title: Fragments of human fibroblast collagenase. Purification and characterization.
abstract: On purification, human fibroblast collagenase breaks down into two major forms (Mr22,000 and Mr 27,000) and one minor form (Mr 25,000). The most likely mechanism is autolysis, although the presence of contaminating enzymes cannot be excluded. From N-terminal sequencing studies, the 22,000-Mr fragment contains the active site; differential binding to concanavalin A shows the 25,000-Mr fragment is a glycosylated form of the 22,000-Mr fragment. These low-Mr forms can be separated by Zn2+-chelate chromatography. An activity profile of this column, combined with data from substrate gels, indicates no activity against collagen in the 22,000-Mr and 25,000-Mr forms, but rather, activity casein and gelatin. The 27,000-Mr form has no activity. The 22,000/25,000-Mr form can act as an activator for collagenase in a similar way to that reported for stromelysin. The activity of the 22,000/25,000-Mr form is not inhibited by the tissue inhibitor of metalloproteinases (TIMP). The 27,000-Mr C-terminal part of the collagenase molecule therefore appears to be important in maintaining the substrate-specificity of the enzyme, and also plays a role in the binding of TIMP.
authors.affiliation: Rheumatology Research Unit, Addenbrooke's Hospital, Cambridge, U.K.
authors.forename: I M
authors.initials: IM
authors.lastname: Clark
authors.forename: T E
authors.initials: TE
authors.lastname: Cawston
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003208:Concanavalin A; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology; D006023:Glycoproteins / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D008666:Metalloendopeptidases; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D019714:Tissue Inhibitor of Metalloproteinases
chemical_list: D006023:Glycoproteins; D019714:Tissue Inhibitor of Metalloproteinases; D003208:Concanavalin A; D008666:Metalloendopeptidases; D003012:Microbial Collagenase