Tyrosine hydroxylase purification from rat PC12 cells.
Tyrosine hydroxylase was purified in high yield from rat PC12 cells. This three-day procedure consisted of differential ammonium sulfate precipitation, anion-exchange chromatography, and heparin-Sepharose affinity chromatography. It yielded an average of 15 mg of purified protein from 100 flasks of PC12 cells, with greater than 40% recovery of tyrosine hydroxylase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single protein band with a molecular weight of approximately 60,000. The protein had a specific activity of 670 nmol/min/mg and had a Km for its reducing cofactor tetrahydrobiopterin of 1.8 mM. The purified protein can be phosphorylated and activated by cyclic AMP-dependent protein kinase.
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70119.
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Gahn
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Roskoski
D000645:Ammonium Sulfate; D000818:Animals; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D007700:Kinetics; D008970:Molecular Weight; D010766:Phosphorylation; D051381:Rats; D014407:Tumor Cells, Cultured / Q000201:enzymology; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D014446:Tyrosine 3-Monooxygenase; D000645:Ammonium Sulfate