Photoaffinity labelling of central-nervous-system myelin. Evidence for an endogenous type I cyclic AMP-dependent kinase phosphorylating the larger subunit of 2′,3′-cyclic nucleotide 3′-phosphodiesterase

Abstract

Endogenous cAMP-stimulated phosphorylation of a 49,700 MW Wolfgram protein component in rabbit CNS was investigated by using photoaffinity labeling and 2'',3''-cyclic nucleotide 3''-phosphodiesterase activity staining after electroblotting on to nitrocellulose paper. Photoaffintiy labeling with 8''-azidoadenosione 3'',5''-cyclic monophosphate showed a cAMP-binding protein that appeared to be intrinsic to the myelin membrane and appeared to represent the R-subunit of a type I cAMP-dependent protein kinase. This photoaffinity-labeled protein was of larger apparent MW than the protein showing cAMP-stimulated phosphorylation. Blotting of 1-dimensional sodium dodecyl sulfate/polyacrylamide-gel electrophoretograms followed by staining for 2'',3''-cyclic nucleotide 3''-phosphodiesterase activity showed 2 activity bands corresponding to the 2 components of the Wolfgram protein doublet. cAMP-stimulated protein phosphorylation corresponded to the upper component of this doublet. Electroblotting of 2-dimensional non-equilibrium pH-gradient electrophoretograms also showed co-migration of cAMP-stimulated protein phosphorylation with enzyme activity. CNS myelin contains an endogenous type I cAMP dependent protein kinase that phosphorylates the larger subunit of 2'',3''-cyclic nucleotide 3''-phosphodiesterase.