Purification and partial characterization of a collagenolytic enzyme from Pseudomonas aeruginosa.
A proteinase from Pseudomonas aeruginosa exhibiting collagenolytic activity was purified 1575-fold with a recovery of 24% by use of chemical and chromatographic technics. The enzyme preparation appeared to be homogeneous when subjected to chromatographic, electrophoretic and ultracentrifugational analyses. A standard state sedimentation coefficient of 2.10 S was calculated and further analyses indicated that the enzyme had a molecular weight of 17 500 and dimerizes under certain conditions to yield an apparent molecular weight of 34 000. In addition to insoluble collagen, the enzyme catalyzed the hydrolysis of congocoll, azocoll, soluble collagen and casein, but did not attack orcein-elastin, azoalbumin, p-toluene eulfonyl-L-arginine methyl ester, benzoyl-L-tyrosine ethyl ester, and the hexapeptide N-benzyloxycarbonyl-glycyl-L-prolyglycylglycyl-L-prolyl-L-alanine. Enzymatic activity against congocoll was 6-fold greater at pH 7.5 in Tris with HCl than in phosphate buffer at the same ionic strength. Cobalt, and to a lesser extent, Zn2+ appeared to activate the enzyme, especially in phosphate buffer. NcCN and p-chloromercuribenzoate did not appreciably inhibit enzyme activity, while (NH4)2 SO4, EDTA and cysteine displayed a significant inhibitory effect under certain conditions.
D000596:Amino Acids / Q000032:analysis; D002413:Cations, Divalent; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003545:Cysteine / Q000494:pharmacology; D004355:Drug Stability; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D003012:Microbial Collagenase / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013997:Time Factors
D000596:Amino Acids; D002413:Cations, Divalent; D002729:Chloromercuribenzoates; D000431:Ethanol; D004492:Edetic Acid; D003012:Microbial Collagenase; D003545:Cysteine