Tyrosine hydroxylase activity and extrinsic fluorescence changes produced by polyanions.
The activity of tyrosine hydroxylase in vitro is affected by many factors, including pH, phosphorylation by several protein kinases, and polyanions. 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 subsaturating (125 microM) tetrahydrobiopterin at pH 6. RNA increased enzyme activity up to 6-fold with an EC50 of 3 micrograms/ml. RNA and DNA each increased tyrosine hydroxylase activity by decreasing the Km of the enzyme for tetrahydrobiopterin from 3 mM to 295 microM in the presence of 100 micrograms/ml RNA or 171 microM in the presence of 100 micrograms/ml DNA. We used the apolar fluorescent probe 8-anilino-1-naphthalenesulphonic acid (1,8-ANS) as a reporter group to provide the first evidence for changes in conformation related to changes in activity. At pH 6.0, 1,8-ANS bound to tyrosine hydroxylase and exhibited a characteristic fluorescence spectrum. At pH 7.2, both enzyme activity and fluorescence decreased. DNA or heparin (another polyanion) activated tyrosine hydroxylase and decreased fluorescence of the reporter group 30% at pH 6.0. This decrease suggests that these polyanions altered the conformation of tyrosine hydroxylase. The activating effects of polyanions were diminished at physiological pH (6.8-7.2) or in the presence of bivalent-cation salts (10 mM) or univalentcation salts (100 mM). These results suggest that polyanions play a minimal role, if any, in the physiological regulation of tyrosine hydroxylase activity.
Gahn
L G
LG
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70119.
Roskoski
R
R
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004247:DNA / Q000494:pharmacology; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D016716:PC12 Cells / Q000201:enzymology; D000071228:Polyelectrolytes; D011108:Polymers / Q000494:pharmacology*; D011487:Protein Conformation; D012313:RNA / Q000494:pharmacology; D051381:Rats; D012492:Salts / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014446:Tyrosine 3-Monooxygenase / Q000187:drug effects / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D001708:Biopterins; D017868:Cyclic AMP-Dependent Protein Kinases; D004247:DNA; D005456:Fluorescent Dyes; D006493:Heparin; D000071228:Polyelectrolytes; D011108:Polymers; D012313:RNA; D012492:Salts; D014446:Tyrosine 3-Monooxygenase; C027132:1-anilino-8-naphthalenesulfonate; C003402:sapropterin