Cyclic AMP-dependent protein kinases play a central role in metabolic regulation. The brain and adrenal medulla contain a phosphorylating enzyme (cyclic AMP-dependent protein kinase), one of its substrates (tyrosine hydroxylase) and the dephosphorylating enzyme (phosphoprotein phosphatase). Protein kinase contains two dissimilar subunits (regulatory and catalytic) which combine to produce an inactive tetramer (R2C2). Cyclic AMP dissociates the regulatory dimer from the catalytic subunits which become active. We have proposed the hypothesis that the R subunit blocks the binding of protein or peptide substrates to C thereby inhibiting its activity. To test this idea, we plan to characterize the binding of a fluorescent substrate (dansylser-peptide) and (3H) N-acetylser-peptide to free C subunit and holoenzyme. We also plan to prepare fluorescent derivatives of C subunit and measure their interaction with physiologic protein substrates as the complexed holoenzyme and as the free C subunit. We plan to characterize the binding of nucleotides to free C subunit and holoenzyme by displacement analysis using lin-benzo ADP as a fluorescent ligand. We will compare the peptide maps of th type II R subunits and C subunits from bovine brain, skeletal and heart muscle. The type II R subunit from neural tissue differs immunochemically from other tissues and we wish to determine the extent of these differences. We will also compare these structures with the type I R and C from bovine muscle. We plan to characterize the phosphorylation activation-inactivation of rat corpus striatal and pheochromocytoma tyrosine hydroxylase. We plan to test the idea that inactivation may be related to hyperphosphorylation, increased lability or proteolysis. We plan to test the feasibility of C subunit affinity chromatography for the purification of tyrosine hydroxylase and other substrates. We plan to characterize and purify brain phosphoprotein phosphatase. We plan to determine its substrate specificty. Our preliminary data indicate the phosphotyrosine hydroxylase is a substrate. We also want to determine the importance of substrate arginyl residues in determining phosphatase substrate specificity.
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