various detergents failed to activate the enzyme. Reductase activity was readily inhibited in intact microsomes by impermeable inhibitors such as trypsin, mercury dextran and anti-reductase IgG. Under the conditions used, these agents did not affect the intactness of microsomes as determined by latency of mannose-6-phosphate phosphohydrolase activity. The sensitivity to these inhibitors was not increased in disrupted microsomes. It is concluded that the domain containing the active site of the reductase is situated on the cytosolic surface of the endoplasmic reticulum.

Animals↗
PubMed1984

Hydroxymethylglutaryl-coenzyme A reductase-containing hepatocytes are distributed periportally in normal and mevinolin-treated rat livers.

Mevinolin is a potent inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase; EC 1.1.1.34), an enzyme that catalyzes the rate-limiting step in cholesterol biosynthesis. We have been studying the hepatic distribution of reductase with immunofluorescence microscopy and liver ultrastructure with electron microscopy in normal and drug-treated rats. In control animals, only about 20% of the hepatocytes were reductase positive. These cells were localized in the periportal lobular zones. The numbers of positive hepatocytes in animals given mevinolin or cholestyramine (or both) were directly proportional to the activities of the HMG-CoA reductase determined biochemically. This induction of HMG-CoA reductase immunofluorescence was centered periportally. Rats given 0.075% mevinolin alone had a homogeneous distribution of reductase staining in their hepatocyte cytoplasm, whereas a combination of 0.25% mevinolin and 3% cholestyramine caused a 150-fold increase in enzyme activity and induced prominent juxtanuclear immunofluorescent globules of HMG-CoA reductase in all hepatocytes. With electron microscopy, these bodies were composed of tightly packed stacks of smooth endoplasmic reticulum cysternae and aggregates of branched smooth endoplasmic reticulum tubules. Our data suggest that a subpopulation of periportal rat hepatocytes may be uniquely specialized for cholesterol synthesis.

Animals↗
PubMed1983

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Within the last few years considerable evidence has accumulated which indicates that changes in HMG-CoA reductase are due primarily, if not solely, to changes in enzyme quantity. These include the changes caused by insulin, glucagon, cholesterol, mevalonolactone, cholestyramine, compactin, cyclic monoterpenes and the diurnal rhythm. Investigations of whether these changes in enzyme quantity primarily represent changes in rates of enzyme synthesis or degradation are just beginning. Regulation of reductase activity by reversible phosphorylation seems unlikely when all the available data are critically examined.

Animals↗
PubMed1980

Characteristics of magnesium-adenosine triphosphate-dependent inactivators of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The inhibition of homogeneous 3-hydroxy-3-methyl-glutaryl coenzyme A reductase by MgATP-dependent inactivators isolated from rat liver cytosol and microsomes was examined. The inhibition was independent of incubation time. The inhibition was readily reversed by dilution or dialysis, by the addition of EDTA, and by incubating the inhibited enzyme with glycerol and glycerol kinase to convert the ATP to ADP. The inactivated enzyme was not reactivated by various phosphatases. When inactivated in the presence of [gamma-32P]ATP, no radioactivity was incorporated into the reductase. These observations indicate that the inactivators do not exert their effects through covalent modification of the reductase.

Adenosine Triphosphate↗
PubMed1979

Studies on the regulation of glycoprotein biosynthesis. An investigation of the rate-limiting steps of dolichyl phosphate biosynthesis.

The possible role of HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase (the rate-controlling enzyme of cholesterol biosynthesis) in regulating the rate of dolichyl phosphate biosynthesis in rat liver was investigated. Rats were either fasted 48 h or fed diets supplemented with the drug cholestyramine. The activity of HMG-CoA reductase was 5000-fold greater in liver from cholestyramine-fed rats as compared to fasted rats. The activity of dolichyl phosphate synthetase, the prenyl transferase responsible for the biosynthesis of dolichyl phosphate from farnesyl pyrophosphate and isopentenyl pyrophosphate, was similar in both nutritional conditions and was markedly less active than HMG-CoA reductase even in the fasted state. Acetate incorporation into cholesterol was 2200-fold greater in liver slices from cholestyramine-fed rats as compared to fasted rats. By contrast, acetate incorporation into dolichyl phosphate was only 6-fold higher. Further studies suggested that the levels of farnesyl pyrophosphate and isopentenyl pyrophosphate are several hundred-fold greater in liver from cholestyramine-treated rats. From these results, it is concluded that the rate of dolichyl phosphate biosynthesis in rat liver is not regulated by the activity of HMG-CoA reductase but is probably regulated at the level of dolichyl phosphate synthetase.

Acetates↗
PubMed1979

Influence of a naturally occurring competing enzymic activity on studies of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.

An enzymic activity which competes with 3-hydroxy-3-methylglutaryl coenzyme A reductase for D-hydroxymethylglutaryl CoA has been found in isolated rat liver microsomes and in microsomal extracts. The presence of this activity in enzyme preparations causes a decrease in the rate of mevalonate formation leading to an underestimation of reductase activity and an overestimation of the apparent Km of the reductase. The product formed by this competing enzymic activity behaves similarly to, but not identically with, mevalonolactone when chromatographed on Bio-Rad AG 1-x8 formate, which is used in many reductase assay procedures to separate mevalonolactone from hydroxymethylglutaryl CoA. Removal of this competing enzymic activity from reductase preparations can be accomplished by gel filtration using Bio-Gel A 1.5m, by washing the microsomes or by incubating the microsomal extract at 37 degrees C. Using enzyme preparations free of this competing enzymic activity, the apparent Km values of the reductase for D-hydroxymethylglutaryl CoA and NADPH were found to be 1.3 and 26 micronM respectively.

Animals↗
PubMed1979

Perinatal development of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in rat lung, liver and brain.

The developmental pattern of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), which catalyzes the rate-limiting step of cholesterol biosynthesis, was studied in lung, liver and brain of Sprague-Dawley rats. Each tissue exhibited a distinct pattern. Reductase activity in the fetal lung reached a peak at 19 days of gestation, which corresponds to the onset of active surfactant production. This observation is consistent with the suggestion that the fetal lung synthesizes all surfactant components including cholesterol. In the liver, reductase activity varied in a reciprocal fashion with serum cholesterol levels. The peak of brain reductase activity occurred at 3 days after birth at the onset of rapid brain growth despite rapidly rising serum cholesterol levels.

Aging↗
PubMed1979

De novo fatty acid synthesis and fatty acid elongation catalyzed by subcellular fractions from hog and human aorta.

De novo synthesis and mitochondrial elongation of fatty acids have been demonstrated in subcellular fractions from hog and human aorta. Microsomal fatty acid elongation has been shown in hog aorta. The activity catalyzing the formation of fatty acids from acetyl and malonyl CoA was associated with a high molecular weight complex in the 6 x 10(6) g x min supernatant fraction. The principal product was palmitic acid. Some myristic and stearic acids were also formed. One elongation system was associated with protein which sedimented between 4500 g x min and 150,000 g x min. It used acetyl CoA but not malonyl CoA, and NADH was the preferred reducing agent. Radioactivity from acetyl CoA was incorporated into many fatty acids. In hog aorta a second elongation system was found associated with protein which sedimented at 6 x 10(6) g x min. It used malonyl CoA preferentially as substrate and either NADH or NADPH as reducing agent.

Acetyl Coenzyme A↗