A new activator protein that activates tryptophan 5-monooxygenase and tyrosine 3-monooxygenase in the presence of Ca2+-, calmodulin-dependent protein kinase. Purification and characterization.
Rat brain tryptophan 5-monooxygenase was activated by incubation with ATP, Mg2+, calmodulin, and micromolar concentrations of Ca2+. The activating activity was resolved into two distinct peaks upon gel filtration on Sepharose CL-6B: one, Ca2+-, calmodulin-dependent protein kinase, and the other, a heat-labile activator protein. The activator protein was purified to apparent homogeneity from rat brain by a procedure involving calmodulin-Sepharose 4B, Sephadex G-150, and phenyl-Sepharose CL-4B column chromatography. The molecular weight of the activator protein was determined to be 70,000 by sedimentation equilibrium and by gel filtration on Sephadex G-150. The protein gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of which was estimated to be 35,000, indicating that the protein might be composed of two identical subunits. Analysis of cross-linked activator protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis also suggested that the protein might be a dimer of identical subunits. Some other molecular properties of the activator protein were: sedimentation coefficient, 4.3 S; Stokes radius, 3.6 nm; diffusion coefficient, 6.0 x 10(-7) cm2/s; frictional ratio, 1.32; and partial specific volume, 0.73 cm3/g. The activator protein activated tyrosine 5-monooxygenase as well as tryptophan 5-monooxygenase in the presence of ATP, Mg2+, Ca2+, calmodulin, and Ca2+-, calmodulin-dependent protein kinase.
D000313:Adrenal Medulla / Q000201:enzymology; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001933:Brain Stem / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D002417:Cattle; D002540:Cerebral Cortex / Q000378:metabolism*; D004789:Enzyme Activation; D046911:Macromolecular Substances; D008970:Molecular Weight; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D014365:Tryptophan Hydroxylase / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D000596:Amino Acids; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D046911:Macromolecular Substances; D014446:Tyrosine 3-Monooxygenase; D014365:Tryptophan Hydroxylase; D011494:Protein Kinases; D002118:Calcium