THE NATURE OF THE AMINO ACID POOL USED FOR PROTEIN SYNTHESIS IN RAT BRAIN SLICES

The incorporation into brain slice protein of externally provided [1-¹⁴C]valine was measured at varying levels of valine in the medium, under conditions of constant protein synthesis and equilibration of intracellular valine specific activity. The results indicate that the valine pool used for protein synthesis is not identical to the pool of total free valine. Neither does the incorporation solely occur from an extracellular pool which is in equilibrium with the incubation medium. The data are compatible with a two-site activation model in which aminoacylation of tRNA occurs at both an internal site utilizing amino acid from the intracellular pool and an external (possibly membranous) site converting extracellular valine directly to valyl-tRNA. A good fit to the experimental observations is also provided by a compartmented intracellular valine pool model.     

IN VIVO INHIBITION OF RAT BRAIN PROTEIN SYNTHESIS BY l-DOPA

Abstract— A study has been made of the effect of a single intraperitoneal dose of l -DOPA on the in vivo metabolism of [¹⁴C]leucine and [¹⁴C]lysine by the brain, and on their uptake into brain protein. Administration of 500 mg DOPA/kg to 40-g rats raised the concentrations of several free amino acids; the only amino acid which underwent a statistically significant increment was alanine. Intracisternally-injected [U-¹⁴C]leucine was rapidly metabolized to other labelled compounds; DOPA administration did not influence significantly the rate of its metabolism. No similar metabolic change was observed after administering [U-¹⁴C]lysine intracisternally.
Incorporation of [¹⁴C]leucine and [¹⁴C]lysine into total brain protein was significantly reduced 45 min after DOPA administration. There was also depression of the uptake of labelled amino acid into a supernatant fraction, obtained by high speed centrifugation of the brain homogenate, and into brain microtubular protein (tubulin). Reduced amino-acid incorporation into brain proteins observed 45 min after l -DOPA injection coincided with extensive disaggregation of brain polyribosomes. At 120 min after DOPA treatment, disaggregation was no longer significant and there was a smaller depression in labelled amino aicd incorporation, which disappeared completely 240 min after l -DOPA injection. It is concluded that disaggregation of brain polysomes following DOPA treatment is an accurate reflection of a change in the intensity of brain protein synthesis in vivo.     

IN VIVO INHIBITION OF RAT BRAIN PROTEIN SYNTHESIS BY d-AMPHETAMINE

Abstract— Between 1 and 4 h after rats received a single injection of d-amphetamine (15 mg/kg)(when brain polysomes are known to be disaggregated), the in vivo incorporation of [¹⁴C]lysine into trichloroacetic acid-precipitable brain protein was reduced by 28–48%. Incorporation of the ¹⁴C label into the protein present in a 100,000 g supernatant extract of whole brain was similarly reduced (by 44%). Amphetamine administration suppressed protein synthesis in rat cerebral cortex, cerebellum, hypothalamus, striatum, and brainstem to an equivalent extent. The drug did not significantly affect lysine pool sizes measured in these brain regions; thus the reduced incorporation of labeled lysine was not the result of an isotope dilution effect. We therefore conclude that the brain polysome disaggregation resulting from amphetamine administration is associated with decreased in vivo synthesis of some brain proteins.     

BIOSYNTHESIS OF RIBONUCLEIC ACID IN RAT BRAIN SLICES

The incorporation of uridine into RNA in brain slices was studied. Optimal conditions for uridine incorporation were determined. The characteristics of the product suggest that de novo DNA-directcd synthesis of fairly high molecular weight material takes place. Incorporation into RNA of several areas of brain was studied. The incorporation was also studied as a function of the age of the animal. Finally, an apparent correlation was observed between the decrease in uridine incorporation with age and the increase of the enzyme uridine nucleosidase which hydrolyses uridine to uracil, a material which cannot be incorporated into RNA.     

