Purification and Characterization of Phenylalanine Hydroxylase-stimulating Protein from Rat Liver

Phenylalanine hydroxylase-stimulating protein markedly stimulates phenylalanine hydroxylase activity under various conditions and thus may serve a critical function in phenylalanine metabolism. At pH values above neutrality, or in the presence of phospholipids such as lysolecithin, when the naturally occurring cofactor, tetrahydrobiopterin, is used, the specific activity of phenylalanine hydroxylase decreases with increasing enzyme concentration. The stimulating effect of phenylalanine hydroxylase-stimulating protein is due to the relief of this decrease in specific activity. The stimulating protein has now been purified from rat liver more than 1000-fold over the crude extract. Electrophoresis of the stimulating protein on polyacrylamide gel reveals two main bands, both of which are active as stimulator and are identical in molecular weight. The molecular weight of this protein has been determined by light scattering, sedimentation-diffusion, and electrophoresis on different percentage acrylamide gels to be 51,500 ± 1,200. The 51,500 molecular weight form consists of four subunits of molecular weight 12,500 ± 400 as determined by gel electrophoresis in 0.1% sodium dodecyl sulfate or in 8 m urea. The extinction coefficient at 280 nm and the amino acid composition of the stimulating protein have also been determined. On storage, higher aggregates of the stimulating protein are formed, which appear to be due to the formation of intermolecular disulfide linkages. Assays of crude extracts from normal and phenylketonuric human livers show that the stimulating protein is present at comparable levels in both livers. There is no species specificity with respect to the stimulation of phenylalanine hydroxylase activity by the stimulating protein; the rat liver hydroxylase can be stimulated by human liver stimulating protein, and the human liver hydroxylase by rat liver stimulating protein. Evidence presented in a subsequent report (Huang, C. Y., and Kaufman, S. (1973) J. Biol. Chem. 248, 4242) indicates that the stimulating protein is an enzyme which catalyzes the conversion of an intermediate to product (or products) in the hydroxylation reaction.