methylglutaryl Coenzyme A Reductase from Rat Liver (1974)

SUMMARY Soluble 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) from rat liver micro- somes has been shown to be inactivated reversibly by cold temperature whereas microsomal enzyme did not show any cold sensitivity. At 0” the soluble enzyme lost 80% of its activity within 30 min and warming at 37” for 20 min restored all the lost activity. Active enzyme preparations did not lose activity if quick frozen at -80’. The simplest explanation of these observations is the hypothesis that the enzyme can exist in two forms, i.e. a cold-inactivated and a heat-activated form. The enzyme had minimum activity between 0 and 19” and the specific activity was less than 0.1 nmole per min per mg. A IO-fold increase in activity was obtained by incubation at 37” for 20 min, but activity was destroyed at 42’. The origi- nal assay method employed for this solubilized enzyme in- volved incubation at 37” which does not differentiate between the two reactions (a) inactive enzyme -+ active enzyme and (b) substrate --) product. However, more readily interpreta- ble results were obtained either by activating the enzyme at 37” prior to assay or by conducting the assay at a tempera- ture at which activation does not occur, i.e. between 16 and 19”. The Arrhenius plot of the activation of the enzyme by heat (log V,,, versus l/T) showed apparent changes in slope at 19 and 28”. The rate of activation was found to be strongly protein concentration-dependent indicating an intermolecular reaction. The enzyme was stable in 40 to 120 mM buffer concentra- tions for at least 5 hours, but addition of 4 M KC1 caused an irreversible loss in activity of cold-inactivated enzyme and did not prevent the cold inactivation of activated enzyme. K,,, for substrate n-3-hydroxy3-methylglutaryl-CoA of the active soluble enzyme was estimated to be 2.2 X lop5 M, similar to that of the microsome-bound enzyme. HMG-COA reductase is the first example of a microsomal enzyme that is reversibly cold-sensitive. The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reduc- tase (HMG-CoAl reductase, EC 1.1.1.34) is of current interest * This investigation was supported by National Institutes of Health Grants l%L 05360 and -GL 08476.- Dr. Heller’s work was sunnorted nartlv bv a Stanlev McCormick Fund Fellowship. by A&&can eanc& gociety Insiitutional Research Grant INl32N, and by General Research Support Grant NIH 5SOl RR 05353-11. 1 The abbreviation used is: HMG-CoA, 3-hydroxy-3-methyl- glutaryl coenzyme A. since this is the rate-limiting enzyme in the biosynthesis of cholesterol. In rat liver the enzyme is located in the microsomes and undergoes a sharp decrease in activity when the animal is fed cholesterol (1). Therefore, the enzyme is subject to end product inhibition by cholesterol. In spite of much attention the molecular mechanism of this regulation is unknown. To elucidate this regulatory process, studies of the physical, chemical, and catalytic properties of HMG-CoA reductase are essential. The solubilization of the enzyme from microsomal membranes was first reported by Linn ‘(2) and subsequently by Kawachi and Rudney (3). Problems have been encountered by investigators in applying Linn’s method of solubilization, and the reported specific activities of enzyme solubilized and purified by Kawachi and Rudney (3) were very low. In 1973, we reported the solubilization of rat liver HMG-CoA reductase by a slow freezing and thawing treatment of the microsomes and also its partial purification (4). Recently, Brown et al. (5) have also reported solubilizing and partially purifying the en- zyme and have noted that the soluble enzyme is cold-labile and completely loses its activity if cooled to 4” unless 4