Purification and state of activation of rat kidney phenylalanine hydroxylase.
Phenylalanine hydroxylase, the enzyme that catalyzes the irreversible hydroxylation of phenylalanine to tyrosine, was purified from rat kidney with the use of phenyl-Sepharose, DEAE-Sephacel, and gel permeation high pressure liquid chromatography. Our most highly purified fractions had a specific activity in the presence of 6-methyltetrahydropterin, of 1.5 mumol of tyrosine formed/min/mg of protein, which is higher than has been reported hitherto. For the rat kidney enzyme, the ratio of specific activity in the presence of 6-methyltetrahydropterin to the specific activity in the presence of tetrahydrobiopterin (BH4) is 5. By contrast, this ratio for the unactivated rat liver hydroxylase is 80. These results indicate that the kidney enzyme is in a highly activated state. The rat kidney hydroxylase could not be further activated by any of the methods that stimulate the BH4-dependent activity of the rat liver enzyme. In addition, the kidney enzyme binds to phenyl-Sepharose without prior activation with phenylalanine. The phenylalanine saturation pattern with BH4 as a cofactor is hyperbolic with substrate inhibition at greater than 0.5 mM phenylalanine, a pattern that is characteristic of the activated liver hydroxylase. The molecular weight of the rat kidney enzyme as determined by gel permeation chromatography is 110,000, suggesting that the enzyme might be an activated dimer. We conclude, therefore, that phenylalanine hydroxylases from rat kidney and liver are in different states of activation and may be regulated in different ways.
D000818:Animals; D004789:Enzyme Activation; D005934:Glucagon / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D010651:Phenylalanine Hydroxylase / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats
D005934:Glucagon; D010651:Phenylalanine Hydroxylase