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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