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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM022125-11S1
Application #
3270945
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1982-04-01
Project End
1987-05-31
Budget Start
1987-04-01
Budget End
1987-05-31
Support Year
11
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
San Diego
State
CA
Country
United States
Zip Code
92037
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