IDENTIFICATION OF MEMBRANE DIPEPTIDASE AS A MAJOR GLYCOSYL-PHOSPHATIDYLINOSITOL-ANCHORED PROTEIN OF THE PANCREATIC ZYMOGEN GRANULE MEMBRANE, AND EVIDENCE FOR ITS RELEASE BY PHOSPHOLIPASE-A

Membrane dipeptidase (EC 3.4.13.19) enzyme activity that is inhibited by cilastatin has been detected in pancreatic zymogen granule membrane s of human, porcine and rat origin. Immunoelectrophoretic blot analysi s of human and porcine pancreatic zymogen granule membranes with polyc lonal antisera raised against the corresponding kidney membrane dipept idase revealed that the enzyme is a disulphide-linked homodimer of sub unit mass 61 kDa in the human and 45 kDa in the pig. Although membrane dipeptidase was, along with glycoprotein-2, one of the only two major components of carbonate high pH-washed membranes, no enzyme activity or immunoreactivity was detected in the zymogen granule contents, Dige stion with bacterial phosphatidylinositol-specific phospholipase C (PI -PLC), and subsequent recognition by antibodies specific for the cross reacting determinant, revealed that membrane dipeptidase in human and porcine pancreatic zymogen granule membranes is glycosyl-phosphatidyli nositol-anchored. Membrane dipeptidase was released from the pancreati c zymogen granule membranes by an endogenous hydrolase, and the releas ed form migrated as a disulphide-linked dimer on SDS/PAGE under non-re ducing conditions. Under reducing conditions it migrated with the same apparent molecular mass as the membrane-bound form, and was still a s ubstrate for bacterial PI-PLC. Treatment of kidney microvillar membran es with phospholipase A(2) resulted in the release of membrane dipepti dase in a form that demonstrated electrophoretic and cilastatin-Sephar ose binding properties identical to those of the endogenously released form of the enzyme from zymogen granule membranes. These results indi cate that the glycosyl-phosphatidylinositol anchor on the pancreatic m embrane dipeptidase is cleaved by an endogenous hydrolase, probably a phospholipase A, and that this cleavage may promote the release of the protein from the membrane.