Purification of p-hydroxyphenylpyruvate hydroxylase from rat liver--requirement for cofactors.
The enzyme p-hydroxyphenylpyruvate hydroxylase (EC 1.13.11.27) from rat liver was studied with the assay method which measures the release of 14CO2 from p-hydroxyphenyl [carboxy-14C]pyruvate. Extensive dialysis of the crude enzyme extract against Tris buffer or purification involving ammonium sulfate, gel filtration, and ion exchange results in loss of enzyme activity that can be reactivated by Fe2+, dichlorophenolindophenol, and various other agents. The effect of these activators depends critically on their final concentration in the assay media. A 70-fold purification of the enzyme fraction yielded a preparation which behaved as a single protein band in Sephadex G-150. It had an isoelectric point at 5.85 and molecular weight of 63 000. The enzyme obtained appears to be different in some respects from those described by other workers from the liver of dog, human, chicken, and frog.
D015086:2,6-Dichloroindophenol / Q000494:pharmacology; D015111:4-Hydroxyphenylpyruvate Dioxygenase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002374:Catalase / Q000494:pharmacology; D002413:Cations, Divalent; D002614:Chelating Agents / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000494:pharmacology; D007501:Iron / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008297:Male; D008628:Mercury / Q000494:pharmacology; D008970:Molecular Weight; D010105:Oxygenases / Q000378:metabolism*; D051381:Rats
D002413:Cations, Divalent; D002614:Chelating Agents; D003300:Copper; D015086:2,6-Dichloroindophenol; D007501:Iron; D002374:Catalase; D010105:Oxygenases; D015111:4-Hydroxyphenylpyruvate Dioxygenase; D008628:Mercury; D005978:Glutathione; D001205:Ascorbic Acid