Purification and characterization of neuron-specific surface antigen defined by monoclonal antibody BM88.
Monoclonal antibody BM88 recognizes a neurospecific surface antigen in the CNS and the PNS. In the present study, the antigen recognized by BM88 was immunopurified from pig brain and shown to be a 22-kDa polypeptide by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nonreducing conditions a protein of 40 kDa was obtained, a result indicating that the antigen is composed of two polypeptide chains of equal molecular weight linked by disulfide bridges. Gel filtration of the purified antigen in the presence of Emulphogene suggested that it may be either a monomeric or a dimeric protein. However, in the presence of Triton X-100 a monomeric structure was implied. N-Glycanase digestion indicated that the protein is probably not glycosylated. The purified antigen was characterized as an integral membrane protein by hydrophobic chromatography and phase-separation experiments with Triton X-114. The antigen, or at least the antibody binding region of the molecule, is very susceptible to protease attack, as judged by protease digestion experiments on brain membranes. By using very low concentrations of papain combined with short incubation times, the antigen was converted to a 16.3-kDa membrane-associated polypeptide as assessed by immunoblotting. This polypeptide contained the BM88 binding epitope. Soluble BM88 immunoreactive polypeptides were not obtained. Bacillus cereus phospholipase C was also unable to solubilize the antigen from the membrane. Our results suggest that the molecule, possessing at least one small extramembranous domain, is attached to the membrane via a polypeptide chain.
Patsavoudi
E
E
Department of Biochemistry, Hellenic Pasteur Institute, Athens, Greece.
Hurel
C
C
Matsas
R
R
D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D002241:Carbohydrates / Q000737:chemistry; D000939:Epitopes; D007158:Immunologic Techniques; D008565:Membrane Proteins / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D011863:Radioimmunoassay; D013552:Swine
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D002241:Carbohydrates; D000939:Epitopes; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins