A PERIPLASMIC INSULIN-CLEAVING PROTEINASE (ICP) FROM ACINETOBACTER-CALCOACETICUS SHARING PROPERTIES WITH PROTEASE-III FROM ESCHERICHIA-COLIAND IDE FROM EUKARYOTES

A periplasmic insulin-cleaving proteinase (ICP)(1)), purified to its e lectrophoretic homogeneity in the SDS-PAGE from the Gram-negative bact erium Acinetobacter calcoaceticus, was examined and compared in its pr operties with the protease III (protease Pi, pitrilysin, EC 3.4.99.44) of Escherichia coli and the insulin-destroying proteinase (IDE, insul inase, EC 3.4.99.45) from eucaryotes. The enzyme was proven to be a me talloprotease like protease III and IDE, as was shown by the inhibitor y effects exerted by EDTA and o-phenanthroline. Furthermore, dialysis against EDTA and o-phenanthroline led to a complete loss of activity, which could be restored by addition of Co2+, and, to a lesser extent, but at a lower metal ion concentration by Zn2+. Similar to protease II I and IDE, ICP prefers the cleavage of small polypeptides (insulin, in sulin B-chain, glucagon) to the cleavage of proteins (casein, human se rum albumin, globin) and was inactive against synthetic amino acid der ivates (esters, p-nitranilides, and furoylacroleyl substrates) of subt ilisin, thermolysin, trypsin, and chymotrypsin. The peptide-bond-speci ficity of the ICP in the cleavage of the oxidized insulin B-chain was investigated and the results were compared to the specificity of prote ase III of E. coli, IDE, protease-24,11, and thermolysin. Cleavage sit es in the oxidized insulin B-chain generated by ICP are Asn3-Gln4, His 10-Leu11, Ala14-Leu15, Leu17-Val18, Gly23-Phe24, Phe24-Phe25, and Phe2 5-Tyr26. Principally, ICP cleaves between hydrophobic amino acids and amides. The ICP shares one of the only two cleavage sites with the pro tease III and four sites with the IDE.