A chronic hyperphenylalanemia was effectively produced in developing mice by daily administrations of phenylalanine (2 mg/g body wt) and a phenylalanine hydroxylase inhibitor alpha-methyl-D,L-phenylalanine (0.43 mg/g body wt). The presence of alpha-methylphenylalanine in newborn mice inhibited 65-70% of hepatic phenylalanine hydroxylase activity within 12 h. Since this maximum inhibition persisted for 24 h or longer, decreased enzyme activity was maintained by daily administrations. Whereas concentrations of phenylalanine increased approximately 40-fold in both plasma and brain following injection of alpha-methylphenylalanine and phenylalanine, plasma levels of tyrosine were not altered significantly. Concomitant with changes in phenylalanine concentrations we observed the brain polyribosomes' disaggregation, which reached a maximum 3 h after injection and persisted as long as 18 h. Polyribosomes did not become refractory to as many as 10 daily injections of alpha-methylphenylalanine and phenylalanine. In addition to polyribosome disaggregation, chronic hyperphenylalanemia reduced the rates of polypeptide chain elongation on polyribosomes isolated from brain homogenates.

The phenylalanine analogues p-chlorophenylalanine and alpha-methylphenylalanine were used to inhibit phenylalanine hydroxylase in animal models for phenylketonuria. The present report examines the affects of these analogues on the metabolism of neuroblastoma cells. p-Chlorophenylalanine inhibited growth and was toxic to neuroblastoma cells. Although in vivo this analogue increased cell monoribosomes by 42%, it did not significantly affect poly(U)-directed protein synthesis in vitro. P-Chlorophenylalanine did not compete with phenylalanine or tyrosine for aminoacylation of tRNA and was therefore not substituted for those amino acids in nascent polypeptides. The initial cellular uptake of various large neutral amino acids was inhibited by this analogue but did not affect the flux of amino acids already in the cell; this suggested that an alteration of the cell's amino acid pools was not responsible for the cytotoxicity of the analogues. In contrast with p-chlorophenylalanine, alpha-methylphenylalanine did not exert these direct toxic effects because the administration of alpha-methylphenylalanine in vivo did not affect brain polyribosomes and a comparable concentration of this analogue was neither growth inhibitory nor cytotoxic to neuroblastoma cells in culture. The suitability of each analogue as an inhibitor of phenylalanine hydroxylase in animal models for phenylketonuria is discussed.

Mouse brain ribosomes were radioactively labelled by a cell-free reductive alkylation reaction with NaBH4 and [14C]formaldehyde. The radioactivity was largely associated with ribosomal proteins, but little, if any, of the rRNA was radioactive after the alkylation procedure. Both ribosomal structural proteins and loosely associated components were successfully labelled by this procedure. The sedimentation properties of the ribosomes were unaltered and their ability to carry out poly(U)-directed protein synthesis, although decreased, was largely retained. Incubation of 14C-labelled ribosomes with brain cytosol resulted in a 17% loss of radioactivity, although treatment of the ribosomes with 1.0 m-KCl to remove the loosely associated factors rendered the ribonucleoprotein particles resistant to cytosol effects. The ribosome-cytosol interactions did not appear to be related to an exchange process, since the released radioactivity was largely degraded to acid-soluble material. In addition, the incubation of native ribosomes with brain cytosol resulted in an almost complete loss in the ability of the ribosomes to participate in cell-free protein synthesis.