Characterization and purification of an outer membrane metalloproteinase from Pseudomonas aeruginosa with fibrinogenolytic activity

A membrane proteinase from Pseudomonas aeruginosa, called insulin-cleaving membrane proteinase (ICMP), was located in the outer membrane leaflet of th e cell envelope. The enzyme is expressed early in the logarithmic phase par allel to the bacterial growth during growth on peptide rich media. It is lo cated with its active center facing to the outermost side of the cell, beca use its whole activity could be measured in intact cells. The very labile m embrane proteinase was solubilized by non-ionic detergents (Nonidet P-40, T riton X-100) and purified in its amphiphilic form to apparent homogeneity i n SDS-PAGE by copper chelate chromatography and two subsequent chromatograp hic steps on Red-Sepharose CL-4B (yield 58.3%, purification factor 776.3). It consisted of a single polypeptide chain with a molecular mass of 44.6 kD a, determined by mass spectrometry. ICMP was characterized to be a metallop rotease with pH-optimum in the neutral range. The ICMP readily hydrolyzed G lu(13)-Ala(14) and Tyr(16)-Leu(17) bonds in the insulin B-chain. Phe(25)-Ty r(26) and His(10)-Leu(11) were secondary cleavage sites suggesting a primar y specificity of the enzyme for hydrophobic or aromatic residues at P-1'-po sition. The ICMP differed from elastase, alkaline protease and LasA in its cleavage specificity, inhibition behavior and was immunologically diverse f rom elastase. The amino acid sequence of internal peptides showed no homolo gies with the known proteinases. This outer membrane proteinase was capable of specific cleavage of alpha and beta fibrinogen chains. Among the p-nitr oanilide substrates tested, substrates of plasminogen activator, complement convertase and kallikrein with arginine residues in the P-1-subsite were t he substrates best accepted, but they were only cleaved at a very low rate. (C) 1999 Elsevier Science B.V. All rights reserved.