title: A precursor form of latent collagenase produced in a cell-free system with mRNA from rabbit synovial cells.
abstract: mRNA extracted from rabbit synovial fibroblasts which had been induced to produce large amounts of collagenase (EC 3.4.23.7) by urate crystals was translated in a cell-free wheat germ system. Collagenase was identified by immunoprecipitation using mono-specific antibody to rabbit synovial collagenase. In the absence of microsomal membranes, a single precursor with Mr = 59,000 was synthesized. This polypeptide was susceptible to proteolytic degradation. In the presence of canine pancreatic microsomes, the nascent protein was processed to a polypeptide with Mr = 57,000 (identical in mobility on sodium dodecyl sulfate-gel electrophoresis to the major latent collagenase secreted from cells) and was protected from tryptic digestion unless a detergent was used to disrupt the membranes. In addition to Mr = 57,000 material, cells secreted immunologically reactive latent collagenase with Mr = 61,000. High molecular weight collagenase was separated from Mr = 57,000 species by binding to concanavalin a-Sepharose, suggesting that this enzyme was a product of post-translational glycosylation. Both latent enzymes were activated by trypsin and human plasma kallikrein to Mr = 45,000 and 49,000. The evidence indicates that rabbit synovial fibroblast collagenase is synthesized and secreted as a single polypeptide zymogen, not as an enzyme-inhibitor complex.
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004792:Enzyme Precursors / Q000235:genetics*; D005347:Fibroblasts / Q000201:enzymology; D003012:Microbial Collagenase / Q000235:genetics* / Q000302:isolation & purification; D008970:Molecular Weight; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D013582:Synovial Fluid / Q000201:enzymology*
chemical_list: D004792:Enzyme Precursors; D012333:RNA, Messenger; D003012:Microbial Collagenase