title: Purification and properties of rat uterine procollagenase.
abstract: A procollagenase from monolayer cultures of postpartum rat uterine cells has been purified. The crucial step in the purification is the binding of the procollagenase from crude, fetal bovine serum-containing culture medium to heparin-Sepharose, followed by elution with extremely low concentrations (5-10 nM) of dextran sulfate. Resultant eluates contain 8-10% procollagenase. Purification is completed by ion-exchange chromatography on DEAE-Sepharose, gel filtration on AcA-44, and chromatography on blue-Sepharose. Rat uterine procollagenase appears as a protein doublet of Mr approximately 58,000, as indicated by two polyacrylamide gel electrophoresis systems, by AcA-44 chromatography, and by equilibrium sedimentation ultracentrifugal analysis. The proenzyme forms are converted by trypsin to an active enzyme doublet of Mr approximately 48,000. Small amounts of active enzyme, which are often generated during the purification, are electrophoretically indistinguishable from trypsin-activated collagenase. Active collagenase can be separated from the zymogen forms by DEAE-Sepharose chromatography. The two forms of the proenzyme doublet can be partially separated by gel filtration on AcA-44 and preliminary analysis indicates each has equal collagenolytic activity. The amino acid analysis of rat uterine collagenase reveals it to be markedly different from two other vertebrate collagenases whose composition is known. The uterine proenzyme is unusually rich in glycine and in the hydroxy amino acids and is considerably more acidic than the human skin fibroblast collagenase, consistent with the different ion-exchange behavior of the two molecules. The specific activity of rat uterine collagenase at 37 degrees C is approximately 3000 micrograms collagen/min/mg, using native reconstituted guinea pig skin type I collagen fibrils as substrate. The enzyme cleaves denatured collagen, but fails to attack a variety of noncollagen proteins.
mesh_terms: D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography / Q000379:methods; D017364:Collagenases; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000302:isolation & purification*; D005260:Female; D003012:Microbial Collagenase / Q000302:isolation & purification*; D008970:Molecular Weight; D051381:Rats; D014599:Uterus / Q000201:enzymology*
chemical_list: D004792:Enzyme Precursors; D017364:Collagenases; C022440:procollagenase; D003012:Microbial Collagenase