Resolution of rat brain synaptic phosphoprotein B-50 into multiple forms by two-dimensional electrophoresis: evidence for multisite phosphorylation.
Phosphoprotein B-50 was extracted from rat brain membranes by alkaline extraction and purified by ammonium sulphate precipitation and flat-bed isoelectric focusing. The purified protein shows microheterogeneity upon isoelectric focusing in a narrow pH gradient (pH 3.5-5.0). As visualized by two-dimensional gel electrophoresis, B-50 resolved into four clearly separated forms which differ slightly in isoelectric point. The forms are in part mutually convertible by exhaustive phosphorylation (using protein kinase C) and dephosphorylation (using Escherichia coli alkaline phosphatase). Proteolysis with Staphylococcus aureus protease yielded two radioactive peptides. Analysis of their molecular weights and the time course of their formation suggests that B-50 was cleaved at only one specific site. Our data indicate the presence of more than one phosphorylatable site. The possibility that the heterogeneity of B-50 was in part due to a glycoprotein nature of B-50 was studied extensively. However, none of the six different methods used revealed the presence of glyco-moieties in B-50.
Zwiers
H
H
Verhaagen
J
J
van Dongen
C J
CJ
de Graan
P N
PN
Gispen
W H
WH
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001923:Brain Chemistry; D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000378:metabolism; D019922:GAP-43 Protein; D006023:Glycoproteins / Q000032:analysis; D007525:Isoelectric Focusing; D007700:Kinetics; D008666:Metalloendopeptidases; D010446:Peptide Fragments / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D013570:Synaptic Membranes / Q000032:analysis*
D019922:GAP-43 Protein; D006023:Glycoproteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011494:Protein Kinases; D011493:Protein Kinase C; D000469:Alkaline Phosphatase; D010450:Endopeptidases; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus