Characterization and partial purification of an insulinase from Neurospora crassa

Abstract

An insulin-binding metal- and thiol-dependent proteinase has been purified 1491-fold from high speed cytosolic fractions of the fungus Neurospora crassa. This enzyme resembles insulin-degrading enzymes (insulinases) present in mammalian cells and in Drosophila melanogaster in the following ways: (i) it degrades radiolabeled insulin with a specificity similar to that of rat muscle insulinase, as demonstrated by HPLC analysis of the degradation products; (ii) it is inhibited by bacitracin, EDTA, 1,10-phenanthroline, and the sulfhydryl-reactive compounds N-ethylmaleimide and p-chloromercuribenzoate, but not by inhibitors of serine proteases or by lysosomal protease inhibitors. Cross-linking with 125I-insulin labels a band of ca. 120 kDa, and several smaller bands which may represent degradation products. The N. crassa insulinase is stimulated by Mn 2+ and strongly inhibited by Zn 2+; Mn 2+ can also reactivate the enzyme after inhibition by EDTA, but Zn 2+ is ineffective. The N. crassa protein differs in this regard from mammalian and insect insulinases which are generally activated by both Mn 2+ and Zn 2+. This finding extends the apparent evolutionary conservation of these metal- and thiol-dependent proteases into the microbial realm.