Chinese hamster ovary cells produce an enzyme that nicks heat-labile enterotoxin from enterotoxigenic Escherichia coli.
Chinese hamster ovary (CHO) cells were examined for production of an enzyme that nicked the polypeptide chain of the heat-labile enterotoxin from enterotoxigenic Escherichia coli between the A1 and A2 fragments of its A subunit. Serum-free culture medium prepared each day after CHO cell inoculation was concentrated 100 times and its proteolytic activity for formation of the A1 fragment was examined by Western blotting with anti-LT A antibody. The A subunit was detected in culture medium on day 6 after cell inoculation, although not in media on day 1 or 3, indicating that CHO cells produced a nicking enzyme. This nicking enzyme had an optimal pH of about 7.5 and an apparent Mr. of 120,000, as seen by Superose 12 TM gel filtration with an FPLC system. The activity of this enzyme was strongly inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, but not by p-chloromercuribenzoic acid, EDTA or ethyleneglycol bis (beta-aminoethylether)-N,N-N',N'-tetraacetic acid, suggesting that this enzyme was a serine protease. The activity was not stimulated by plasminogen or fibrin. These findings suggest that the nicking enzyme was different from proteases such as elastase, collagenase and plasminogen activator, which are probably also secreted by fibroblast-like CHO cells.
Tsuji
T
T
Department of Microbiology, School of Medicine, Fujita Health University, Aichi, Japan.
Miyama
A
A
D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D015153:Blotting, Western; D016466:CHO Cells / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D004768:Enterotoxins / Q000378:metabolism*; D004791:Enzyme Inhibitors; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*
D001427:Bacterial Toxins; D016895:Culture Media, Serum-Free; D004768:Enterotoxins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; C027538:heat-labile enterotoxin, E coli; D012697:Serine Endopeptidases