Purification and characterization of the blue-green rat phaeochromocytoma (PC12) tyrosine hydroxylase with a dopamine-Fe(III) complex : reversal of the endogenous feedback inhibition by phosphorylation of serine-40

• Published in: Biochemical journal Vol. 284; no. 3; pp. 687 - 695
• Main Authors: ANDERSSON, K. K, VASSORT, C, BRENNAN, B. A, QUE, L. JR, HAAVIK, J, FLATMARK, T, GROS, F, THIBAULT, J
• Format: Journal Article
• Language: English
• Published: Colchester Portland Press 15.06.1992
• Subjects: Adrenal Gland Neoplasms - enzymology; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Chromatography; Chromatography, Affinity; Dopamine - analysis; Durapatite; Electron Spin Resonance Spectroscopy; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Escherichia coli - genetics; Feedback; Fundamental and applied biological sciences. Psychology; Humans; Hydroxyapatites; Indicators and Reagents; Iron - analysis; Kinetics; Molecular Weight; Oxidoreductases; PC12 Cells; Pheochromocytoma - enzymology; Phosphorylation; Rats; Recombinant Proteins - metabolism; Spectrum Analysis, Raman; Tyrosine 3-Monooxygenase - genetics; Tyrosine 3-Monooxygenase - isolation & purification; Tyrosine 3-Monooxygenase - metabolism; Phosphorylation; Pheochromocytoma; Purification; Rat; Enzyme; Rodentia; Biosynthesis; Malignant tumor; Catecholamine; Tyrosine 3-monooxygenase; Vertebrata; Regulation(control); Mammalia; Cell line; Enzymatic activity; Iron III Complexes
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2840687

Tyrosine hydroxylase (TH) was purified from tumours of rat phaeochromocytoma (PC12) cells by a three-step purification procedure giving 30 mg of pure enzyme in 3 days. The enzyme sedimented with an S(eo),w value of 9.2 S and revealed an apparent subunit molecular mass of 62 kDa with a minor 60 kDa component. Two-dimensional gel isoelectric focusing/electrophoresis and tryptic digestion revealed that the heterogeneity could be accounted for by limited proteolysis of the 62 kDa component and the presence of covalently bound phosphate. The enzyme had a strong blue-green colour (epsilon 700 = 3.1 +/- 0.2 mM-iron-1.cm-1). The resonance Raman spectrum obtained with lambda excitation = 605 nm revealed the presence of an Fe(III)-catecholamine complex in the isolate enzyme, similar to that observed in the bovine adrenal enzyme [Andersson, Cox, Que, Flatmark & Haavik (1988) J. Biol. Chem. 263, 18621-18626]. In the rat PC12 enzyme, all of the iron present (0.53 +/- 0.03 atom per subunit) seems to be chelated by the feedback inhibitors (0.49 +/- 0.05 mol of dopamine and 0.10 +/- 0.03 mol of noradrenaline per mol of subunit). The e.p.r. spectra at 3.6 K show g-values at 7.0, 5.2 and 1.9 as observed for other catecholate-complexed enzymes. After phosphorylation of serine-40 and addition of L-tyrosine a new rhombic (magnitude of E/D = 0.33) e.p.r. species could be observed. Phosphorylation of serine-40 by cyclic AMP-dependent protein kinase increased the catalytic activity; depending on assay conditions, up to 80-110-fold activation could be observed when measured at high TH (i.e. high endogenous catecholamine) concentration.