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Purification and Characterization of P 400 Protein, a Glycoprotein Characteristic of Purkinje Cell, from Mouse Cerebellum
Author(s) -
Maeda Nobuaki,
Niinobe Michio,
Nakahira Kensuke,
Mikoshiba Katsuhiko
Publication year - 1988
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1988.tb01151.x
Subject(s) - cerebellum , glycoprotein , monoclonal antibody , microbiology and biotechnology , sepharose , epitope , affinity chromatography , biochemistry , purkinje cell , biology , protein a/g , chemistry , antibody , fusion protein , enzyme , immunology , neuroscience , gene , recombinant dna
P 400 protein is a concanavalin A (Con A)‐binding, 250–kilodalton glycoprotein characteristic of cerebellum. Extraction conditions for P 400 protein were investigated, and complete solubilization of P 400 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 P 400 protein. Endo‐β‐ N ‐acetylglucosaminidase F digestion of P 400 protein revealed that P 400 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 P 400 protein was investigated by immunoblot analysis using 18A10 monoclonal antibody. P 400 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. Immunohis‐tochemical studies indicated that P 400 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.