Partial characterization of endogenous phosphorylation conditions for hen brain cytosolic and membrane proteins

The optimal conditions for endogenous protein phosphorylation with 5 microM [gamma-32P]ATP, 10 mM MgCl2 in preparations containing synaptosomal cytosol or membranes (shocked crude mitochondrial fraction P2) from adult hen brains were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography and microdensitometry. Phosphate incorporation increased linearly with protein concentration from 75-125 micrograms/200 microliters in brain cytosol and was maximal at 75 micrograms/200 microliters in brain membranes. Optimal incubation times were 60-90 s for brain cytosol and 10-15 s for brain membranes. With the exception of the 20 kilodalton myelin basic protein in the membrane fraction, pH 6.5 is generally optimal. While temperature optima varied considerably with different bands, most of them were found between 35 and 45 degrees C. When identical preparations from hen and rat brain were co-electrophoresed, one of the most striking differences was that the enhancement of phosphorylation of a 55 kilodalton doublet, which may be tubulin, by addition of 50 microM Ca2+ was at least 3 times greater in rat than in hen brain cytosol. Another species difference was apparent in the membrane fractions in which the 20 kilodalton hen brain presumptive myelin basic protein (MBP) was phosphorylated to approximately the same extent as that of the 16 and 20 kilodalton rat brain MBPs combined.