A STUDY OF PROTEINS WITHIN THE CNS BY TWO-DIMENSIONAL GEL ELECTROPHORESIS

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The identification of interesting proteins within the CNS utilizing 2-dimensional gel electrophoresis (2DE) continues. 1) One of the largest proteins on our brain gels, "protein 36" has been isolated and purified from homogenates of rat brain. Protein 36 is a homodimer with a molecular weight of 64 kD and a monomeric weight of 37 kD with a pI of 6.5. Immunocytochemical studies using a rabbit antibody raised to this protein revealed that protein 36 is localized in large pyramidal cells, dendrites and axons of layer V of the cerebral cortex. 2) A second protein ("protein 94") has been isolated from the bovine brain and purified. This is a cAMP-stimulated phosphoprotein (M.W. 94 kD) which was originally identified in the rat neostriatum. Purification was monitored by autoradiography of 32P-phosphorylated samples separated by SDS-PAGE. Immunocytochemical studies revealed that protein 94 was localized in astrocytes. 3) We have detected at least 3 Ca++ -binding proteins in the rat cortex by 45Ca++ autoradiography. Two proteins were identified as calmodulin and the B subunit of calcineurin. In addition, an unidentified Ca++ - binding protein of molecular weight about 18 kD and pI 5.4 was revealed. 4) Seven high affinity calcium binding proteins have been detected following ammonium sulfate precipitation. Three of the proteins have been identified (calmodulin, B subunit of calcineurin and vitamin D-dependent calcium binding protein. The identity of the other 4 proteins are unknown. 5) We have studied phosphoproteins present in seven discrete microdissected brain areas. Marked regional differences in the pattern of phosphorylation among the brain studied under basal conditions and following activation of cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase C were noted. 6) A series of studies examining proteins within auditory nuclei are under progress. Results indicate several differences in proteins visible on two-dimensional gels across auditory nuclei or more generally between sensory nuclei and other brain areas.