Binding properties of detergent-solubilized NCAM.
An assay has been designed for the identification of NCAM-binding proteins present in an NP-40 detergent extract of brain membranes. This method, which is capable of analyzing both heterophilic and homophilic interactions, uses species-specific antibodies against NCAM in combination with radioiodination, so that after unlabeled chicken and iodinated frog brain membrane proteins were allowed to interact, the chicken NCAM could be specifically isolated by immunoaffinity adsorption. The radiolabeled frog proteins coisolated with chicken NCAM were then characterized by one- and two-dimensional gel electrophoresis in combination with immunoblotting. The only detectable NCAM-binding proteins were identified as the 140- and 180-kD forms of NCAM. The presence and absence of polysialic acid on NCAM did not change the amount or nature of the frog proteins immunopurified under these conditions. As an alternative for detecting heterophilic ligands, a simplified immunoprecipitation method was employed using either iodine or sulfate radiolabels. Again under these conditions only NCAM was detected. These results are consistent with the hypothesis that the major binding protein for NCAM is NCAM itself, and suggest that differences in polysialic acid content do not directly alter the properties of NCAM's homophilic binding site.
Hall
A K
AK
Department of Genetics and Center for Neuroscience, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106.
Nelson
R
R
Rutishauser
U
U
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015816:Cell Adhesion Molecules, Neuronal / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols; D011894:Rana pipiens; D012995:Solubility
D002352:Carrier Proteins; D015816:Cell Adhesion Molecules, Neuronal; D003902:Detergents; D008565:Membrane Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40