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.
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.
Andersson
K K
KK
Department of Biochemistry, University of Bergen, Norway.
Vassort
C
C
Brennan
B A
BA
Que
L
L
Haavik
J
J
Flatmark
T
T
Gros
F
F
Thibault
J
J
D000310:Adrenal Gland Neoplasms / Q000201:enzymology*; D000818:Animals; D002845:Chromatography; D002846:Chromatography, Affinity; D004298:Dopamine / Q000032:analysis*; D017886:Durapatite; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005246:Feedback; D006801:Humans; D006882:Hydroxyapatites; D007202:Indicators and Reagents; D007501:Iron / Q000032:analysis*; D007700:Kinetics; D008970:Molecular Weight; D016716:PC12 Cells; D010673:Pheochromocytoma / Q000201:enzymology*; D010766:Phosphorylation; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013059:Spectrum Analysis, Raman; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D006882:Hydroxyapatites; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D017886:Durapatite; D007501:Iron; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine