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 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 gamma-glutamyltransferase (gamma-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 or similar 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, gamma-GT) was analyzed. A considerable interindividual variation of enzyme activities was observed, suggesting some heterogeneity in the membrane composition of these fragments.
Deng
J T
JT
Department of Nephrology-Hypertension, University of Antwerp, Wilrijk, Belgium.
Hoylaerts
M F
MF
Nouwen
E J
EJ
De Broe
M E
ME
Van Hoof
V O
VO
D015720:5'-Nucleotidase / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D002779:Cholestasis / Q000097:blood / Q000201:enzymology; D006801:Humans; D007150:Immunohistochemistry; D007931:Leucyl Aminopeptidase / Q000276:immunology* / Q000378:metabolism; D008099:Liver / Q000201:enzymology / Q000648:ultrastructure*; D008854:Microscopy, Electron; D010740:Phospholipases / Q000378:metabolism; D005723:gamma-Glutamyltransferase / Q000378:metabolism
D000911:Antibodies, Monoclonal; D005723:gamma-Glutamyltransferase; D010740:Phospholipases; D000469:Alkaline Phosphatase; D015720:5'-Nucleotidase; D007931:Leucyl Aminopeptidase