Resolution of TIMP-free and TIMP-complexed 70kDa progelatinase from culture medium of Rous sarcoma virus-transformed chicken embryo fibroblasts.
Chicken embryo fibroblasts (CEF) produce a 70kDa progelatinase, a member of the matrix metalloproteinase family, and secrete elevated levels of the enzyme upon transformation by Rous sarcoma virus (RSV). This enzyme can be purified by affinity chromatography complexed with a 21kDa tissue inhibitor of metalloproteinases (TIMP)-like molecule. Gel-filtration of the purified progelatinase suggests the presence of a mixed population of enzyme: a TIMP-complexed and a TIMP-free progelatinase. These two species were separated by Mono Q FPLC in the absence of denaturants. Quantitation of the purified progelatinase reveals that the transformed RSVCEF produce more TIMP-free enzyme than the normal CEF. Native PAGE analysis indicates that purified TIMP-free progelatinase is capable of binding to TIMP and generating a TIMP-complexed progelatinase. Treatment of the TIMP-free gelatinase with organomercurials results in a rapid conversion to the active 62kDa species and indicates that the TIMP-free progelatinase is more susceptible to activation than the TIMP-complexed progelatinase.
Department of Biochemistry, State University of New York, Stony Brook 11794.
R T
RT
Aimes
S M
SM
Nielsen-Preiss
J P
JP
Quigley
D000818:Animals; D001358:Avian Sarcoma Viruses; D002461:Cell Line, Transformed; D002642:Chick Embryo; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000302:isolation & purification*; D005109:Extracellular Matrix / Q000737:chemistry; D005347:Fibroblasts / Q000378:metabolism; D005780:Gelatin / Q000032:analysis; D018093:Gelatinases; D006023:Glycoproteins / Q000302:isolation & purification*; D008666:Metalloendopeptidases / Q000302:isolation & purification*; D019714:Tissue Inhibitor of Metalloproteinases
D003470:Culture Media; D004792:Enzyme Precursors; D006023:Glycoproteins; D019714:Tissue Inhibitor of Metalloproteinases; D005780:Gelatin; D018093:Gelatinases; D008666:Metalloendopeptidases; C069860:progelatinase