Production and purification of prostromelysin and procollagenase from IL-1 beta-stimulated human gingival fibroblasts.
Conditions were established to stimulate human gingival fibroblast explant cultures to synthesize milligram quantities of the metalloproteinase proenzymes, prostromelysin and procollagenase. To stimulate enzyme production, cells were treated with 1 nM recombinant human IL-1 beta for approximately 7 days under serum free conditions. Using a combination of rapid column chromatography steps, approximately 10 milligrams of prostromelysin and 5 milligrams of procollagenase were purified from 1 liter of conditioned media. Prostromelysin electrophoresed as a doublet with molecular weights of 55,57 kD, whereas, procollagenase migrated with slightly lower molecular weights of 52, 54 kD. Both proenzymes were treated with trypsin or aminophenylmercuric acetate to generate active species. The molecular weights of the active enzymes were approximately 10 kD smaller than the proenzymes. Active enzymes were inhibited by metal chelators and the natural metalloproteinase inhibitor, tissue inhibitor of metalloproteinase (TIMP), but not by the serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF). Activated stromelysin degraded a number of substrates including transferrin, proteoglycan monomer, proteoglycan aggregated with hyaluronic acid, and substance P. By contrast, collagenase degraded interstitial type I collagen and the peptide thioester, Ac-Pro-Leu-Gly-SCH(iBu)Co-Leu-GlyOEt. Identity of both enzymes were confirmed by amino-terminal protein sequence analysis as well as by immunoblot analysis using monoclonal antibodies.
Lark
M W
MW
Department of Biochemical and Molecular Pathology, Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey 07065.
Walakovits
L A
LA
Shah
T K
TK
Vanmiddlesworth
J
J
Cameron
P M
PM
Lin
T Y
TY
D000595:Amino Acid Sequence; D002460:Cell Line; D002845:Chromatography; D017364:Collagenases; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005347:Fibroblasts / Q000201:enzymology*; D005881:Gingiva / Q000201:enzymology*; D006801:Humans; D015151:Immunoblotting; D007375:Interleukin-1 / Q000494:pharmacology*; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D003012:Microbial Collagenase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D014162:Transfection
D004792:Enzyme Precursors; D007375:Interleukin-1; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004247:DNA; D017364:Collagenases; D008666:Metalloendopeptidases; C022440:procollagenase; C048047:prostromelysin; D003012:Microbial Collagenase