Purification and characterization of a novel calcium-dependent serine proteinase secreted from malignant hamster embryo fibroblast Nil2C2

• Published in: Biochimica et biophysica acta Vol. 990; no. 2; pp. 156 - 161
• Main Authors: Sakiyama, Hisako, Nishino, Yoko, Tanaka, Tsutomu, Tomosawa, Toshikazu, Kinoshita, Hirohisa, Nagata, Koichi, Chiba, Keiko, Sakiyama, Shigeru
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 24.02.1989; Elsevier; North-Holland
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Calcium; Calcium - metabolism; Cell Line; Collagen; Collagen - metabolism; Cricetinae; Enzymes and enzyme inhibitors; Extracellular matrix; Fibroblasts - enzymology; Fibronectin; Fibronectins - metabolism; Fundamental and applied biological sciences. Psychology; Gelatin - metabolism; Hydrolases; Isoflurophate - pharmacology; Malignant hamster embryo fibroblast; Molecular Weight; Neoplasms, Experimental - enzymology; Nil2C2; Serine Endopeptidases - isolation & purification; Serine Endopeptidases - metabolism; Serine proteinase; Nil2C2; Calcium; AGLNE; MCA; Fibronectin; DFP; SDS-PAGE; PMSF; Collagen; Malignant hamster embryo fibroblast; Boc; Extracellular matrix; Serine proteinase; Cell culture; Purification; Gel electrophoresis; Enzyme; Proteinase; Rodentia; Subunit; Structure function relationship; Hydrolysis; Vertebrata; Mammalia; Fibroblast; Hamster
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/S0304-4165(89)80028-5

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 ( M r 66000 and 33000) 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, Ca 2+ at millimolar concentrations was essential but Ca 2+ 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.