Purification of circulating liver plasma membrane fragments using a monoclonal antileucine aminopeptidase antibody

Hepatology. 1996 Mar;23(3):445-54. doi: 10.1002/hep.510230308.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5'-Nucleotidase / metabolism
  • Alkaline Phosphatase / metabolism
  • Antibodies, Monoclonal / pharmacology*
  • Cell Membrane / enzymology
  • Cell Membrane / ultrastructure
  • Cholestasis / blood
  • Cholestasis / enzymology
  • Humans
  • Immunohistochemistry
  • Leucyl Aminopeptidase / immunology*
  • Leucyl Aminopeptidase / metabolism
  • Liver / enzymology
  • Liver / ultrastructure*
  • Microscopy, Electron
  • Phospholipases / metabolism
  • gamma-Glutamyltransferase / metabolism

Substances

  • Antibodies, Monoclonal
  • gamma-Glutamyltransferase
  • Phospholipases
  • Alkaline Phosphatase
  • 5'-Nucleotidase
  • Leucyl Aminopeptidase