Forms Of Phenylalanine Hydroxylase Research Articles

Degradation rates differ between mutant and wild-type forms of phenylalanine hydroxylase expressed in vitro.

Degradation rates differ between mutant and wild-type forms of phenylalanine hydroxylase expressed in vitro.

Two apparent molecular weight forms of human and monkey phenylalanine hydroxylase are due to phosphorylation.

Two-dimensional polyacrylamide gel analyses of purified human and monkey liver phenylalanine hydroxylase reveal that the enzyme consists of two different apparent molecular weight forms of polypeptide, designated H (Mr = 50,000) and L (Mr = 49,000), each containing three isoelectric forms. The two apparent molecular weight forms, H and L, represent the phosphorylated and dephosphorylated forms of phenylalanine hydroxylase, respectively. After incubation of purified human and monkey liver enzyme with purified cAMP-dependent protein kinase and [gamma-32P]ATP, only the H forms contained 32P. Treatment with alkaline phosphatase converted the phenylalanine hydroxylase H forms to the L forms. The L forms but not the H forms could be phosphorylated on nitrocellulose paper after electrophoretic transfer from two-dimensional gels. Phosphorylation and dephosphorylation of human liver phenylalanine hydroxylase is not accompanied by significant changes in tetrahydrobiopterin-dependent enzyme activity. Peptide mapping and acid hydrolysis confirm that the apparent molecular weight heterogeneity (and charge shift to a more acidic pI) in human and monkey liver enzyme results from phosphorylation of a single serine residue. However, phosphorylation by the catalytic subunit of cAMP-dependent protein kinase does not account for the multiple charge heterogeneity of human and monkey liver phenylalanine hydroxylase.

The two molecular weight forms of rat phenylalanine hydroxylase are encoded by different messenger RNAs.

Immunoprecipitation of the phenylalanine hydroxylase formed by translation of rat liver RNA in a rabbit reticulocyte cell-free protein synthesis system was used to examine the origin of the molecular weight heterogeneity of the enzyme. Sodium dodecyl sulfate-polyacrylamide electrophoresis of the immunoprecipitated products showed that in most cases a single specifically immunoprecipitated polypeptide was produced which corresponded to the higher molecular weight (H) form of phenylalanine hydroxylase (Mr = 50,000). The identity of the product was confirmed by immunological competition and peptide mapping. RNA from other rats, however, coded for both the H-form and the lower molecular weight (L) form of phenylalanine hydroxylase or for only the L-form. The evidence suggests that the L-form derives from a different mRNA, rather than by proteolysis of the H-form, an interpretation which is supported by the isolation of the lower form of phenylalanine hydroxylase from livers of some rats.

Hydrocortisone induction of phenylalanine hydroxylase isozymes in cultured hepatoma cells

The hydrocortisone stimulation of phenylalanine hydroxylase activity in Reuber H4 hepatoma cells is shown to be associated with an alteration in phenylalanine hydroxylase isozyme composition. Three forms of phenylalanine hydroxylase were identified in H4 cells which have been treated with hydrocortisone; however, only one of these forms appears to be present prior to glucocorticoid treatment. The relative amounts, as well as the total amount, of the three forms and their chromatographic behavior on hydroxylapatite are nearly identical to the three phenylalanine hydroxylase isozymes found in adult rat liver. The hydroxylase isozyme composition in 2 day old rats is similar to that found in adult rats and in H4 cells treated with hydrocortisone.

Demonstration of two forms of phenylalanine hydroxylase in human liver obtained at autopsy

1. 1. Phenylalanine hydroxylase ( l-phenylalanine tetrahydropteridine: O 2 oxidoreductase (4-hydroxylating), EC 1.14.3.1) has been demonstrated to be present in human liver obtained at autopsy. 2. 2. The enzyme is shown to be present in 2 forms: (a) a ‘soluble’ form, which remains in the 6000 × g supernatant; (b) a ‘particulate’ form which sediments at 6000 × g. 3. 3. The particulate form has been solubilised and its properties suggest that it is bound to a membrane via a lipid linkage. 4. 4. Some properties of the two forms have been studied and these suggest that they are closely related. 5. 5. It should now be possible to use human livers obtained at autopsy as a source of phenylalanine hydroxylase for purification and study.