Expression of the neuron-specific glycoprotein GP50 by granule cell cultures.
The expression and properties of the neuron-specific glycoprotein, GP50, by neonatal rat granule cells grown in primary tissue culture were studied using the monoclonal antibody MabSM-GP50. GP50 was expressed by granule cells in culture but not by astrocytes or PC12 cells that had been induced to differentiate with nerve growth factor. Immunocytochemical staining of granule cell cultures demonstrated that immunoreactivity was concentrated in the cell body. Although the amount of GP50 increased slowly throughout the culture period the maximum level of expression in vitro was markedly lower than that observed in cerebellum of the comparable age. Radiolabelling of cell surface proteins by lactoperoxidase-catalyzed iodination demonstrated that GP50 was expressed on the surface of granule cells. Following phase-partitioning of granule cells in the presence of Triton X-114, 75% of GP50 was found to be present in the detergent phase, indicating that it is an integral membrane protein. Sucrose density gradient centrifugation of Triton X-100 extracts of granule cells resolved GP50 into two components with sedimentation coefficients of 3.6S and 7.3S. The 3.6S species (Mr 42,000 Da) accounted for greater than 80% of the total, indicating that GP50 is present predominantly in a monomeric form within the membrane. These properties are similar to those of GP50 expressed in P12 cerebellum but contrast with the behavior of GP50 from mature brain, in which the 7.3S, hydrophilic form is the predominant species. The results suggest that the mature expression of GP50 may be dependent upon the histotypic pattern of development which occurs in vivo.
Department of Biochemistry, University of Toronto, Ont., Canada.
T
T
Paladino
P W
PW
Beesley
S
S
Nicholson
J W
JW
Gurd
D000818:Animals; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D007150:Immunohistochemistry; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051381:Rats
D009419:Nerve Tissue Proteins; C052914:brain synaptic membrane glycoprotein gp 50