BIOLOGICAL ROLE OF NEUROCATIN--NOVEL BRAIN PEPTIDE

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The aim of this research program is to characterize a neuroregulatory factor (neurocatin) isolated from mammalian brain. Neurocatin modulates both the release and metabolism of dopamine and norepinephrine in synaptosomes isolated from rat brain. It appears to act, at least in part, by increasing the calcium permeability of the plasma membrane. The specific goals of this project are: 1. Characterization of purified neurocatin. Neurocating has been purified to >90% purity and its amino acid composition determined. Since current evidence indicates it is a peptide, the procedures employed will include: a). Determination of the amino acid sequerice. b). Synthesis of neurocatine from amino acids by standard methods or peptide synthesis. c). Verification that the activity of the synthetic compound is the same as that of the native compound. Following determination of the chemical structure, radiolabeled neurocatin will be prepared by incorporation of radioactive amino acid in the synthesis. This radiolabeled compound will be used to measure the number of high affinity binding sites present in the membranes of synaptosomes, to characterize the receptor binding and to autoradiographically map the distribution of the receptors (high affinity binding sites) in the rat brain. Antibodies will be developed to neurocatin and these will be used to screen brain tissue for the presence of other molecules expressing cross reactivity with the antibodies to neurocatin. Immunohistochemical mapping will be used to determine, in rat brain the distribution of species reacting with antibodies to neurocatin. 2. Determination of the biological role of purified factor. Evidence has already been obtained that neurocatin increase release of catecholamines from synaptosomes and induces major alterations in their metabolism. The alteration include inhibition of oxidative deamination of dopamine, decreased formation of 3,4-dihydroxyphenylacetic acid (DOPAC), and increased formation of norepinephrine and its N-methyl derivatives. We will determine the step of the metabolic pathways at which control by neurocatine is expressed and the mechanism(s) by which the activity of each reaction is modulated. Our studies will include characterization of the Ca2+ permeability changes in the plasma membrane and of the proteins phosphorylated in the presence of neurocatine. With respect to the latter, we will: 1. quantitate the relative labelling of the proteins by densitometry after SDS-PAGE gel electrophoresis followed by autoradiography, 2. measure the dose-response of phosphorylation induced by addition of factor PW, 3. determine the type of protein kinase which is activated (whether phosphorylation occurs at the serine, tyrosine or threonine residues) and 4. identify enzymes in the pathway for catecholamine synthesis and catabolism which may be phosphorylated.