PURIFICATION OF CIRCULATING LIVER PLASMA-MEMBRANE FRAGMENTS USING A MONOCLONAL ANTILEUCINE AMINOPEPTIDASE ANTIBODY

Membrane-bound liver alkaline phosphatase (Mem-LiALP, EC 3.1.3.1.) is a high-molecular-mass Liver alkaline phosphatase (ALP) present in meta static, infiltrative and cholestatic liver disease. Shedding of hepato cyte plasma membrane fragments (LiPMF) is thought to be responsible fo r the appearance of Mem-LiALP in the circulation. Several other membra ne-bound enzymes, such as gamma-glutamyltransferase (gamma-GT), leucin e aminopeptidase (LAP), and 5'-nucleotidase (5'-Nu) are present in the membrane of the shedded LiPMF. By means of immunohistochemical and im munoassay procedures, we presently show that AD-1, a specific monoclon al antibody originally produced against MemliALP, reacts with LAP, a c onstituent of the human liver plasma membrane. Using AD-1 as an immuno sorbant, we isolated circulating LiPMF from cholestatic sera to a high level of purity and separated it from other high-molecular-mass mater ial, such as liver ALP similar to Lipoprotein-X complexes. These purif ied membrane fragments retained their biochemical characteristics. Gly cosyl-phosphatidylinositol anchor bearing liver ALP (Anch-LiALP) could be released from the LiPMF by Triton X-100. Whereas ALP was released upon treatment of AD-1 purified LiPMF with phospholipase C, phospholip ase D only cleaved the glycosyl-phosphatidylinositol anchor following detergent solubilization of the enzyme. Serum LiPMF from patients with different kinds of cholestatic liver disease were bound onto AD-1 coa ted nitrocellulose disks and the activity of four membrane-bound enzym es (LAP, ALP, 5'Nu, gamma-GT) was analyzed. A considerable interindivi dual variation of enzyme activities was observed, suggesting some hete rogeneity in the membrane composition of these fragments.