Kinetic properties of brain tyrosine hydroxylase and its partial purification by affinity chromatography

Abstract

Summary The tyrosine hydroxylase activity of sheep brain caudate nuclei homogenates was solubilized by treatment with 0.2% Triton X-100 and concentrated by ultra-filtration. Although the enzyme shows an absolute requirement for a reduced tetrahydropteridine (DMPH 4 ) as cofactor, concentrations > 2 mM are inhibitory. The maximal reaction velocity is stimulated two-fold by the inclusion of 0.5 mM Fe 2+ in the assay. However, the apparent Michaelis constants, K m , for L-tyrosine and DMPH 4 are not affected by Fe 2+ and values of 0.1 and 0.33 mM were obtained for these parameters. A five-fold purification of the enzyme concentrate was achieved by affinity chromatography on iodotyrosine-substituted agarose. Active eluate fractions were distinctly turbid, suggesting that intermolecular aggregation of the tyrosine hydroxylase protein occurs as a concomitant of the purification procedure.