The rate-limiting enzyme in catecholamine synthesis is tyrosine hydroxylase. It is phosphorylated at serine (Ser) residues Ser8, Ser19, Ser31 and Ser40 in vitro, in situ and in vivo. A range of protein kinases and protein phosphatases are able to phosphorylate or dephosphorylate these sites in vitro. Some of these enzymes are able to regulate tyrosine hydroxylase phosphorylation in situ and in vivo but the identity of the kinases and phosphatases is incomplete, especially for physiologically relevant stimuli. The stoichiometry of tyrosine hydroxylase phosphorylation in situ and in vivo is low. The phosphorylation of tyrosine hydroxylase at Ser40 increases the enzyme's activity in vitro, in situ and in vivo. Phosphorylation at Ser31 also increases the activity but to a much lesser extent than for Ser40 phosphorylation. The phosphorylation of tyrosine hydroxylase at Ser19 or Ser8 has no direct effect on tyrosine hydroxylase activity. Hierarchical phosphorylation of tyrosine hydroxylase occurs both in vitro and in situ, whereby the phosphorylation at Ser19 increases the rate of Ser40 phosphorylation leading to an increase in enzyme activity. Hierarchical phosphorylation depends on the state of the substrate providing a novel form of control of tyrosine hydroxylase activation. Keywords: activity, catecholamine synthesis, phosphorylation, protein kinases, protein phosphatases, tyrosine hydroxylase. The catecholamines (CAs) dopamine (DA), noradrenaline and adrenaline are physiologically important neurotransmitters and hormones. It has been established that when the CAs are secreted there is generally no decrease in their levels within tissues (Zigmond et al. 1989). This is because there is a concomitant increase in the rate of CA synthesis that is closely coupled to secretion. Tyrosine hydroxylase (TH; tyrosine 3-monooxygenase; E.C. 1.14.16.2) is the first and rate-limiting enzyme in CA synthesis and it catalyses the hydroxylation of L-tyrosine to DOPA. The activity of TH can be modulated by two mechanisms: medium-to long-term regulation of gene expression (enzyme stability, transcriptional regulation, RNA stability, alternative RNA splicing and translational regulation) and short-term regulation of enzyme activity (feedback inhibition, allosteric regulation and phosphorylation) (Kumer and Vrana 1996). TH activation by phosphorylation is the primary mechanism responsible for the maintenance of CA levels in tissues after CA secretion. TH can be phosphorylated at serine residues (Ser) 8, 19, 31 and 40 by a variety of protein kinases. The regulation of the phosphorylation of these sites and the consequences in terms of TH activity in vitro have been extensively investigated. In situ and in vivo it is less clear which protein kinase(s), and/or phosphatase(s), modulates TH phosphorylation and which mechanism(s) leads to the activation of TH and CA synthesis. The major focus of this review is therefore the regulation and consequences of the (Kaufman 1995;Nagatsu 1995;Kappock and Caradonna 1996;Kumer and Vrana 1996;...

Homogenization of fresh brain tissue in isotonic medium shears plasma membranes causing nerve terminals to become separated from their axons and postsynaptic connections. The nerve terminal membranes then reseal to form synaptosomes. The discontinuous Percoll gradient procedure described here is designed to isolate synaptosomes from brain homogenates in the minimum time to allow functional experiments to be performed. Synaptosomes are isolated using a medium-speed centrifuge, while maintaining isotonic conditions and minimizing mechanically damaging resuspension steps. This protocol has advantages over other procedures in terms of speed and by producing relatively homogeneous synaptosomes, minimizing the presence of synaptic and glial plasma membranes and extrasynaptosomal mitochondria. The purified synaptosomes are viable and take up and release neurotransmitters very efficiently. A typical yield of synaptosomes is between 2.5 and 4 mg of synaptosomal protein per gram rat brain. The procedure takes approximately 1 h from homogenization of the brain until collection of the synaptosomal suspension from the Percoll gradient.

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