A Method for the Estimation of Esterase Synthesis and Degradation and its Application to Evaluate the Influence of Insulin and Glucagon

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Abstract
The irreversible reaction between liver esterases and the active-site-directed inhibitor bis(4-nitrophenyl)phosphate can be used in vivo both for the estimation of the esterase contents and for the measurement of the esterase degradation rates. A method based on this reaction is described which allows the simultaneous estimation of the rate constants of degradation and synthesis of esterases during a period of change in protein concentration. Rat liver contained about 1 mg of organophosphate-binding esterases per g of fresh tissue while the microsomal fraction contains about 30 mg of esterases per g of microsomal protein. Esterase degradation and de novo synthesis remained in equilibrium for a period of at least 5 days following the injection of 10 mg bis(4-nitro[14C]phenyl)phosphate per kg. The decrease of the relative amount of labeled esterases with time followed 1st-order kinetics yielding an average esterase degrading constant of 0.0165 h-1 which corresponds to a half-life of 42 h. These data were confirmed by an independent experiment using 1 of the standard procedures for the estimation of degradation rates; [14C]Leu was incorporated and 1 of the esterases was subsequently isolated by immuno-precipitation. Using isoelectric focusing and dodecyl sulfate electrophoretic methods, the various esterase isoenzymes appeared to have very similar, if not identical turnover rates. This method for the estimation of the turnover characteristics was applied to evaluate hormone effects on liver esterases. The time course of the contents and the turnover of liver esterases was measured under the influence of glucagon treatment in diabetic rats and under the influence of high doses of insulin. The esterase content decreased faster than the average content of microsomal protein under the influence of glucagon. The reverse effect was observed with insulin-treated rats. Both insulin and glucagon apparently reduced the intracellular esterase turnover in rat liver. Kinetic analysis of the results revealed that insulin mainly lowered the esterase degradation rate, though the rate of esterase synthesis might also have been restricted. In the glucagon-treated rats the de novo synthesis of esterases was strongly reduced.