Purification and characterization of P400 protein, a glycoprotein characteristic of Purkinje cell, from mouse cerebellum.
P400 protein is a concanavalin A (Con A)-binding, 250-kilodalton glycoprotein characteristic of cerebellum. Extraction conditions for P400 protein were investigated, and complete solubilization of P400 protein from a submicrosomal fraction (P31 fraction) of mouse cerebellum was attained by the combination of 4% Zwittergent 3-14 and 4 M guanidinium chloride. The solubilized P400 protein was purified using Sepharose CL-4B and Con A-Sepharose chromatography. A monoclonal antibody (18A10) was prepared against P400 protein. Endo-beta-N-acetylglucosaminidase F digestion of P400 protein revealed that P400 protein has a small number of asparagine-linked oligosaccharide chains and that the epitope that is recognized by 18A10 monoclonal antibody is not on the asparagine-linked oligosaccharide portion. Tissue distribution of P400 protein was investigated by immunoblot analysis using 18A10 monoclonal antibody. P400 protein was abundant in the cerebellum, but a very small amount of P400 protein or related antigen was also detected in other parts of the nervous system and in nonneural tissues. Immunohistochemical studies indicated that P400 protein was distributed abundantly in the soma, the dendritic arborization, and the axon of the Purkinje cell. No immunoreaction was observed in the other types of cells.
D000118:Acetylglucosaminidase; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000941:Antigens / Q000276:immunology; D015220:Calcium Channels; D002531:Cerebellum / Q000032:analysis*; D002845:Chromatography; D003902:Detergents; D006023:Glycoproteins / Q000032:analysis* / Q000276:immunology; D015151:Immunoblotting; D007150:Immunohistochemistry; D053496:Inositol 1,4,5-Trisphosphate Receptors; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008562:Membrane Glycoproteins; D051379:Mice; D011689:Purkinje Cells / Q000032:analysis*; D018160:Receptors, Cytoplasmic and Nuclear; D012995:Solubility; D014018:Tissue Distribution
D000911:Antibodies, Monoclonal; D000941:Antigens; D015220:Calcium Channels; D003902:Detergents; D006023:Glycoproteins; D053496:Inositol 1,4,5-Trisphosphate Receptors; C025389:Itpr1 protein, mouse; D008562:Membrane Glycoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase