Reversible Cold Inactivation of Microsomal 31Hvdroxv-31

Soluble 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) from rat liver microsomes 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 original assay method employed for this solubilized enzyme involved incubation at 37” which does not differentiate between the two reactions (a) inactive enzyme -+ active enzyme and (b) substrate --) product. However, more readily interpretable results were obtained either by activating the enzyme at 37” prior to assay or by conducting the assay at a temperature 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 concentrations 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.