Active and inactive forms of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the liver of the rat. Comparison with the rate of cholesterol synthesis in different physiological states

Hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-controlling enzyme in cholesterol synthesis, exists in a phosphorylated (inactive) and dephosphorylated (active) form. The current studies were undertaken to determine whether long term regulation of reductase in rat liver is achieved through changes in the proportion of enzyme in the 2 forms. Rates of cholesterol synthesis were varied 50-fold by diurnal light cycling, fasting, stress, and feeding of cholesterol and cholestyramine. Portions of each liver were used to measure: (1) rate of cholesterol synthesis in tissue slices, and (2) reductase activity in microsomes prepared in 4 different ways: after isolation in absence and presence of 50 mM sodium fluoride and after preincubation in absence and presence of escherichia coli alkaline phosphatase. Sodium fluoride was added to the homogenization buffer because it inhibits dephosphorylation of the enzyme during the microsomal isolation. E. coli alkaline phosphatase was used to maximally activate the enzyme by removing a phosphate group. In livers of animals at midpoint of the dark cycle, reductase activity was 7-fold higher in microsomes prepared in absence of fluoride than in microsomes prepared in presence of fluoride. Alkaline phosphatase activated reductase of fluoride-treated microsomes 13-fold, but it produced only a 1.4-fold activation of microsomes isolated in absence of fluoride. Using enzyme activity after phosphatase treatment as a measure of total reductase, the authors calculated that 75-90% of reductase under all physiological conditions was in a phosphorylated (inactive) form at the time the homogenates were prepared. The proportion of phosphorylated enzyme remained constant under conditions in which total reductase activity and the rate of cholesterol synthesis varied as much as 50-fold. Thus, long term alterations in cholesterol synthesis in rat liver are due not to changes in the state of phosphorylation of reductase, but rather to changes in the total amount of enzyme protein.