Purification and some properties of tyrosine 3-monooxygenase from rat adrenal.
Tyrosine 3-monooxygenase was purified to homogeneity, as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, from rat adrenal. The specific activity of the final preparation was approximately 1,600 nmol min-1 mg protein-1, which was much higher than the highest yet reported. The enzyme was markedly stabilized in the presence of glycerol, Tween 80 and EDTA. As judged by gel filtration on Ultrogel AcA 34, sodium dodecyl sulfate/polyacrylamide gel electrophoresis and cross-linking studies, the enzyme appeared to be composed of four identical subunits, each possessing a molecular weight of 59,000. The isoelectric point of the enzyme was estimated to be 6.7 in the presence of 8 M urea and 6.6 in its absence. Amino acid analysis of the enzyme revealed a fairly high content of serine residues in this protein. Purification of the enzyme caused changes in the kinetic properties of the enzyme. The Km for 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine decreased from 220 microM to 58 microM. The pH profile for the enzyme activity became more broad and the pH optimum was changed from an acid pH to a neutral pH. Although polyanions, such as heparin and dextran sulfate, markedly stimulated the activity of crude enzyme by increasing the V, they were much less effective in the activation of purified enzyme. A marked stimulation of the enzyme activity by phospholipids, such as phosphatidylserine, phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine, were not observed in both pure and crude preparations even at low concentrations of the pterin cofactor.
D000313:Adrenal Medulla / Q000201:enzymology*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography; D002850:Chromatography, Gel; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D010766:Phosphorylation; D051381:Rats; D014446:Tyrosine 3-Monooxygenase / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D014446:Tyrosine 3-Monooxygenase