TYROSINE-HYDROXYLASE ACTIVITY AND EXTRINSIC FLUORESCENCE CHANGES PRODUCED BY POLYANIONS

The activity of tyrosine hydroxylase in vitro is affected by many fact ors, including pH, phosphorylation by several protein kinases, and pol yanions. We investigated the activation of tyrosine hydroxylase by RNA or DNA (polyanions), using purified rat PC12 cell enzyme. RNA and DNA each increased tyrosine hydroxylase activity in the presence of subsa turating (125 muM) tetrahydrobiopterin at pH 6. RNA increased enzyme a ctivity up to 6-fold with an EC50 of 3 mug/ml. RNA and DNA each increa sed tyrosine hydroxylase activity by decreasing the K(m) of the enzyme for tetrahydrobiopterin from 3 mM to 295 muM in the presence of 100 m ug/ml RNA or 171 muM in the presence of 100 mug/ml DNA. We used the ap olar fluorescent probe 8-anilino-1-naphthalenesulphonic acid (1,8-ANS) as a reporter group to provide the first evidence for changes in conf ormation related to changes in activity. At pH 6.0, 1.8-ANS bound to t yrosine hydroxylase and exhibited a characteristic fluorescence spectr um. At pH 7.2, both enzyme activity and fluorescence decreased. DNA or heparin (another polyanion) activated tyrosine hydroxylase and decrea sed fluorescence of the reporter group 30% at pH 6.0. This decrease su ggests that these polyanions altered the conformation of tyrosine hydr oxylase. The activating effects of polyanions were diminished at physi ological pH (6.8-7.2) or in the presence of bivalent-cation salts (10 mM) or univalent-cation salts (100 mM). These results suggest that pol yanions play a minimal role, if any, in the physiological regulation o f tyrosine hydroxylase activity.