Phenylalanine-induced phosphorylation and activation of rat hepatic phenylalanine hydroxylase in vivo.
Rats were given intraperitoneal injections of 2 mCi of carrier-free 32Pi and substances known to activate liver phenylalanine hydroxylase. After 30 min, these animals were anesthetized and their livers removed for analysis of enzyme activity, 32Pi incorporation into immunoprecipitated phenylalanine hydroxylase and [gamma-32P]ATP specific activity. Following glucagon treatment, rat liver phenylalanine hydroxylase activity was stimulated more than 6-fold when assayed in the presence of the natural cofactor, tetrahydrobiopterin (BH4). Glucagon injection also resulted in an incorporation of 0.41 mol of 32Pi/mol of hydroxylase subunit (approximately 50,000 Da). In vivo stimulation of phenylalanine hydroxylase activity and 32Pi incorporation by glucagon had been previously observed in this laboratory (Donlon, J., and Kaufman, S. (1978) J. Biol. Chem. 253, 6657-6659). However, we show for the first time in the present study that in vivo treatment with phenylalanine alone results in a 4-fold increase in the BH4-dependent activity of phenylalanine hydroxylase concomitant with a significant incorporation of phosphate into phenylalanine hydroxylase (0.51 mol of 32Pi/mol of hydroxylase subunit). It is further demonstrated in vivo that the combined treatment with phenylalanine and glucagon results in a greater than 10-fold stimulation of BH4-dependent activity and the greatest level of 32Pi incorporation (0.75 mol of 32Pi/mol of hydroxylase subunit). Phenylalanine did not produce an elevation in plasma glucagon in these animals. A model is, thereby, proposed with respect to the ligand binding effects of phenylalanine on the state of phosphorylation and activation of phenylalanine hydroxylase. The significance of these regulatory roles are considered in light of the probable physiological environment of the enzyme.
Tipper
J
J
Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, Maryland 20892.
Kaufman
S
S
D000818:Animals; D001345:Autoradiography; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005934:Glucagon / Q000097:blood / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D009943:Organophosphorus Compounds / Q000378:metabolism; D010649:Phenylalanine / Q000097:blood / Q000494:pharmacology*; D010651:Phenylalanine Hydroxylase / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats
D009943:Organophosphorus Compounds; D001708:Biopterins; D010649:Phenylalanine; D005934:Glucagon; D010651:Phenylalanine Hydroxylase; C003402:sapropterin