Chinese hamster ovary cells produce an enzyme that Nicks heat-labile enterotoxin from enterotoxigenic Escherichia coli

Abstract

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 AI and A1 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 AI 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 phenxlmethylsulfonyl fluoride, but not by p-chloromercuribenzoic acid, EDT A or ethyleneglycol bis (β - aminoethylether)-N,N-N′, N’t-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. © 1992, Kluwer Academic Publishers. All rights reserved.