CHARACTERIZATION OF AN ANTIBODY TO THE CROSS-REACTING DETERMINANT OF THE GLYCOSYL-PHOSPHATIDYLINOSITOL ANCHOR OF HUMAN MEMBRANE DIPEPTIDASE

A polyclonal antiserum raised to the phospholipase C-solubilized form of membrane dipeptidase (EC 3.4.13.11) purified from human kidney was found to cross-react with unrelated trypanosomal and porcine glycosyl-phosphatidylinositol anchored proteins. Those antibodies recognising the cross-reacting determinant (CRD) were isolated by chromatography on a column of immobilized phospholipase C-solubilized porcine aminopeptidase P (EC 3.4.11.9), and the epitopes involved in the recognition were then characterized by immunoclectrophoretic blot analysis and by a competitive ELISA. The phospholipase C-solubilized forms of human and porcine membrane dipeptidase, porcine aminopeptidase P and trypanosome variant surface glycoprotein were recognised by the anti-CRD antiserum, and this recognition was abolished by prior treatment of the proteins with either mild acid or nitrous acid. In contrast, the detergent-solubilized, membrane-forms of human and porcine membrane dipeptidase were not recognised. Of a range of components of the glycosyl-phosphatidylinositol anchor, only inositol 1,2-cyclic monophosphate and the insulin-mimetic disaccharide, glucosaminyl-1,6-inositol 1,2-cyclic monophosphate, inhibited in the micromolar range the binding of the anti-CRD antiserum to immobilized porcine aminopeptidase P. These results indicate that the major epitope recognised by this anti-CRD antiserum is the inositol 1,2-cyclic monophosphate formed on phospholipase C cleavage of the glycosyl-phosphatidylinositol anchor.