INVESTIGATION OF 2 GLYCOSYLATED FORMS OF BILE-SALT-DEPENDENT LIPASE IN HUMAN PANCREATIC-JUICE

Pure human pancreatic bile-salt-dependent lipase, devoid of its oncofe tal glycoform [Mas, E., Abouakil, N., Roudani, S., Miralles, F., Guy-C rotte, O., Figarella, C., Escribano, M. J. & Lombardo, D. (1993) Bioch em. J. 289, 609-615], was analyzed on immobilized concanavalin A (ConA ). Two variants were separated: an unabsorbed ConA-unreactive fraction ; and an absorbed ConA-reactive fraction. Carbohydrate compositions of ConA-reactive and ConA-unreactive fractions were not significantly di fferent, and analysis of H-3-labelled oligosaccharides liberated from these fractions on the ConA-Sepharose column indicated that the fracti onation of the bile-salt-dependent lipase on this column depends upon oligosaccharide structures. The activity of the ConA-reactive fraction was however much lower, independent of the substrate (4-nitrophenyl h exanoate or cholesteryl esters), than that of the ConA-unreactive frac tion. Therefore, catalytic constants for the hydrolysis of 4-nitrophen yl hexanoate were determined; both fractions had quite similar K-m, wh ile the k(cat) for the ConA unreactive fraction was 3-4-fold higher th an that of the ConA-reactive fraction. ConA-reactive and ConA unreacti ve fractions were shown to have slightly different molecular masses an d different amino acid compositions. Cleavage patterns after cyanogen bromide treatment of the ConA-reactive and ConA-unreactive fractions s uggested that the ConA-reactive (high M(r) form) and ConA-unreactive ( low M(r) form) forms could be different isoforms of the bile-salt-depe ndent lipase secreted by the human pancreas.