[55] 3-Hydroxy-3-methylglutaryl-CoA reductase from rat liver

Kleinsek, Don A.; Dugan, Richard E.; Baker, T.A.; Porter, John W.

doi:10.1016/0076-6879(81)71057-7

Cited by 35 publications

(13 citation statements)

References 30 publications

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“…mg-'. This is comparable with values previously obtained for catalytic fragments purified from various mammalian species [20], and for the bacterially expressed catalytic fragment of Syrian hamster enzyme [21]. The K,,, values for HOMeGlt-CoA and NADPH (Table 2) are also of the same order as those reported previously.…”

Section: Construction Of An Expression Vector For Wild-type Ho-supporting

confidence: 79%

Analysis of the Specificity of the AMP‐Activated Protein Kinase by Site‐Directed Mutagenesis of Bacterially Expressed 3‐hydroxy 3‐methylglutaryl‐CoA Reductase, Using a Single Primer Variant of the Unique‐site‐elimination Method

Ching

Davies

Hardie

1996

European Journal of Biochemistry

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The specificity of protein kinases is usually examined using synthetic peptide substrates, either designed variants, or, more recently random peptide libraries. However not all protein kinases utilize synthetic peptides efficiently as substrates. Even among those that do, these approaches neglect effects caused by three-dimensional protein conformation, or the existence of determinants remote from the phosphorylation site. To follow up our previous peptide studies on the specificity of the AMP-activated protein kinase (AMPK) [Dale, S., Wilson, W. A., Edelman, A. M., & Hardie, D. G. (1995) FEBS Lett. 361, 191-1951, we have expressed the C-terminal, catalytic domain of Chinese hamster hydroxymethylglutaryCoA reductase in Escherichia coli. The domain was expressed with an N-terminal His, tag which allowed rapid purification on Ni'+-agarose. The purified protein retained full enzymic activity, and, as with the native enzyme, was totally inactivated by phosphorylation by AMPK at a single site corresponding to Ser871. Using a novel modification of the unique-site elimination method (which allowed direct mutagenesis of the double-stranded expression vector using a single oligonucleotide primer) we expressed 18 mutations involving residues around Ser871. The results broadly confirmed the recognition motif previously proposed on the basis of peptide studies. Three of the mutants were better substrates for AMPK than the wild type, and one of these (K872A) had hydroxymethylglutaryl-CoA reductase kinetic parameters virtually indistinguishable from the wild type. This suggests that hydroxymethylglutaryl-CoA reductase may have been selected to be a sub-optimal substrate for AMPK.Keywords: AMP-activated protein kinase; hydroxymethylglutaryl-CoA reductase ; protein-kinase specificity ; site-directed mutagenesis ; unique-site elimination.Protein kinases and phosphatases catalyze cycles of phosphorylation and dephosphorylation which appear to represent the major switching mechanism in eukaryotic cells [l 1. Predictions from current partial genome sequences suggest that vertebrate genomes encode of the order of 2000 protein kinases 121. Some protein kinases appear to be specific for a single protein target, but many phosphorylate multiple targets, although even in the latter cases only specific serine, threonine or tyrosine side chains are modified. A key question therefore is, how do protein kinases recognize the correct side chains on the correct protein targets?Previous studies on the specificity of protein kinases have generally used variant synthetic peptide substrates whose sequences were based on phosphorylation sites on known target proteins (e.g. . While this approach has been very informative (reviewed in [6]), it does require prior identification of protein substrates, and sequencing of their phosphorylation sites.

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Section: Construction Of An Expression Vector For Wild-type Ho-supporting

confidence: 79%

Analysis of the Specificity of the AMP‐Activated Protein Kinase by Site‐Directed Mutagenesis of Bacterially Expressed 3‐hydroxy 3‐methylglutaryl‐CoA Reductase, Using a Single Primer Variant of the Unique‐site‐elimination Method

Ching

Davies

Hardie

1996

European Journal of Biochemistry

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“…HMG CoA Reductase Inhibition Activity HMG-CoA reductase was partially purified according to the method of Kleinsek et al 19) Briefly, male Sprague-Dawley rats (180-200 g) were fed an AIN-96 diet containing 2% cholestyramine for 5 d prior to killing at the mid-dark period, which is the diurnal high point of enzyme activity. After the animals were decapitated, the livers were excised and immediately homogenized in 2 ml/g liver of ice-cold buffer A (potassium phosphate 50 mM, sucrose 0.2 M, and dithiothreitol 2 mM, pH 7.0) with a glass-Teflon Potter-Elvejhem homogenizer.…”

