Kinetics of Peptide Hydrolase Activity in Crude Wheat Leaf Extracts

Abstract

The protein present in crude wheat leaf extracts causes serious deviations from simple Michaelis-Menten kinetics for soluble peptide hydrolases. A theoretical analysis indicated that the usual approach, of adjusting activities observed at various substrate concentrations for the activity observed in the absence of added substrate, gave erroneous estimates of kinetic parameters. The size of the error depends on the concentration of endogenous protein and the Km for its degradation. A technique is suggested to allow an estimate to be made of the true values of the kinetic parameters. The method also allows estimation of the Km for the degradation of the endogenous protein. The Km for haemoglobin degradation by crude extracts of wheat primary leaves at pH 4.5 was 0.802 mg ml-1 and at pH 6.5 was 3.35 mg ml-1. The Km for endogenous protein degradation at pH 4.5 was 2.24 mg ml-1 and at pH 6.5 was 1.49 mg ml-1. The implications of these findings for the possible role of soluble peptide hydrolases in intracellular protein degradation is discussed.