Soluble tyrosine hydroxylase (tyrosine 3-monooxygenase) from bovine adrenal medulla: large-scale purification and physicochemical properties.
A new procedure that permits large-scale purification of tyrosine 3-monooxygenase (tyrosine hydroxylase) (L-tyrosine,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) from the cytosolic fraction of bovine adrenal medulla is described. The homogenous enzyme revealed a subunit Mr of 60,000 and a specific activity of 425 nmol.min-1.mg-1. The N-terminal amino-acid sequence (27 residues) revealed 89% homology with the human pheochromocytoma enzyme as deduced from its cDNA sequence. The pure enzyme contained 0.66 +/- 0.09 mol iron, 0.13 mol zinc and 0.62 +/- 0.04 mol phosphate per mol subunit of Mr = 60,000. A broad light absorption band with its maximum around 700 nm (epsilon 700 nm = 1.3 (mM monomer)-1.cm-1) explains its blue-green color. EPR spectra at 3.6 K revealed high-spin Fe(III) (S = 5/2) in an environment of nearly axial symmetry (g values at 7.2-6.7, 4.7-5.3 and 1.9-2.0). A close correlation was observed between the absorbance at 700 nm and the intensity of the axial type of EPR spectrum. The absorption peak at 700 nm is compatible with a ligand-to-iron charge-transfer transition as a result of catecholate coordination to the iron. Physicochemical studies suggest that the enzyme does not undergo such major substrate- or cofactor-induced conformational changes as have been reported for the related enzyme, phenylalanine hydroxylase.
D000313:Adrenal Medulla / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D002845:Chromatography; D002846:Chromatography, Affinity; D003600:Cytosol / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D005453:Fluorescence; D006801:Humans; D007525:Isoelectric Focusing; D008670:Metals / Q000032:analysis / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010710:Phosphates / Q000032:analysis; D010766:Phosphorylation; D011487:Protein Conformation; D051381:Rats; D013053:Spectrophotometry; D014364:Tryptophan; D014446:Tyrosine 3-Monooxygenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism
D002614:Chelating Agents; D008670:Metals; D010710:Phosphates; D014364:Tryptophan; D014446:Tyrosine 3-Monooxygenase