Section: Preparation Of Kiwifruit Extractsmentioning

confidence: 99%

Cardiovascular Protective Properties of Kiwifruit Extracts in Vitro

Jung

Song

Han

et al. 2005

Biological & Pharmaceutical Bulletin

104

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It is currently accepted that the consumption of fruit-derived antioxidants such as vitamin C, carotenoids, and flavonoids provides a preventive effect against cardiovascular disease. The purpose of the present study was to investigate potential cardiovascular protective properties of aqueous and 70% ethanol extracts from kiwifruit by analyzing the antioxidative, antihypertensive, hypocholesterolemic, and fibrinolytic activities in vitro. Aqueous and 70% ethanol extracts at 50 mg/ml showed DPPH-radical scavenging activities of 72.31% and 70.75%, respectively. Total antioxidant activity in linoleic acid emulsion was 85-88% at 10 mg/ml and 96-98% at 50 mg/ml of kiwifruit extract. Inhibitory activities against angiogensin I-converting enzyme of kiwifruit extracts were 21-26% at 10 mg/ml and 46-49% at 50 mg/ml, and inhibitory activities on HMG-CoA reductase were 13-14% at 10 mg/ml and 19-30% at 50 mg/ml. Fibrinolytic activity of kiwifruit was also observed at a high concentration of 100 mg/ml in both aqueous and 70% EtOH extracts. Based on our results, kiwifruit have potential cardiovascular protective properties in vitro.

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“…Radioassay was performed as described by Kleinsek et al [22] with the inclusion of 0.4 M KCI, in the assay mixture. The activity of hepatic HOMeGlt-CoA reductase was measured, either in microsomal preparations or in protein solubilized from the microsomes, as indicated in the individual experiments.…”

Section: Assay Of Homeclt-coa Reductusementioning

confidence: 99%

“…Solubilized HOMeGlt-CoA reductase was purified to homogeneity by the back-to-back dye-ligand HOMeGlt-CoA affinity gel procedure described under method 5 by Kleinsek et al [22].…”

Section: Purifkation Of' Homeclt-coa Recluctasementioning

confidence: 99%

Regulation of Short‐Term Changes in Hepatic β‐Hydroxy‐β‐methylglutaryl‐CoA Reductase Activity

Dugan

Baker

Porter

1982

European Journal of Biochemistry

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Immunotitrations of rat liver hydroxymethylglutaryl-CoA (HOMeGlt-CoA) reductase activity were performed before and after short-term changes in the nutritional or hormonal state of the animals. Changes in enzyme activity (increase or decrease) within 1 h following cholesterol feeding or glucagon or mevalonolactone administration to normal rats, or insulin administration to diabetic rats were accompanied by no change in the specific activity of the enzyme, as determined from the quantity of enzyme activity inactivated by a fixed quantity of antibody. These results support the conclusion that the loss in enzyme activity was due to conversion of the enzyme to immunounreactive products. In agreement with this conclusion the enzyme activity lost after these short-term physiological changes was not restorable by phosphoprotein phosphatase action. On the other hand, incubation of rat liver microsomes with ATP and Mg2+ decreased the specific activity of HOMeGlt-CoA reductase about tenfold, as determined by immunotitration. The low specific activity produced under these conditions was increased by phosphatase action to nearly the original level. The above evidence suggests that the changes in HOMeGlt-CoA reductase activity that resulted from short-term physiological changes in hormonal or nutritional states of an animal were brought about by a change in the quantity of enzyme, and not by reversible phosphorylation of preexisting enzyme.The rate-limiting enzyme for cholesterol synthesis in rat liver, hydroxymethylglutaryl-CoA (HOMeGlt-CoA) reductase, undergoes a large and rapid change in catalytic activity in response to a number of nutritional and hormonal factors [I]. Early studies on the regulation of this enzyme [2,3] indicated that its large diurnal variation in activity is due to changes in the rate of biosynthesis of the enzyme, and not to a change in the rate of degradation. More recently short-term experiments have provided evidence that reversible inactivation precedes the degradation of HOMeGlt-CoA reductase to immunoinactive fragments [4 -71. Furthermore, evidence from a number of laboratories has conclusively demonstrated that microsomal HOMeGlt-CoA reductase can be phosphorylated and inactivated by a cyclic-AMP-independent kinase in the presence of ATP and Mg2+ [8-131. The phosphorylated enzyme can be dephosphorylated and reactivated by a cytosolic phosphatase [8 -131.Recent reports from three laboratories have presented evidence for a short-term physiological phosphorylation/dephosphorylation mechanism of regulation of HOMeGltCoA reductase activity via a response to hormones, such as insulin and glucagon [14,15], and pathway metabolites, such as cholesterol and mevalonic acid [16,17]. However, other investigations have brought forth evidence that phosphorylation/dephosphorylation is not the mechanism by which marked changes in enzyme activity are produced after longterm changes in the physiological state of the animal [18 -201.

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[55] 3-Hydroxy-3-methylglutaryl-CoA reductase from rat liver

Cited by 35 publications

References 30 publications

Analysis of the Specificity of the AMP‐Activated Protein Kinase by Site‐Directed Mutagenesis of Bacterially Expressed 3‐hydroxy 3‐methylglutaryl‐CoA Reductase, Using a Single Primer Variant of the Unique‐site‐elimination Method

Analysis of the Specificity of the AMP‐Activated Protein Kinase by Site‐Directed Mutagenesis of Bacterially Expressed 3‐hydroxy 3‐methylglutaryl‐CoA Reductase, Using a Single Primer Variant of the Unique‐site‐elimination Method

Cardiovascular Protective Properties of Kiwifruit Extracts in Vitro

Regulation of Short‐Term Changes in Hepatic β‐Hydroxy‐β‐methylglutaryl‐CoA Reductase Activity

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