Abstract
Tyrosine hydroxylase (TH) was purified from pheochromocytoma PC-12 cloned cells by a short and gentle procedure. The enzyme was isolated in pure form and has a molecular weight of approximately 210,000-220,000. SDS electrophoresis yields one single protein band with a molecular weight of approximately 62,000. Antiserum to rat pheochromocytoma TH was obtained in rabbits and immunotitration data show that the antiserum to rat TH reduces the activity of the homologous enzyme more effectively than the activity of the heterologous enzyme. The activity of purified TH is stimulated by a cAMP-dependent protein kinase phosphorylating system (PKP system), and the highest percentage of stimulation is obtained when the enzyme activity is measured at physiological pH’s. The stimulation of the purified enzyme by the PKP system results in a reduction of the apparent Km for the cofactor 6-MePH4 and in an increase of the Ki for dopamine. Incubation of purified TH with the PKP system and [32P]ATP, resulted in incorporation of radioactivity into the 62,000 subunit of the enzyme.
ACKNOWLEDGMENTS The authors wish to thank Drs. P. Greengard, D. Aswad, and L. DeGenarro from Yale University Medical School, Department of Pharmacology, for their advice on the purification of protein kinase and phosphorylation reaction.
- Copyright © 1980 by The American Society for Pharmacology and Experimental Therapeutics
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Purification and Characterization of Tyrosine Hydroxylase from a Clonal Pheochromocytoma Cell Line
