A Radioimmunoassay for the Phosphoprotein B‐50: Distribution in Rat Brain

Abstract

A radioimmunoassay (RIA) for the B‐50 protein was developed to determine B‐50 in total homogenates of rat tissues. A tracer of purified B‐50 was prepared at high activity (10–30 μCi/μg protein) by phosphorylating B‐50 with carrier‐free [γ‐³²P]ATP, catalyzed by purified protein kinase C. The RIA was performed using affinity‐purified anti‐B‐50 immunoglobulins G in a detergent containing medium and detected B‐50 at levels of 0.1–10 ng. Specificity of the antibodies was ascertained by immunoprecipitation of B‐50 from a crude mitochondrial membrane fraction from rat brain and by immunoblotting. For the B‐50 content in rat brain the following distribution pattern was found: medulla spinalis < cerebellum < hippocampus; cerebral cortex < periaqueductal gray < septum. The septum contained 80 μg/g tissue weight. The level in liver homogenates was below detection. The regional distribution is in fair agreement with the pattern of the endogenous B‐50 phosphorylation in rat brain synaptosomal plasma membranes previously reported.