Tyrosine Hydroxylase Molecules Research Articles

Effects of polyamines on tyrosine hydroxylase activity in adrenals

The ability of polyamines (putrescine, spermidine, and spermine) to modify tyrosine hydroxylase (TH) activity was examined in crude or purified enzyme preparation and in adrenal tissue slices. Polyamines showed biphasic effects on TH activity in vitro at physiological pH 7.0, with an inhibitory effect at low concentrations (<1 mM) and a stimulatory effect at high concentrations. The degree of both inhibition and stimulation produced by polyamines at low and high concentrations, respectively, were proportional to the number of the amino group in the polyamines (putrescine < spermidine < spermine). The degree of inhibition by polyamines was much greater with purified enzyme than with crude enzyme preparations. Tyrosine hydroxylation in situ in adrenal tissue slices was stimulated by polyamines without inhibition at any concentrations tested. This evidence suggests that TH molecules in vivo could interact with polyamines or polyamine-like substances which inhibit the TH activity at physiological concentrations less than 1 mM.

Molecular and physiological properties of tyrosine hydroxylase induced by reserpine in rat adrenal gland

Newly synthesized tyrosine hydroxylase ( TH ) induced by reserpine was compared with the enzyme in control rats in terms of the molecular and physiological properties. When repeated doses of reserpine were given at daily intervals for three days, the enzyme activity measured in homogenates of the adrenal glands was increased 3-fold. Furthermore, when TH in the adrenal glands from both control and reserpine-treated rats was purified, both total activity of the enzyme and the enzyme protein content purified from reserpine-treated rats were also about 3-fold higher than those of the control rats. The two purified enzymes revealed similar properties; a single subunit with a Mr of 60,000 was observed by SDS polyacrylamide gel electrophoresis, and the Km value for a pterin cofactor, 6-methyl-tetrahydropterin was about 300 μM. In contrast, in situ TH activity measured under physiological conditions at pH 7.2 in adrenal tissue slices was elevated 6-fold by reserpine pretreatment for 3 days, and was stimulated by carbachol (0.1 mM) and elevated K +(52 mM) in a roughly proportional rather than additive way relative to slices from untreated rats. These results indicate that newly synthesized TH induced by reserpine in rat adrenal gland had similar properties as the enzyme in control rats and that reserpine increased not only the amount of TH molecules but also the in situ activity of TH. Since reserpine also increases the biosynthesis of tetrahydrobiopterin as demonstrated by Viveros and co-workers, this 6-fold increase in in situ TH activity may depend both upon the 3-fold increase in the amount of enzyme molecules and upon the increase of the physiologically available tetrahydrobiopterin in the adrenal gland.

Monoclonal antibodies to tyrosine hydroxylase from rat pheochromocytoma PC12h cells with special reference to nerve growth factor-mediated increase of the immunoprecipitable enzymes

Twelve hybridomas, which secrete the monoclonal antibodies to rat pheochromocytoma tyrosine hydroxylase (TH), were obtained. All of these antibodies immunoprecipitated the TH molecules from rat pheochromocytoma PC12h cells, adrenal medulla and brain. Two antibodies, namely PCTH-3 and -7 (both IgG1), directly inhibited the catalytic activity of TH. Another antibody, PCTH-4 (IgG1), bound to the enzyme without inhibition of the catalytic activity. The antibodies PCTH-3 and -4 immunostained the 60 K bands on a nitrocellulose sheet, which were electrotransfered from the gel after the SDS-polyacrylamide gel electrophoresis of PC12h cell and rat adrenal medulla homogenates. The antibody PCTH-4 immunocytochemically labeled specific neurons in the locus ceruleus, hypothalamus and substantia nigra of rat brain. Immunotitration using PCTH-4 antibody revealed that the nerve growth factor-mediated increase of TH activity in PC12h cells is due to the increase of the enzyme molecules.

Cell contact-mediated regulation of tyrosine hydroxylase synthesis in cultured bovine adrenal chromaffin cells.

The specific activity of tyrosine hydroxylase (TH) in bovine adrenal chromaffin cells can be controlled by changing cell density. Chromaffin cells initially plated at low density (2-3 X 10(4) cells/cm2), and subsequently replated at a 10-fold higher density showed a sixfold increase in specific TH activity within 48 h, resulting from enhanced synthesis (increased number of TH molecules as demonstrated by immunotitration and blockade by cycloheximide) rather than activation. The density-mediated TH induction was blocked by inhibitors of both messenger RNA synthesis (alpha-amanitin) and processing (9-beta-arabinofuranosyladenine), indicating a transcriptional level of regulation. Medium conditioned by high density replated cells could not mimic the effect of high density plating itself, thus direct cell contact, rather than a diffusible factor, is responsible for the density-mediated TH induction. Since neither acetylcholinesterase nor lactate dehydrogenase specific activities were increased by high cell density, it can be concluded that the contact-mediated induction of TH is rather specific, and not the result of a general process of enzyme induction.

Tyrosine hydroxylase activation: Comparison of in vitro phosphorylation and in vivo administration of haloperidol

Rat striatal tyrosine hydroxylase (TH) was assayed 2 hr following treatment with 1 mg/kg haloperidol. TH activity in striata from haloperidol-treated rats (haloperidol TH) was increased significantly relative to control when assayed at pH 7.0, but not at pH 6.0, in the presence of 175 μM tetrahydrobiopterin (BH 4). TH was also phosphorylated in vitro, catalyzed by sufficient quantities of the catalytic subunit of bovine heart protein kinase to cause greater than 90% activation after 10 min. TH was activated by phosphorylation at both pH 6.0 and pH 7.0, but the activation was greater pH 7.0 Haloperidol TH, activated relative to control TH at pH 7.0, was activated by phosphorylation, but there was no difference between haloperidol TH and control TH activity at either pH 6.0 or 7.0 following phosphorylation. Comparison of Lineweaver-Burk plots of nonphosphorylated and phosphorylated TH indicated that activation by phosphorylation was due to a 5-fold change in K m for BH 4 and a 2-fold change in V max at pH 7.0. Haloperidol TH kinetics were intermediate between those of nonphosphorylated and phosphorylated TH at pH 7.0. Analysis by Lineweaver-Burk, Hanes-Woolf, and Eadie-Scatchard plots suggested that the haloperidol TH kinetic data were the result of a mixture of two forms of the enzyme, with different affinities for cofactor. Theoretical calculations of TH activity of mixtures of nonphosphorylated and phosphorylated TH suggested that the haloperidol data could be explained by postulating a mixture of 25–35% phosphorylated TH molecules with 35–75% nonphosphorylaed TH molecules. An hypothesis of the role of TH phosphorylation during conditions of increased neuronal firing rate, such as may occur with haloperidol treatment, is presented.