Cerebral ribosomal protein phosphorylation in experimental hyperphenylalaninaemia

Abstract

Investigations were carried out on the effects of phenylalanine loading on ribosomal protein phosphorylation in cerebral cortices of infant rats. Administration of L-phenylalanine intraperitoneally, in doses of 1 or 2 mg/g body wt., resulted within 30 min in a significant decrease in incorporation of radioactivity from intracisternally administered [32P]Pi into constitutive ribosomal proteins of the cerebral 40S subunit. This phenomenon was not accompanied by significant variations in 32P uptake into the cerebral cytosol. Incorporation of radioactivity into ribosomal proteins of the cerebral 60S subunit exhibited only minor variations under these circumstances. Alterations in the phosphorylation state of cerebral 40S ribosomal proteins induced by phenylalanine loading involved principally the S6 protein, which exists in multiple states of phosphorylation. The proportions of the more highly phosphorylated congeners of this protein were markedly decreased, as detected by two-dimensional electrophoretograms and autoradiographs of the cerebral 40S ribosomal proteins. Phenylalanine loading also altered the relative extent of phosphorylation of the S6 protein in cerebral polyribosomes and monoribosomes. In control animals, the specific radioactivity of 40S proteins in cerebral polyribosomes was five to ten times that of 40S proteins in the monoribosome population. At 1 h after phenylalanine administration, the specific radioactivities of 40S proteins in the two ribosome populations tended to approach equality. These alterations in ribosomal protein phosphorylation were accompanied by a decrease in the proportion of polyribosomes in purified ribosome preparations isolated from cerebral cortices of phenylalanine-treated infant rats. In animals given the higher dose of phenylalanine (2 mg/g body wt.), subsequent administration of a mixture of seven neutral amino acids, which resulted in partial recovery of polyribosomes, also tended to reverse the changes in ribosomal protein phosphorylation. Variations in the activities of ribonuclease enzymes in the cerebral cytosol were also observed under these conditions. Administration of phenylalanine increased the activities of cerebral ribonucleases, whereas subsequent treatment with the amino acid mixture partly reversed this effect. The results suggest that alterations in cerebral ribosomal protein phosphorylation, ribosome aggregation and ribosome function are interrelated in experimental hyperphenylalaninaemia.

Inhibition of cerebral protein kinase activity and cyclic AMP-dependent ribosomal-protein phosphorylation in experimental hyperphenylalaninaemia

“…The methods employed to prepare purified cerebral ribosomes and ribosomal subunits for sucrosedensity-gradient analysis and isolation of ribosomal proteins have been described in detail (Roberts & Morelos, 1980). Cerebral tissue was gently homogenized in medium (1:3, w/v) containing 0.25Msucrose, 12 mM-MgCl2, 100 mM-KCl, 6 mM-JJ-mercaptoethanol, 50mM-Tris/HCl, pH 7.6 at 4 0C, and ribonuclease inhibitor protein (50,g/ml) (Roberts & Morelos, 1976).…”

“…Samples (O.1ml) of the postmitochondrial-supernatant preparation were treated with 0.9ml of 10% (w/v) trichloroacetic acid. After centrifugation, radioactivity in the supernatant fraction was determined as a measure of 32p uptake into the cerebral cytosol (Roberts & Morelos, 1980).…”

“…Ribosomal proteins were isolated from purified 40 S and 60 S subunits and subjected to twodimensional slab-gel electrophoresis as described previously (Roberts & Ashby, 1978;Roberts & Morelos, 1980). Samples were also taken for measurement of protein and radioactivity (Roberts & Morelos, 1980). The slabs were stained with Coomassie Brilliant Blue.…”

“…Incorporation of radioactivity from intracisternally injected [32p]p; into cerebral 40S ribosomal protein of infant rats in vivo has been shown to be selectively stimulated by parenteral administration of dibutyryl cyclic AMP or the potent inhibitor of cyclic nucleotide phosphodiesterase, 3-isobutyl-1methylxanthine (Roberts & Morelos, 1979). In contrast, intraperitoneal administration of loading doses of L-phenylalanine markedly decreased incorporation of 32p into cerebral 40S ribosomal protein (Roberts & Morelos, 1980). The present investigations reveal that this inhibitory effect of phenylalanine loading on cerebral ribosomal-protein phosphorylation could not be overcome by intracisternal administration of doses of dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine that stimulated this phosphorylation process in control animals (Table 1).…”

“…Investigations in our laboratory revealed that phosphorylation of the S6 protein of the cerebral 40S ribosomal subunit is acutely decreased by phenylalanine loading in infant rats (Roberts & Morelos, 1980). Phosphorylation of this protein in eukaryotic cells is normally stimulated by cyclic AMP (Wool, 1979), and also appears to be closely coupled with the activation of protein synthesis (Martini & Kruppa, 1979;Wettenhall & Howlett, 1979;Thomas et al, 1980).…”