Biosynthesis and secretion of procollagenase by rabbit synovial fibroblasts. Inhibition of procollagenase secretion by monensin and evidence for glycosylation of procollagenase.
Monolayer cultures of rabbit synovial fibroblasts stimulated with phorbol myristate acetate to produce large amounts of collagenase (EC 3.4.24.7) were used to study the biosynthesis and secretion of this enzyme. [3H]Leucine was added to cell cultures for pulse-chase and continuous-labelling experiments. The labelled procollagenase synthesized was identified by immunoprecipitation followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. The amounts of intracellular and extracellular proenzyme were quantified by measuring radioactivity incorporated into the proteins. procollagenase was synthesized as doublet proteins of Mr 57 000 and Mr 61 000. Immunoprecipitable proenzyme proteins were first detected in culture medium 35 min after [3H]leucine was added to the cells. Monensin treatment of the cells inhibited procollagenase secretion and led to intracellular accumulation of the proenzyme. Cells treated with tunicamycin produced only the 57 000-Mr form, indicating that in rabbit synovial cells the 61 000-Mr form was post-translationally modified by addition of oligosaccharides to asparagine residues. The ratios of glycosylated to unglycosylated forms in cell lysates and in culture medium were 0.22:1 and 0.07:1 respectively.
D000818:Animals; D002478:Cells, Cultured; D017364:Collagenases; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D005663:Furans / Q000494:pharmacology*; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D008985:Monensin / Q000494:pharmacology*; D011817:Rabbits; D013583:Synovial Membrane / Q000187:drug effects / Q000201:enzymology*; D014415:Tunicamycin / Q000494:pharmacology
D004792:Enzyme Precursors; D005663:Furans; D014415:Tunicamycin; D008985:Monensin; D017364:Collagenases; C022440:procollagenase; D003012:Microbial Collagenase