Molecular characterization of GP50: a neuron-specific, synaptic-enriched glycoprotein.
The molecular properties of the neuron-specific, synaptic-enriched glycoprotein GP50 have been investigated with the aid of the monoclonal antibody MabSM-GP50. GP50 immunoreactivity was detected in the brains of the frog, trout, pigeon, snake, rabbit, mouse, cow, and human, although variation in quantity and electrophoretic mobility of the immunoreactive protein between species was apparent. Deglycosylation of synaptic membranes (SMs) with endoglycosidase H, peptide:N-glycosidase F, trifluoromethane-sulfonic acid, and alkaline sodium borohydride indicated that GP50 is associated primarily, if not exclusively, with high-mannose and/or hybrid-type oligosaccharides and lacks complex N-linked and O-linked sugar chains. GP50 remained associated with the membrane fraction following extraction of SMs with alkaline sodium carbonate, was partially (55%) present in the detergent phase following the phase partitioning of SMs in the presence of Triton X-114, and was resistant to proteolytic digestion with trypsin when present as a component of intact membranes. Taken together, these results indicate that GP50 is an integral component of the SM. Sucrose gradient centrifugation of Triton X-100 extracts of SMs or of forebrain and cerebellar homogenates resolved GP50 into two fractions with sedimentation coefficients of 3.6S and 7.3S, which accounted for 45 and 55% of the total, respectively. The 7.3S form occurred exclusively in the aqueous phase following partitioning with Triton X-114, whereas the 3.6S species was found in both the aqueous and detergent phases.(ABSTRACT TRUNCATED AT 250 WORDS)
Paladino
T
T
Department of Biochemistry, University of Toronto, West Hill, Ontario, Canada.
Beesley
P W
PW
Gurd
J W
JW
D000818:Animals; D015153:Blotting, Western; D001923:Brain Chemistry; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins / Q000302:isolation & purification*; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*; D009844:Oligosaccharides / Q000032:analysis; D010750:Phosphoproteins / Q000302:isolation & purification*; D051381:Rats; D012995:Solubility
D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D009844:Oligosaccharides; D010750:Phosphoproteins