title: The use of the natural cofactor, (6R)-l-erythrotetrahydrobiopterin in the analysis of nonphosphorylated and phosphorylated rat striatal tyrosine hydroxylase at pH 7.0.
abstract: The kinetics of tyrosine hydroxylase from the desalted high-speed supernatants of rat striatal homogenates were examined at pH 7.0 using different concentrations of the natural cofactor, (6R)-l-erythrotetrahydrobiopterin, ranging from 4 ?M to 1.5 mM. All analyses were performed using two different buffering solutions and their appropriate reducing systems for maintaining cofactor in the reduced state. In the presence of phosphate buffer the results show that tyrosine hydroxylase exists in two kinetically different forms with apparent K(m) values for the cofactor of 16 ?M (low K(m)) and 2.3 mM (high K(m)). Similar results were obtained using MOPS buffer. A comparative analysis of the appropriate V(max) values indicates that tyrosine hydroxylase as obtained by our standard preparation procedures is predominately (95%) in the high K(m) form. When the striatal supernatant was exposed to phosphorylating conditions and subsequently analyzed it appeared that the enzyme now existed totally in the low K(m) form with very little change in the overall V(max). A comparison of the results using the two different buffering systems, phosphate and MOPS, revealed that following phosphorylation a large percentage of enzyme was maintained in the phosphorylated state only when using phosphate buffer. In light of the present results, we can for the first time suggest a functional significance not only for the two apparently different kinetic forms of the enzyme but also for a supporting role for phosphorylation. In vivo dopamine synthesis may be accomplished to a significant extent by the phosphorylated form of the enzyme while the non-phosphorylated form may constitute a relatively inactive reservoir which can be recruited for increased dopamine synthesis by phosphorylation.
authors.affiliation: Section on Biochemical Pharmacology, National Institutes of Health, NHLBI, Bethesda, MD 20205, U.S.A.
authors.forename: L P
authors.initials: LP
authors.lastname: Miller
authors.forename: W
authors.initials: W
authors.lastname: Lovenberg