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

@article{citeulike:2678050, abstract = {A membrane proteinase from Pseudomonas aeruginosa, called insulin-cleaving membrane proteinase (ICMP), was located in the outer membrane leaflet of the cell envelope. The enzyme is expressed early in the logarithmic phase parallel to the bacterial growth during growth on peptide rich media. It is located with its active center facing to the outermost side of the cell, because its whole activity could be measured in intact cells. The very labile membrane proteinase was solubilized by non-ionic detergents (Nonidet P-40, Triton X-100) and purified in its amphiphilic form to apparent homogeneity in SDS-PAGE by copper chelate chromatography and two subsequent chromatographic 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 kDa, determined by mass spectrometry. ICMP was characterized to be a metalloprotease with pH-optimum in the neutral range. The ICMP readily hydrolyzed Glu13-Ala14 and Tyr16-Leu17 bonds in the insulin B-chain. Phe25-Tyr26 and His10-Leu11 were secondary cleavage sites suggesting a primary specificity of the enzyme for hydrophobic or aromatic residues at P'1-position. The ICMP differed from elastase, alkaline protease and LasA in its cleavage specificity, inhibition behavior and was immunologically diverse from elastase. The amino acid sequence of internal peptides showed no homologies with the known proteinases. This outer membrane proteinase was capable of specific cleavage of [alpha] and [beta] fibrinogen chains. Among the p-nitroanilide substrates tested, substrates of plasminogen activator, complement convertase and kallikrein with arginine residues in the P1-subsite were the substrates best accepted, but they were only cleaved at a very low rate.}, author = {Fricke, Beate and Parchmann, Olaf and Kruse, Katharina and R\"{u}cknagel, Peter and Schierhorn, Angelika and Menge, Sieglinde}, citeulike-article-id = {2678050}, citeulike-linkout-0 = {http://dx.doi.org/10.1016/S0925-4439(99)00040-X}, citeulike-linkout-1 = {http://www.sciencedirect.com/science/article/B6T1Y-3X64N7P-4/1/64866723e916348eddf687dfc9760ac6}, day = {30}, doi = {10.1016/S0925-4439(99)00040-X}, journal = {Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease}, keywords = {icmp, iron, protease, virulence}, month = {August}, number = {3}, pages = {236--250}, posted-at = {2008-04-16 14:59:27}, priority = {2}, title = {Characterization and purification of an outer membrane metalloproteinase from Pseudomonas aeruginosa with fibrinogenolytic activity}, url = {http://dx.doi.org/10.1016/S0925-4439(99)00040-X}, volume = {1454}, year = {1999} }

TY - JOUR ID - citeulike:2678050 L3 - citeulike-article-id:2678050 N2 - A membrane proteinase from Pseudomonas aeruginosa, called insulin-cleaving membrane proteinase (ICMP), was located in the outer membrane leaflet of the cell envelope. The enzyme is expressed early in the logarithmic phase parallel to the bacterial growth during growth on peptide rich media. It is located with its active center facing to the outermost side of the cell, because its whole activity could be measured in intact cells. The very labile membrane proteinase was solubilized by non-ionic detergents (Nonidet P-40, Triton X-100) and purified in its amphiphilic form to apparent homogeneity in SDS-PAGE by copper chelate chromatography and two subsequent chromatographic 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 kDa, determined by mass spectrometry. ICMP was characterized to be a metalloprotease with pH-optimum in the neutral range. The ICMP readily hydrolyzed Glu13-Ala14 and Tyr16-Leu17 bonds in the insulin B-chain. Phe25-Tyr26 and His10-Leu11 were secondary cleavage sites suggesting a primary specificity of the enzyme for hydrophobic or aromatic residues at P'1-position. The ICMP differed from elastase, alkaline protease and LasA in its cleavage specificity, inhibition behavior and was immunologically diverse from elastase. The amino acid sequence of internal peptides showed no homologies with the known proteinases. This outer membrane proteinase was capable of specific cleavage of [alpha] and [beta] fibrinogen chains. Among the p-nitroanilide substrates tested, substrates of plasminogen activator, complement convertase and kallikrein with arginine residues in the P1-subsite were the substrates best accepted, but they were only cleaved at a very low rate. IS - 3 JF - Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease EP - 250 TI - Characterization and purification of an outer membrane metalloproteinase from Pseudomonas aeruginosa with fibrinogenolytic activity VL - 1454 SP - 236 KW - icmp KW - iron KW - protease KW - virulence AU - Fricke, Beate AU - Parchmann, Olaf AU - Kruse, Katharina AU - Rücknagel, Peter AU - Schierhorn, Angelika AU - Menge, Sieglinde PY - 1999/08/30/ UR - http://dx.doi.org/10.1016/S0925-4439(99)00040-X ER -