PROTEIN SYNTHESIS IN ISOLATED NUCLEI FROM ADULT RAT BRAIN

Nuclei from adult rat brains isolated with isotonic sucrose were incubated with [³H]leucine and later purified by centrifugation through hypertonic sucrose solutions. It was found that under these conditions, tritiated leucine was incorporated into TCA precipitable material. Protein synthesis was impaired if the nuclei were treated with the nonionic detergent Triton X-100 or hypertonic sucrose. The presence of puromycin or cycloheximide markedly inhibited the incorporation of the radioactive amino acid. Actinomycin D and RNase did not have any effect on the incorporation. Autoradiography indicated the presence of labelled material within the nuclei and not in cytoplasmic contaminants. Glial nuclei were more actively involved in protein synthesis than neuronal nuclei.     

PROTEIN SYNTHESIS IN VITRO IN RAT BRAIN AFTER SHORT-TERM PROTEIN STARVATION AND REFEEDING

Rats were fed a protein-free diet for 4 or 6 days. They were compared with rats kept on the same diet for 3 or 5 days and on adequate protein for one additional day. The incorporation of ¹⁴C-labelled amino acid into protein was studied in systems containing ATP, GTP, phosphoenolpyruvate, pyruvate kinase and if required, a mixture of unlabelled amino acids and either the 6000 g supernatant fraction of a brain homogenate or microsomes and soluble enzymes. The 6000 g supernatant fraction showed variation in amino acid incorporating activity as well as in RNase activity as measured by breakdown of labelled polyuridylic acid. There was no difference in RNase activity in isolated microsomes, but the amino acid incorporating activity was significantly higher in preparations obtained from rats fed one meal of protein after 5 days of protein-starvation.     

THE EFFECT OF TRIETHYLLEAD ON TOTAL AND MYELIN PROTEIN SYNTHESIS IN RAT FOREBRAIN SLICES

Rats (20-day-old) were acutely intoxicated with triethyllead and their forebrains were studied during the following 14 days. All the lead in the tissue was found in the form of triethyllead, proving that the toxin per se was responsible for the pathological changes observed in the organ. The incorporation of [¹⁴C]leucine into the acid-insoluble protein was suppressed in the forebrain slices prepared from the intoxicated animals as well as in the slices, to which PbEt₃ was added in vitro. In both systems the synthesis of myelin protein was inhibited more than the total protein synthesis. The results suggest a specificity of triethyllead toward processes involved in the furnishing of the myelin membrane proteins.     

HIGH AFFINITY UPTAKE OF l-GLUTAMINE IN RAT BRAIN SLICES

—l -Glutamine is taken up into rat brain slices by a specific‘high affinity’uptake system (K_m 52 μm ) which is not influenced by high concentrations of l -glutamate and l -asparagine. The uptake system appears to be associated with cellular structures that do not survive homogenization under conditions which yield synaptosomes. The‘high affinity’uptake of glutamine is dependent on the external sodium ion concentration and can be inhibited by p-chloromercuriphenylsulphonate, amino-oxyacetic acid, ouabain, dibenamine and allylglycine. The effects of several inhibitors indicate that l -asparagine uptake is mediated by a system different from the‘high affinity’system mediating l -glutamine uptake.     

