Purification and characterization of rat striatal tyrosine hydroxylase. Comparison of the activation by cyclic AMP-dependent phosphorylation and by other effectors.
Purification of rat striatal tyrosine hydroxylase in the presence of protease inhibitors effected a high yield of apparently homogeneous enzyme which is stable to prolonged storage. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 61,300. Removal of protease inhibitors results in the appearance of additional bands with molecular weights of 59,800 and 57,000. Cyclic AMP-dependent protein kinase incorporates 1 mol of phosphate/61,000-Da subunit of tyrosine hydroxylase, and concomitantly decreases the apparent Km of the enzyme for cofactor. Phosphorylated tyrosine hydroxylase is unstable at 37 degrees C, exhibiting a 50% decrease in apparent Vmax in 40 min with no change in Km for cofactor. Levels of incorporated phosphate remain constant over this time period. Tyrosine hydroxylase activated by and in the presence of phosphatidylinositol or high concentrations of NaCl exhibited a similar loss of activity at 37 degrees C, whereas enzyme activated by heparin was relatively stable. The rate of phosphorylation of tyrosine hydroxylase is markedly increased in the presence of any of these effectors, suggesting that they promote a common conformation. Further, heparin appears to bind to tyrosine hydroxylase at a site distant from the phosphorylation site. Physiological effectors of tyrosine hydroxylase may act in concert with cyclic AMP-dependent phosphorylation, perhaps by binding to an allosteric site, to regulate enzyme activity in vivo.
Richtand
N M
NM
Inagami
T
T
Misono
K
K
Kuczenski
R
R
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003342:Corpus Striatum / Q000201:enzymology*; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D014446:Tyrosine 3-Monooxygenase / Q000302:isolation & purification*
D000596:Amino Acids; D000242:Cyclic AMP; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases