Abstract: | A secretory granule-associated enzymatic activity that converts mono-125I]-D-Tyr-Val-Gly into mono-125I]-D-Tyr-Val-NH2 has been studied. The activity is primarily soluble and shows optimal activity at pH 7 to pH 8. Amidation activity was stimulated 9-fold by addition of optimal amounts of copper (3 μM). In the presence of optimal copper, ascorbate stimulated the reaction 7-fold; none of the other reduced or oxidized cofactors tested was as effective. Taking into account the dependence of the reaction on ascorbate and molecular oxygen and the production of glyoxylate 2], it is suggested that the α-amidation enzyme is a monooxygenase. Lineweaver Burk plots with D-Tyr-Val-Gly as the varied substrate demonstrated Michelis-Menten type kinetics with the values of Km and Vmax increasing with the addition of ascorbate to the assay. A variety of peptides ending with a COOH-terminal Gly residue act as inhibitors of the reaction. Two synthetic peptides, γ2MSH and ACTH(1–14), with carboxyl termini similar to the presumed physiological substrates for the enzyme, act as competitive inhibitors with similar K1 values. It is likely that this secretory granule α-amidation activity is involved in the physiological biosynthetic α-amidation of a wide range of bioactive peptides. |