THE EFFECT OF ISCHAEMIA AND RECIRCULATION ON PROTEIN SYNTHESIS IN THE RAT BRAIN

Abstract— Rats were subjected to cerebral compression ischaemia for 15min and were subsequently recirculated with blood for periods up to 3 h. In vivo incorporation of intravenously administered L-[1–¹⁴C]valine into total brain proteins was found to be severely inhibited (about 20% of controls) after 45 min of recirculation. After 3 h, protein synthesis had increased, the specific radioactivity of proteins then being about 40% of controls. The post-ischaemic inhibition of protein synthesis was accompanied by a breakdown in polyribosomes and a concomitant increase in ribosomal subunits. In vitro incorporation of L-[1–¹⁴C]phenylalanine by a postmitochondrial supernatant system derived from animals subjected to 15 min ischaemia and 15 min recirculation was also severely reduced and showed, in contrast to control animals, no response to the addition of a specific inhibitor of polypeptide chain initiation (Poly(I)). Together with the in vivo accumulation of ribosomal subunits this indicates a block in peptide chain initiation during the early stages of recirculation.
Polyribosomes from animals subjected to 15 min ischaemia without recirculation showed a normal rate of in vitro protein synthesis which was inhibited by Poly(I) to a similar extent as polyribosomes from control animals. These results suggest that the post-ischaemic inhibition in chain initiation develops during the early stages of recirculation rather than during the ischaemic period itself.     

PROTEIN SYNTHESIS IN THE MEDULLA OF THE RAT BRAIN. REPRODUCIBILITY OF THE RESULTS

Abstract— [¹⁴C]Leucine was injected intracranially into the brainstem reticular formation at the level of the upper medulla by the stereotaxic method. Subcellular fractions prepared 3 hr after injection showed that the specific activities of leucine-incorporated proteins decreased in the order soluble, microsomal, nuclear and mitochondrial fractions. Specific activities of proteins in the sera were 2·2 per cent of whole homogenate proteins.
The results from 27 experiments showed that 66·6 per cent of the mean specific activities of proteins extracted from whole homogenates fell within ·1 s.d . and 100 per cent within ± 2 s.d . (close to a normal distribution). The coefficients of variation were between 40 and 50 per cent for whole homogenates, sera and all subcellular fractions. Reproducibility of results and factors concerned with possible errors in the technique are discussed.     

MEASUREMENTS OF IN VIVO RATES OF PROTEIN SYNTHESIS IN BRAIN, SPINAL CORD, HEART AND LIVER OF YOUNG VERSUS ADULT RATS, INTACT VERSUS HYPOPHYSECTOMIZED RATS

Abstract— In vivo protein synthesis rates in rats were estimated by single i.p. injections of large quantities of [1-¹⁴C]valine. This method gives reliable estimates of the precursor specific activity and average protein synthesis rates. In the brain, spinal cord, heart and liver, the average rates for adults were 0.65, 0.42, 0.49 and 1.92% replacement of protein-bound amino acid per h. In the brain and liver of 10-day olds the average rates were estimated to be 1.46 and 3.12% per h respectively. Hypophysectomy decreased synthesis rates by 25% or more in all tissues studied. The disadvantages of the method are that applying large amounts of valine i.p. appeared to constitute a stress and that the valine solution required for injection was hypertonic, causing withdrawal of body fluids of the animal.     

SYNTHESIS OF RNA IN DEVELOPING RAT BRAIN IN VITRO

—Incorporation of [8-¹⁴C]adenine into a rapidly-labelled fraction of RNA derived from the nucleus, and into a cytoplasmic RNA of high molecular weight was studied in brain slices from new born rats. The kinetic behaviour of the two fractions of RNA was compatible with a precursor-product relationship between them. The change in the specific activity of adenine and the reduction of radioactivity in prelabelled RNA of brain slices in the presence of actinomycin D, suggest that the observed degradation of nuclear RNA is not due to random changes, but is limited to a relatively small fraction, presumably messenger RNA.     

