title: Purification and characterization of a novel calcium-dependent serine proteinase secreted from malignant hamster embryo fibroblast Nil2C2.
abstract: A novel serine proteinase was purified from the conditioned medium of malignant hamster embryo fibroblasts, Nil2C2. The molecular weight of the purified enzyme was estimated to be 88,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions. The enzyme was split into two subunits (Mr 66,000 and 33,000) with a reducing agent. The enzyme hydrolyzed not only synthetic peptides which are susceptible to trypsin digestion but also extracellular matrix proteins such as type I and IV collagen, fibronectin and gelatin. For the digestion of these proteins, Ca2+ at millimolar concentrations was essential but Ca2+ or chelators did not affect the esterase activity for synthetic peptides. The proteolytic activity was inhibited by diisopropyl fluorophosphate (DFP) and also by phenylmethylsulfonyl fluoride. DFP was shown to bind to the 33 kDa subunit, indicating that the catalytic machinery of the enzyme is located in this subunit.
authors.affiliation: Division of Physiology and Pathology, National Institute of Radiological Sciences, Chiba, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Sakiyama
authors.forename: Y
authors.initials: Y
authors.lastname: Nishino
authors.forename: T
authors.initials: T
authors.lastname: Tanaka
authors.forename: T
authors.initials: T
authors.lastname: Tomosawa
authors.forename: H
authors.initials: H
authors.lastname: Kinoshita
authors.forename: K
authors.initials: K
authors.lastname: Nagata
authors.forename: K
authors.initials: K
authors.lastname: Chiba
authors.forename: S
authors.initials: S
authors.lastname: Sakiyama
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003094:Collagen / Q000378:metabolism; D006224:Cricetinae; D005347:Fibroblasts / Q000201:enzymology*; D005353:Fibronectins / Q000378:metabolism; D005780:Gelatin / Q000378:metabolism; D007531:Isoflurophate / Q000494:pharmacology; D008970:Molecular Weight; D009374:Neoplasms, Experimental / Q000201:enzymology*; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D005353:Fibronectins; D007531:Isoflurophate; D005780:Gelatin; D003094:Collagen; D012697:Serine Endopeptidases; D002118:Calcium