ISOLATION AND CHARACTERIZATION OF A DIBASIC SELECTIVE METALLOENDOPEPTIDASE FROM RAT TESTES THAT CLEAVES AT THE AMINO-TERMINUS OF ARGININE RESIDUES

A metalloendopeptidase that selectively cleaves doublets of basic amin o acids on the amino-terminal side of arginine residues was purified t o homogeneity from rat testes and analyzed further. Two catalytically active forms with apparent relative molecular masses of 110,000 and 14 0,000 Da, respectively, were present in the purified preparation of th e enzyme. Antibodies raised against the purified testis endopeptidase revealed by immunoblot both the 110- and 140-kDa forms in both rat tes tis and brain cortex extracts. The isolated enzyme was inhibited by me tal chelators and divalent cations. Its activity, lost after preincuba tion with EDTA, was restored by low concentrations of Zn2+ and Mn2+, t hus demonstrating the metallopeptidase nature of the enzyme. This endo peptidase also exhibited a high sensitivity to amastatin (100% inhibit ion at 20 muM), an aminopeptidase inhibitor. A substrate specificity s tudy using physiologically important or synthetic peptides containing a processing dibasic site indicated that cleavage occurred selectively at the amino-terminal side of an arginine residue, independent of the nature of the basic doublet. The enzyme produced such a cleavage at t he Arg-Lys doublet of somatostatin 28 (K(m) = 43 muM), at the Arg-Arg doublet of dynorphin A (K(m) = 6.45 muM) and atrial natriuretic factor (K(m) = 6.25 muM), and at the Lys-Arg doublet of preproneurotensin-(1 54-170) (K(m) = 17.3 muM). Moreover, cleavage efficiency was found to be higher for the larger substrates. The distinctive properties of thi s endopeptidase imply that this protein is a member of a novel class o f proteolytic enzymes that may be involved in the endoproteolytic matu ration of hormonal precursors.