SYNTHESIS AND METABOLISM OF l-KYNURENINE IN RAT BRAIN

Abstract— A method for the quantitative analysis of femtomole amounts of kynurenine (along with tryptophan, 3-hydroxykynurenine and kynuramine) in rat brain using high pressure liquid chroma-tography and electron-capture GLC is described. Endogenous concentrations of these substances in rat brain regions were measured, and their formation after the injection of radioactive tryptophan or kynurenine was determined. Kynurenine was formed from tryptophan in brain and was also taken up from the periphery. Extracerebral kynurenine was calculated to account for 60% of the cerebral pool of kynurenine. The cerebral rates of synthesis of kynurenine and 3-hydroxykynurenine were 0.29 and 0.17nmol/g/h. The turnover rate of kynurenine in the brain was 1.02 nmol/g/h measured from [¹⁴C]tryptophan or 1.14 nmol/g/h from [³H]kynurenine injected intraperitoneally. Kynuramine levels in different areas of the brain were similar to those of tryptamine. Following intraperitoneal injection of [¹⁴C]tryptophan, the presence of anthranilic, 3-hydroxyanthranilic, xanthurenic, kynurenic and quinaldic acids was demonstrated in the brain.     

THE SYNTHESIS OF MYELIN IN DEVELOPING RAT BRAIN

Abstract— —The synthesis of myelin proteins has been studied in the grey and white matter slices of developing rat brain by measuring the incorporation of [³H]lysine and [¹⁴C]arginine into polypeptide. The incorporation was sensitive to cycloheximide and puromycin at 1 mM concentration. Developing rat optic nerve slices, free of retinal ganglion cells, were able to synthesize myelin basic and proteolipid proteins, but rat retinal preparation failed to synthesize myelin basic protein. Rabbit retinae were able to synthesize myelin basic and proteolipid proteins. Significant activity of the myelin marker enzyme 2',3'-cyclic nucleotide-2'-phosphodiesterase has been found in the rabbit retina but not in rat retina. The results presented in this communication suggest that myelin proteins in the rat CNS are synthesized by the oligodendroglial cells and that neurons probably do not participate.     

A METHOD FOR MEASURING BRAIN PROTEIN SYNTHESIS RATES IN YOUNG AND ADULT RATS

The injection of large quantities of radioactive amino acid precursor is proposed as a technique for determining rates of cerebral protein synthesis in vivo. In this way the specific radioactivity of the amino acid precursor in the brain is maintained at a relatively constant level for at least 2 h. Injections of 10–15 μ mol of valine per g body weight result in nearly constant rates of incorporation of radioactivity and do not appear to inhibit cerebral protein synthesis in adult or young (2–6 day old) rat brain. Similar rates were obtained in young rat brain with lysine and histidine. Rates of protein synthesis in cerebral hemisphere were for 2-day-olds 2·1 per cent replacement of protein bound amino acid per h and for adult 0·62 per cent per h. Advantages and disadvantages of the procedure are discussed.     

FEEDBACK REGULATION OF 5-HT SYNTHESIS IN RAT STRIATAL SLICES

The effects of changes in intraneuronal levels of 5-HT induced by monoamine oxidase inhibitors (MAOI) given in vivo or exogenous 5-HT added in vitro on 5-HT synthesis in striatal slices of the rat have been examined. The synthesis of 5-HT was estimated by the measurement of the total formation of [³H]5-HT and [³H]5-hydroxyindole acetic acid from [³H]tryptophan and by calculation of the conversion index (CI) of tryptophan into 5-HT. The small formation of [³H]tryptamine and [³H]indole acetic acid from [³H]tryptophan was taken into account in the estimation of 5-HT synthesis. Both MAOI pretreament (180 min) and 5-HT (2·8 μM) inhibited synthesis. The latter effect persisted in catecholamine depleted tissues and was related to intraneuronal changes in 5-HT levels, since it could be prevented by chlorimipramine. The inhibition of 5-HT synthesis was related to the decreased conversion of tryptophan into 5-hydroxytryptophan and could not be prevented by p-chloro-phenylalanine pretreatment which depleted 5-HT levels or by dibutyryl cyclic AMP which normally stimulated 5-HT synthesis. Tryptophan uptake in slices was not affected by exogenous 5-HT. The various mechanisms possibly involved in the end product regulation process of 5-HT synthesis are discussed.     

