These studies have focused on the regulatory properties of tyrosine hydroxylase, tryptophan hydroxylase, nitric oxide synthase (NOS) and their cofactor tetrahydrobiopterin (BH4) in the biosynthesis and release of catecholamine and serotonin in the brain. We have confirmed that nitric oxide (NO) derived from L-arginine through the action of NOS enhances dopamine release in striatal slices. This effect can be inhibited by the NOS inhibitor-nitro arginine. We have also confirmed that BH4 also enhances dopamine release in striatal slices. Our studies show that the stimulating effect of L-arginine in enhancing dopamine release can be decreased in the slices in which the intracellular BH4 levels have been depleted. BH4 depletion and inhibition of NOS are equally effective in blocking the L-arginine- mediated stimulation of dopamine release. This finding supports the conclusion that the L-arginine-mediated stimulation of dopamine release is BH4 dependent. We have purified the 14-3-3 protein from rat and bovine brains and investigated the role of 14-3-3 in the regulation of human recombinant tryptophan hydroxylase (fused with maltose binding protein) expressed in E.coli. Fusion tryptophan hydroxylase was phosphorylated in the presence of both cAMP-dependent protein kinase and calmodulin-dependent protein kinase II. Preliminary results showed that gel shift assay using native polyacrylamide gel indicated no complex formation. Affinity chromatography experiments where fusion enzyme was covalently coupled with the activated ester of Affi gel-10 indicated no binding of 14-3-3 with the coupled enzyme.
