Tyrosine Hydroxylase in “Leaky” Adrenal Medullary Cells: Evidence for In Situ Phosphorylation by Separate Ca2+ and Cyclic AMP‐Dependent Systems

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Abstract
The systems responsible for phosphorylating tyrosine hydroxylase, the rate-limiting enzyme or catecholamine biosynthesis, were investigated in situ in adrenal medullary cells made permeable to solutes of up to 1000 dalton by exposure to brief intense electric fields. Two different phosphorylation systems were found. One is dependent on Ca2+, the other on cAMP. The Ca2+-dependent system is half-maximally activated by 1-2 .mu.M Ca2+ and 0.5 mM ATP and follows a time course similar to that of secretion of catecholamines. Trifluoperazine (0.1 mM) does not inhibit significantly Ca2+-dependent phosphorylation of tyrosine hydroxylase in situ. The cAMP-dependent system is half-maximally activated by addition of 0.5 .mu.M cAMP and .apprx. 0.3 mM ATP. Ca2+-dependent and cAMP-dependent phosphorylations of tyrosine hydroxylase have roughly the same time course and are additive under conditions where 1 system is already saturated. Peptide maps of immunoprecipitated tyrosine hydroxylase, after in situ phosphorylation of the enzyme either in the presence of 10-8 M Ca2+ plus 2 .times. 10-5 M cAMP or of 10-5 M Ca2+, show a marked difference indicating that the enzyme contains several phosphorylation sites. At least 1 of these sites is phosphorylated only by the Ca2+-dependent system, whereas the other site(s) are phosphorylated by both the Ca2+- and cAMP-dependent systems. The effect of in situ phosphorylation of tyrosine hydroxylase on its enzymatic activity was also investigated.

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