Open AccessA Method for the Estimation of Esterase Synthesis and Degradation and its Application to Evaluate the Influence of Insulin and Glucagon
Author(s) -
HEYMANN Eberhard,
MENTLEIN Rolf,
SCHMALZ Rainer,
SCHWABE Christian,
WAGENMANN Frank
Publication year - 1979
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1979.tb04267.x
Subject(s) - esterase , chemistry , chromatography , glucagon , isoelectric focusing , microsome , degradation (telecommunications) , biochemistry , isoelectric point , leucine , enzyme , hormone , amino acid , telecommunications , computer science
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 was found to contain 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 were shown to remain in equilibrium for a period of at least five days following the injection of 10 mg bis(4‐nitro‐[ 14 C]phenyl)phosphate per kg. The decrease of the relative amount of labeled esterases with time was found to follow first‐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 one of the standard procedures for the estimation of degradation rates: [ 14 C]leucine was incorporated and one of the esterases was subsequently isolated by immunoprecipitation. Using isoelectric focussing 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.