Properties of tyrosine hydroxylation in living mouse neuroblastoma clone N1E-115.
Tyrosine hydroxylation was studied in intact cells of mouse neuroblastoma clone N1E-115 which have high levels of tyrosine 3-monooxygenase (EC 1.14.16.2) and which have been fully characterized for tyrosine transport. Measurement of [3H]OH formed from L-[3,5(-3)H]tyrosine in the medium was the method of assay and [3H]OH formed was stoichiometric with the formation of L-[3H]3,4-dihydroxyphenylalanine. Tyrosine hydroxylation was dependent on time of incubation, cell number, and the concentration of [3H]tyrosine in the medium. From velocity vs. [3H]tyrosine concentration experiments, two apparent Km values were obtained: Km1 = 10 +/- 2 microM; Km2 = 140 +/- 10 microM. Substrate inhibition occurred with tyrosine concentrations between 20 and 50 microM. The reaction was twice as fast at pH 5.5 as at pH 7.4. alpha,alpha'-Dipyridyl (1 mM) caused major inhibition (75%) when [3H]tyrosine concentration was 10 microM. L-3-Iodotyrosine was a competitive inhibitor with Ki = 0.3 microM. Dopamine was a non-competitive inhibitor with Ki = 500 microM. 1-Norepinephrine had no effect. These results show that the hydroxylation of tyrosine by living N1E-115 cells has many of the properties of the reaction catalyzed by purified tyrosine 3-monooxygenase from normal tissue.
Richelson
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Departments of Psychiatry and Pharmacology, Mayo Foundation, Rochester, MN 55901, USA.
D000818:Animals; D001681:Biological Assay / Q000379:methods; D002452:Cell Count; D004295:Dihydroxyphenylalanine / Q000096:biosynthesis; D004298:Dopamine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation / Q000187:drug effects; D007477:Ions / Q000494:pharmacology; D051379:Mice; D009447:Neuroblastoma / Q000188:drug therapy / Q000378:metabolism*; D015203:Reproducibility of Results; D014316:Tritium; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D014446:Tyrosine 3-Monooxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism
D004791:Enzyme Inhibitors; D007477:Ions; D014316:Tritium; D014443:Tyrosine; D004295:Dihydroxyphenylalanine; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine