This proposal continues to explore the mechanisms whereby tetrahydropteridines in conjunction with pteridine reductase and phenylalanine or tyrosine hydroxylase cause the insertion of one atom of molecular oxygen as a hydroxyl substituent into the aromatic nucleus of the latter enzymes' substrates. An integral part of the project requires the efficient isolation and characterization of the three enzymes. By novel affinity chromatographic techniques both pteridine reductase and phenylalanine hydroxylase from rat liver have already been isolated to homogeneity and partially characterized. The high specific activity of the recovered hydroxylase has necessitated that many of the previously reported properties of the enzyme be re-examined. Therefore, in addition to typical protein characterization procedures such as amino acid, N-terminal and sequence analyses, PAG electrophoresis, gel filtration, spectral and circular dichroic properties, specific amino-acid labelling, kinetic parameters, etc., both the iron and phosphate content of the protein are to be determined. Further characterization of the active sites of both enzymes will be attempted by employing analogs of the amino acid and nucleotide substrates containing photoaffinity or fluorescent labels. Tyrosine hydroxylase, recalcitrant to large scale purification by conventional techniques, will be isolated from bovine adrenal medulla using affinity matrices containing a rabbit anti-hydroxylase ligand, generated by injection of PAG-electrophoretically purified enzyme. This enzyme will then also be characterized by the above procedures. The accessibility of the reductase and hydroxylases in mg quantities will allow extensive spectral and kinetic analyses of substrate and enzyme interactions, which coupled with an examination of the oxidation pathways of model tetrahydropteridines, will allow further interpretation of the oxygenation process.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physiological Chemistry Study Section (PC)
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Scripps Research Institute
San Diego
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Stuhmiller, L M; Nazarbaghi, R; Webber, S et al. (1989) Methotrexate 5-aminoallyl-2'-deoxyuridine 5'-monophosphate: a potential bifunctional inhibitor of thymidylate synthase. Adv Enzyme Regul 29:141-57
Webber, S; Baumgartner, K; Blair, J N et al. (1988) Interaction of a photolabile NADH analog with rat liver dihydropteridine reductase. Biochem Pharmacol 37:2869-72
Boschelli, D H; Webber, S; Whiteley, J M et al. (1988) Synthesis and biological properties of 5,10-dideaza-5,6,7,8-tetrahydrofolic acid. Arch Biochem Biophys 265:43-9
Webber, S; Hural, J A; Whiteley, J M (1988) The estimation of dihydropteridine reductase in human blood cells. Clin Chim Acta 173:117-26
Webber, S; Hural, J; Whiteley, J M (1987) Preliminary studies on the primary structure of rat liver dihydropteridine reductase. Biochem Biophys Res Commun 143:582-6
Shahbaz, M; Hoch, J A; Trach, K A et al. (1987) Structural studies and isolation of cDNA clones providing the complete sequence of rat liver dihydropteridine reductase. J Biol Chem 262:16412-6
Whiteley, J M; Webber, S; Kerwar, S S (1986) Synthesis and efficient isolation procedure for gamma-linked fluorescein methotrexate. Prep Biochem 16:143-54
Webber, S; Hural, J A; Whiteley, J M (1986) Multiple forms of rat-liver dihydropteridine reductase identified by their differing isoelectric points. Arch Biochem Biophys 248:358-67
Matthews, D A; Webber, S; Whiteley, J M (1986) Preliminary x-ray diffraction characterization of crystalline rat liver dihydropteridine reductase. J Biol Chem 261:3891-3
Webber, S; Whiteley, J M (1985) Comparative activity of rat liver dihydrofolate reductase with 7,8-dihydrofolate and other 7,8-dihydropteridines. Arch Biochem Biophys 236:681-90