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PROPERTIES OF TYROSINE HYDROXYLATION IN LIVING MOUSE NEUROBLASTOMA CLONE N1E‐115
Author(s) -
Richelson E.
Publication year - 1976
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1976.tb00317.x
Subject(s) - clone (java method) , hydroxylation , tyrosine , neuroblastoma , biology , chemistry , genetics , biochemistry , gene , enzyme , cell culture
— 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 [ 3 H]OH formed from L‐[3.5‐ 3 H]tyrosine in the medium was the method of assay and [ 3 H]OH formed was stoichiometric with the formation of L‐[ 3 H]3.4‐dihydroxyphenylalanine. Tyrosine hydroxylation was dependent on time of incubation, cell number, and the concentration of [ 3 H]tyrosine in the medium. From velocity vs. [ 3 H]tyrosine concentration experiments. two apparent K m , values were obtained: K m1 = 10 ± 2μM: K m2 = 140 ± 10μM. Substrate inhibition occurred with tyrosine concentrations between 20 and 50 μM. The reaction was twice as fast at pH 5.5 as at pH 7.4. α,α′‐Dipyridyl (1 mM) caused major inhibition (75%) when [ 3 H]tyrosine concentration was 10 μM. L‐3‐Iodotyrosine was a competitive inhibitor with K i = 0.3 μM. Dopamine was a non‐competitive inhibitor with K i = 500 μM. 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.