Interaction of tyrosine hydroxylase with ribonucleic acid and purification with DNA-cellulose or poly(A)-sepharose affinity chromatography.
Tyrosine hydroxylase in bovine adrenal medulla was activated up to fourfold by incubation with low concentrations (15 micrograms/ml) of ribonucleic acids. At higher RNA concentrations, enzyme activity was inhibited. This interaction with RNA was exploited with the use of poly(A)-Sepharose and DNA-cellulose to effect a rapid purification of stable tyrosine hydroxylase from rat brain and bovine adrenal medulla in high yield (up to 58%). With the purified rat brain enzyme, RNA acted as an uncompetitive inhibitor, a concentration of 15 micrograms/ml lowering the Vmax of tyrosine hydroxylase from 1050 to 569 nmol min-1 mg-1 and lowering the Km for tyrosine from 6.1 to 3.6 microM. With the natural cofactor, tetrahydrobiopterin (BH4), two Km values were obtained, indicating the presence of two forms of the enzyme. Both Km values were decreased only slightly by RNA. The purified brain and adrenal enzymes both contained about 0.07 mol of phosphate/63,000-Da subunit; in both cases, cyclic AMP-dependent protein kinase catalyzed the incorporation of an additional 0.8 mol of phosphate/subunit. The purified enzyme also contains ribonucleic acid, which comprises about 10% of the total mass and appears to be important for full activity.
D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002482:Cellulose / Q000031:analogs & derivatives; D002846:Chromatography, Affinity; D004247:DNA / Q000031:analogs & derivatives; D004996:Ethidium / Q000494:pharmacology; D005453:Fluorescence; D006493:Heparin / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000494:pharmacology; D012313:RNA / Q000032:analysis / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D012118:Resorcinols; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification*
C033190:DNA-cellulose; D012118:Resorcinols; C005282:orcinol; D012313:RNA; D002482:Cellulose; D006493:Heparin; D004247:DNA; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; D004996:Ethidium