In vivo and in vitro mechanisms regulating rat liver beta-hydroxy-beta-methylglutaryl coenzyme A reductase activity.

Abstract

When equivalent units of enzyme activity are titrated with antibody to that enzyme, the endpoints are the same if all molecules have the same activity, but are different if the enzyme population is made up of active and inactive species.Such titrations have been carried out with antibody to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase for enzyme obtained from animals subjected to different hormonal or nutritional treatments.These titrations were carried out to determine whether the mode of regulation of HMG-CoA reductase in rat liver involves an alteration in the catalytic efficiency of pre-existing enzyme or a change in enzyme concentration.The antisera used in these studies was specific, as shown by the failure of either nonimmune or preadsorbed antiserum to react with HMG-CoA reductase.In vitro enzymatically active microsomal HMG-CoA reductase is modified to an inactive species in the presence of Mg", ATP, and a cytosolic protein fraction.The inactivator is nondialyzable, heat-labile, and has a molecular weight of 230,000 as determined by gel filtration.Immunotitrations of microsomes before and after inactivation resulted in different antisera endpoints per unit of HMG-GOA reductase activity.This demonstrates that the inactivation process involves a modification of pre-existing active enzyme.However, the in vivo significance of this reaction has not been established.In contrast to the above, immunotitrations of microsomal HMG-CoA reductase obtained from animals at the diurnal high and low points and for enzyme from cholestyramine-or cholesterol-fed animals yielded similar antisera endpoints per unit of enzyme activity.These studies indicate that HMG-CoA reductase activity is modulated in vivo in these nutritional and hormonal states by a change in the quantity of enzyme.In microsomes isolated in the presence of potassium fluoride, HMG-CoA reductase activity is 7-fold lower than that obtained in the absence of this substance.However, the ratio of inactive HMG-CoA reductase to active enzyme under this condition remained constant in the physiological states that were examined.The rate-determining step in hepatic cholesterol biosyn-