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Purification of circulating liver plasma membrane fragments using a monoclonal antileucine aminopeptidase antibody
Author(s) -
Deng J T,
Hoylaerts M F,
Nouwen E J,
De Broe M E,
Van Hoof V O
Publication year - 1996
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1002/hep.510230308
Subject(s) - alkaline phosphatase , aminopeptidase , monoclonal antibody , biochemistry , chemistry , microbiology and biotechnology , 5' nucleotidase , enzyme , hepatocyte , phosphatidylinositol , nucleotidase , antibody , biology , leucine , amino acid , kinase , immunology , in vitro
Membrane‐bound liver alkaline phosphatase (Mem‐LiALP, EC 3.1.3.1) is a high‐molecular‐mass liver alkaline phosphatase (ALP) present in metastatic, infiltrative and cholestatic liver disease. Shedding of hepatocyte plasma membrane fragments (LiPMF) is thought to be responsible for the appearance of Mem‐LiALP in the circulation. Several other membrane‐bound enzymes, such as γ‐glutamyltransferase (γ‐GT), leucine aminopeptidase (LAP), and 5′‐nucleotidase (5′‐Nu) are present in the membrane of the shedded LiPMF. By means of immunohistochemical and immunoassay procedures, we presently show that AD‐1, a specific monoclonal antibody originally produced against Mem‐LiALP, reacts with LAP, a constituent of the human liver plasma membrane. Using AD‐1 as an immunosorbant, we isolated circulating LiPMF from cholestatic sera to a high level of purity and separated it from other high‐molecular‐mass material, such as liver ALP ∼ lipoprotein‐X complexes. These purified membrane fragments retained their biochemical characteristics. Glycosyl‐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, phospholipase 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 coated nitrocellulose disks and the activity of four membrane‐bound enzymes (LAP, ALP, 5′Nu, γ‐GT) was analyzed. A considerable interindividual variation of enzyme activities was observed, suggesting some heterogeneity in the membrane composition of these fragments.