A precursor form of latent collagenase produced in a cell-free system with mRNA from rabbit synovial cells

• Published in: The Journal of biological chemistry 1981-12, Vol.256 (23), p.11951-11954
• Main Authors: H Nagase, R C Jackson, C E Brinckerhoff, C A Vater, E D Harris, Jr
• Format: Article
• Language: English
• Subjects: Animals; Cells, Cultured; Enzyme Precursors - genetics; Fibroblasts - enzymology; Microbial Collagenase - genetics; Microbial Collagenase - isolation & purification; Molecular Weight; Protein Biosynthesis; Rabbits; RNA, Messenger - genetics; Synovial Fluid - enzymology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)43210-3

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.