Chymotrypsinogen · inositol phosphatide complexes and the transport of digestive enzyme across membranes

Abstract

Chymotrypsinogen A was almost quantitatively extracted from aqueous solution in the presence of inositol phosphatides at relatively low concentrations of both ligands. Calcium ion facilitated the interaction at concentrations of 10 −4–10 −5 M. A water-insoluble chymotrypsinogen · Ca 2+ · inositol phosphatide complex was formed with an apparent stochiometry of 3 mol phospholipid : 3 mol Ca 2+ : 1 mol protein. Small changes in the structure of the protein prevented complex formation; in particular, the almost identical α-chymotrypsin, did not form complexes under the conditions studied. On the other hand, an homologous, but structurally substantially different, secretory protein, trypsinogen, did form complexes. Water-insoluble complexes were not formed with albumin, carbonic anhydrase or lactic dehydrogenase under the same circumstances. Neither phosphatidylethanolamine nor phosphatidylcholine formed complexes with chymotrypsinogen. Phosphatidylserine formed complexes, but was less effective than inositol phosphatides. Complex formation and stability was dependent upon “critical” concentrations of both Ca 2+ and H +. Extraction of the protein from solution increased from neglible to complete when the calcium concentration of the medium was raised slightly from 1.0 · 10 −4 M to 1.5·10 −4 M. Conversely, dissociation was complete when H + concentration was decreased slightly from pH 6.5 to 7.0. The complex is apparently formed as the result of specific electrostatic interactions between the polar head group of the inositol phosphatide and the protein, with the nonpolar alipathic fatty acid chains of the phospholipid providing a hydrophobic microenvironment for the protein. It is proposed that such complexes could account for the movement of digestive enzyme through membranes.