ARTICLE

A HISTOCHEMICAL ENZYME KINETIC SYSTEM APPLIED TO THE TRYPSIN-LIKE AMIDASE AND ESTERASE ACTIVITY IN HUMAN MAST CELLS

¹ From The Section on Histochemistry, Laboratory of Experimental Pathology, Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland

A method for the determination of enzyme kinetic constants V_m, K_m, and K_i in a histochemical system has been devised. As a substitute for the reciprocal of the reaction velocity, the times necessary to reach a fixed amount of end product (the initial visible color) in a tissue site at various substrate concentrations are plotted, according to the method of Lineweaver and Burk, against the reciprocal of the substrate concentrations. The technique as applied to trypsin-like esterase and amidase activities in human mast cells indicates that a single enzyme or closely related enzymes in this site are responsible for the hydrolysis of both the amide and ester substrates and that typical trypsin substrates act as competitive inhibitors of their hydrolysis. Parallel biochemical studies were performed to evaluate the effect of certain aspects of the experimental histochemical method on a purified homospecific enzyme. The relative kinetic constants derived by the histochemical method afford a further means of characterizing enzymic activity in a histochemical system.

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• Hanks, C.T., Chakrabarti, S.G. (1975). Biochemical and Morphological Studies of Rat Submandibular Gland: I. Centrifugal Fractionation of Granule-Rich Fraction. JDR 54: 938-947 [Abstract]  

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