Properties of tyrosine hydroxylation in living mouse neuroblastoma clone N1E-115 |
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Authors: | Richelson E |
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Institution: | Departments of Psychiatry and Pharmacology, Mayo Foundation, Rochester, MN 55901, USA. |
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Abstract: | 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. |
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