A periplasmic insulin‐cleaving proteinase (ICP) from Acinetobacter calcoaceticus sharing properties with protease III from Escherichia coli and IDE from eucaryotes

Abstract

A periplasmic insulin-cleaving proteinase (ICP), purified to its electrophoretic homogeneity in the SDS-PAGE from the Gram-negative bacterium Acinetobacter calcoaceticus, was examined and compared in its properties with the protease III (protease Pi, pitrilysin, EC 3.4.99.44) of Escherichia coli and the insulin-destroying proteinase (IDE, insulinase, EC 3.4.99.45) from eucaryotes. The enzyme was proven to be a metalloprotease like protease III and IDE, as was shown by the inhibitory 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 III and IDE, ICP prefers the cleavage of small polypeptides (insulin, insulin B-chain, glucagon) to the cleavage of proteins (casein, human serum albumin, globin) and was inactive against synthetic amino acid derivates (esters, p-nitranilides, and furoylacroleyl substrates) of subtilisin, thermolysin, trypsin, and chymotrypsin. The peptide-bond-specificity of the ICP in the cleavage of the oxidized insulin B-chain was investigated and the results were compared to the specificity of protease III of E. coli, IDE, protease-24,11, and thermolysin. Cleavage sites in the oxidized insulin B-chain generated by ICP are Asn3-Gln4, His10-Leu11, Ala14-Leu15, Leu17-Val18, Gly23-Phe24, Phe24-Phe25, and Phe25-Tyr26. Principally, ICP cleaves between hydrophobic amino acids and amides. The ICP shares one of the only two cleavage sites with the protease III and four sites with the IDE.

A Role for a Protease in Morphogenic Responses during Yeast Cell Fusion

“…It is interesting to note that N -arginine dibasic convertase has been proposed to be involved in spermatid morphogenesis ( 14 ). Proteases of this family usually process small peptides or clip the NH 2 terminus of larger proteins ( 10 , 24 , 44 ). It is possible that Axl1p is required to proteolitically process the NH 2 terminus of a larger protein involved in morphogenesis or cell fusion.…”

Exoproteome Analysis of the Seaweed Pathogen Nautella italica R11 Reveals Temperature-Dependent Regulation of RTX-Like Proteins

“…For example, a putative zinc-dependent M16 peptidase domain (pfam00675) protein (GenBank: EEB72304) (Supplementary Table S2) that was up-regulated two-fold at 24°C has homology to an uncharacterized family of (insulinase-like) metallopeptidases that are hypothesized to be involved in the degradation of small polypeptides (Fricke et al, 1995). …”

Pitrilysin

“…4 Glucagon + Hp [3,5,8,9] 5 (7-Methoxycoumarin-4-yl)acetyl-N le-Ala-Val-Lys-Tyr-Leu-Asn-Ser-( 2, 4-dinitrophenyl)Lys-Leu-Asp-O-Lys + H 2 0 [5,6] 6 Secretin + H 2 0 [6] 7 Tyrocalcitonin + Hp [6] 8 Angiotensinogen(1-14) + Hp [6] 9 More (the activity of the enzyme is confined to substrates smaller than proteins [5], larger proteins are accepted as substrates to a low extent [9], prefers the cleavage of small polypeptides to the cleavage of proteins, inactive against synthetic amino acid derivatives of subtilisin, thermolysin, trypsin and chymotrypsin [8]) [5,8,9] Product spectrum 1 Hydrolyzed insulin Inhibitor(s)…”

“…Chelating agents [2]; Zincov [5]; on [5,8]; Zn 2 + (1 mM inhibits, but reactivates after inhibition with chelating agents) [5,6]; C0 2+ (1 mM [5,6]) [5,6,8]; Antipain [8]; Chymostatin [8]; Mn 2 + (1 mM, weak, reactivates after inhibition with chelating agents) [5,6]; Ca 2 + (1 mM, weak, reactivates after inhibition with chelating agents) [5,6]; Tetraethylenepentamine [5]; 2,6-Pyridinedicarboxylic acid [5]; EOTA (C0 2+, and to a lesser extent but at lower metal ion concentration Zn 2 + restores activity [8]) [1,5,8]; 1, 10-Phenanthroline (C0 2+, and to a lesser extent but at lower metal ion concentration Zn 2 + restores activity [8]) [1,5,8]; Bacitracin (especially in the presence of zinc [5,6]) [2,5,6]; Oynorphin A(1-13) [5,6]; PCMB [8]; Phosphoramidon [8J; More (not: sulfhydryl-modifying agents [3], alpha-macroglobulin [5]) [3,5] Cofactor(s)/prosthetic group(s)/activating agents Metal compounds/salts Zinc (contains 0.6 mol [2], 1.02-1.14 mol (depending on mutant) [3] of zinc per mol of enzyme) [2,3]; Zn 2 + (most effective [5,…”

“…Enzyme Handbook © Springer·Verlag Berlin Heidelberg 1998Duplication, reproduction and storage in data banks are only allowed with the prior permission of the publishers E. coli[1,6); Acinetobacter calcoaceticus (similar enzyme)[8,9) Storage -20°C, 50 mM Tris-HCI, pH 7.5, 40% v/v glycerol(6)…”