Purification and characterization of a protein antigen (p94) from human brain and its identification in other species.
A protein antigen was chromatographically purified from human brain by its immunoaffinity to 44E3 monoclonal IgG and its chemical nature was investigated. The yield of antigen was estimated at 71%, and a 3160-fold purification was achieved relative to the homogenate. The antigen preparation from brain showed a very high degree of purity when analysed by SDS/polyacrylamide-gel electrophoresis and was composed of a single polypeptide of Mr 94,000. Amino-sugar and neutral-sugar analyses indicated that the protein was not glycosylated. The amino acid composition of the purified protein from brain was compared with that of the analogous protein purified from an acute-lymphoblastic-leukaemic cell line, HOON. The compositions were very similar, suggesting that the two proteins were closely related. Both purified proteins were equivalent in their ability to inhibit the reactivity of monoclonal antibodies 44E3 and 44H4 with leukaemic cells. These two antibodies appear to recognize spatially related, if not identical, epitopes on the same molecule. The antibodies were shown to cross-react with a polypeptide of Mr 94,000 in homogenates of human, bovine and guinea-pig brain white matter. Indirect immunoperoxidase staining of human grey- and white-matter acetone-fixed tissue sections incubated with either antibody indicated that the antigen was present on neuronal and glial cells; the staining was seen as clusters in the cytoplasm, starting at the plasma membrane, but leaving the nucleus unstained. The concentration of the protein in human brain was shown to be similar throughout postnatal development and aging.
Division of Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada.
T F
TF
Cruz
N
N
Girgrah
E J
EJ
Quackenbush
M
M
Letarte
M A
MA
Moscarello
D000293:Adolescent; D000328:Adult; D000367:Age Factors; D000368:Aged; D000369:Aged, 80 and over; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D001923:Brain Chemistry; D002417:Cattle; D002648:Child; D002675:Child, Preschool; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D006801:Humans; D007223:Infant; D007938:Leukemia / Q000378:metabolism; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*; D013045:Species Specificity; D014018:Tissue Distribution
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D009419:Nerve Tissue Proteins; C054199:brain protein antigen p94