PANCREATIC COLIPASE:STRUCTURAL AND IMMUNOLOGICAL STUDIES

Abstract

Lipases hydrolyze triacylglycerols in the form of micellar aggregates or emulsions. Adsorption of the enzyme to lipid particles is a key step in interfacial catalysis. Pancreatic lipase is inhibited by bile salt and phospholipid which prevent enzyme binding to interface.Inhibition is specifically reversed by colipase,a protein of 10.5 KD found with lipase in the pancreatic secretion.Human,porcine and equine colipases have been sequenced.The amino acid sequence of the human and rat proteins has been deduced from the nucleotide sequence of cloned cDNA.Comparison of the primary structures reveals extensive homology.Colipase is secreted aS a precursor form (procolipase) and activated by tryptic cleavage at one single bond (Arg,-Gly,). Limited proteolysis increases the lipid binding capacity of colipase.Kinetic studies of the activation of bile salt inhibited pancreatic lipase by colipase have provided evidence that the cofactor forms a stoechiometric active complex with enzyme at interface.Results of binding studies with model substrates are consistent with the view that colipase binds to interface coated with bile salt and further anchors lipase to its substrate.It has been postulated that colipase possesses two specific surface domains for binding to lipid and lipase.Attempts to identify the lipid and lipase binding sites were made using a physico-chemical approach.Spectroscopic studies have shown that the region containing the three tyrosine residues is involved in the binding to lipid-water interface.Chemical modification of the free carboxyl groups of residues Glu), and/or Asp, which has no effect on lipid binding prevents interaction with lipase.Polyclonal and monoclonal (MAb) antibodies have been raised against porcine colipase and used for the characterization of the binding sites in spatial relationship with antigenic regions.Results of studies carried out with eight MAb and fractions of polyclonal antibodies separated on immobilized synthetic peptides bring evidence that the tyrosine containing region and the N-terminal fragment are involved in lipid binding and that Glu, is implicated in interaction with lipase.