Immobilized colipase affinities for lipases B, A, C and their terminal peptide (336-449): the lipase recognition site lysine residues are located in the C-terminal region.

Zonal high-performance affinity chromatography has been used in order to study the interactions between pig isolipases A, B and C and the terminal peptide chain fragment 336-449 of the pig lipase on the one hand, and the homolog colipase bound to the inert LiChrosorb diol support on the other. A mathematical treatment led the to assessment of the dissociation constant of the lipase-colipase complex using isolipases or the terminal peptide as eluted acceptors and colipase as silica-bound ligand (Mahé, N., Léger, C.L., Linard, A. and Alessandri, J.-M. (1987) J. Chromatogr. 395, 511-521). A higher affinity of isolipase B as compared to isolipases A and C towards colipase was observed (KD, respectively, of 0.68, 11 and 12 microM) at pH 6.5. Under the same chromatographic conditions, the terminal peptide chain interacted with the bound colipase (KD 0.70 microM, close to that of isolipase B). The chromatographic behaviors of both native and chemically modified lipase and terminal peptide were very similar. In particular, guanidination of lysine residues of both peptide and isolipase B led to the loss of interactions with colipase. The same result was observed with the peptide preincubated in the presence of increasing amounts of free colipase. Accordingly, it is suggested that, firstly, a preferential association of isolipase B to colipase could take place and, secondly, the colipase recognition site of lipase could be located in the C-terminal region, the conformational structure of the terminal peptide not being affected by the enzymic cleavage and, therefore, being largely independent of the rest of the polypeptide molecule. On the other hand, a lower colipase affinity for isolipases A or C than for isolipase B or the C-terminal peptide could tentatively be attributed to a non-local (distant) disturbing effect of the negatively charged glycan chain, as sialic acid is present in both isoforms A and C. Finally, the present paper confirms and extends earlier studies on lipase-colipase interactions.