Proteinases in human polymorphonuclear leukocytes. Purification and characterization of an enzyme which cleaves denatured collagen and a synthetic peptide with a Gly-Ile sequence.
Polymorphonuclear leukocytes have been shown to contain proteolytic enzymes which are capable of degrading connective tissue proteins such as native collagen. In this study, proteolytic enzymes were extracted from human polymorphonuclear leukocytes and a neutral proteinase was extensively purified and characterized. The activity of this enzyme was monitored by degradation of denatured [ 3H ]proline-labeled type I collagen or by cleavage of a synthetic dinitrophenylated peptide with a Gly-Ile sequence. The enzyme was readily separated from leukocyte collagenase by concanavalin-A--Sepharose affinity chromatography and further purified by QAE-Sephadex ion-exchange chromatography and gel filtration on Sephacryl S-200. The purified enzyme had a molecular weight of approximately 105000, its pH optimum was about 7.8, and it was inhibited by Na2EDTA and dithiothreitol, but not by fetal calf serum. The enzyme degraded genetically distinct type I, II, III, IV and V collagens, when in a non-helical form, but not when in native triple-helical conformation. Dansyl-monitored end-group analyses, combined with digestion by carboxypeptidase A, indicated that the enzyme cleaved denaturated type I collagen at Gly-Xaa sequences, in which Xaa can be leucine, isoleucine, valine, phenylalanine, lysine, or methionine. Thus, the purified enzyme referred to here as Gly-Xaa proteinase, is a neutral proteinase, which may be of importance in inflammatory disease processes by degrading further collagen peptides which have been rendered non-helical as a result of collagenase cleavage.
D000595:Amino Acid Sequence; D002384:Catalysis; D003094:Collagen / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000097:blood / Q000145:classification / Q000302:isolation & purification*; D006801:Humans; D006868:Hydrolysis; D009504:Neutrophils / Q000201:enzymology*; D010455:Peptides / Q000378:metabolism*; D011489:Protein Denaturation; D013379:Substrate Specificity
D010455:Peptides; D003094:Collagen; D010450:Endopeptidases