PROTEIN METHYLATION IN RAT BRAIN IN VITRO

Abstract— Protein-methylation activity in various organs of the rat was studied with S-adenosyl-L-[methyl-¹⁴C]methionine ([methyl-¹⁴C](SAM) as methyl donor. Activity of the enzyme was highest in brain and lowest in liver. Histones comprised approximately 20 per cent of the total radioactivity incorporated, and lysine-rich histone was the most active. Analysis of amino acids of the methylated proteins of rat brain showed arginine to be the amino acid most extensively methylated, but some methylation occurred in lysine residues. An additional [methyl-¹⁴C]-labelled amino acid was found near histidine on the amino acid column chromatogram.     

ALTERATION OF TRICARBOXYLIC ACID CYCLE METABOLISM IN RAT BRAIN SLICES BY HALOTHANE

Abstract—

• 1 Metabolism of [2-¹⁴C]pyruvate, [1-¹⁴C]acetate and [5-¹⁴C]citrate in the rat cerebral cortex slices was studied in the presence of halothane. Metabolites assayed include acetylcholine (ACh), citrate, glutamate, glutamine, γ-aminobutyrate (GABA) and aspartate. The trichloroacetic acid soluble extract, the trichloroacetic acid insoluble precipitate and its lipid extract were also studied.
• 2 In control experiments, pyruvate preferentially labelled ACh, citrate, glutamate, GABA and aspartate. Acetate labeled ACh, but to a lesser extent than pyruvate. Acetate also labeled lipids and glutamine. Citrate labeled lipids but not ACh and served as a preferential precursor for glutamine. These data support a three-compartment model for cerebral tricarboxylic acid cycle metabolism.
• 3 Halothane caused increases in GABA and aspartate contents and a decrease in ACh content. It has no effect on the contents of citrate, glutamate and glutamine.
• 4 Halothane preferentially inhibited the metabolic transfer of radioactivity from pyruvate into almost all metabolites, an effect probably not related to pyruvate permeability. This is interpreted as halothane depression of the‘large metabolic compartment’ which includes the nerve endings.
• 5 Halothane increased the metabolic transfer of radioactivity from acetate into lipids but did not alter such a transfer into the trichloracetic acid extract.
• 6 Halothane increased the metabolic transfer of radioactivity from citrate into the trichloroacetic acid precipitate, lipids and especially glutamine. Transfer of citrate radioactivity into GABA was somewhat decreased.
• 7 The differential effects of halothane on acetate and citrate utilization suggest that the ‘small metabolic compartment’ should be subdivided. Therefore, at least three metabolic compartments are demonstrated.
• 8 Halothane did not interfere with the dicarboxylic acid portion of the tricarboxylic acid cycle.

     

SYNTHESIS OF GLYCOPROTEINS AND GANGLIO-SIDES IN DEVELOPING RAT BRAIN

Abstract— Intracerebral injections of radioactive fucose into developing rats resulted in specific labelling of the brain glycoproteins in their fucose moieties. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed that the radioactive glycoproteins were very heterogeneous with regard to molecular weight. A procedure utilizing [³H]fucose and [¹⁴C]fucose together with double-label counting techniques was developed for comparing the electrophoretic patterns of newly synthesized glycoproteins from different samples of tissue. By the use of this procedure we showed that the incorporation of radioactive fucose into the glycoproteins of high mol. wt. was relatively greater in the brains of 5-day-old rats than in those of 25-day-old rats. Intracerebral injection of N -[ Ac -³H]acetyl- d -mannosamine resulted in a high degree of specificity for the labelling of sialic acid moieties in glycoproteins and gangliosides. The ratio of the d.p.m. of N -[³H]acetylmannosamine incorporated into glycoproteins to the d.p.m. incorporated into gangliosides was higher in 5-day-old rats than in 15- or 25-day-old rats. Experiments in which 15-day-old rats were injected with a mixture of [¹⁴C]fucose and N -[³H]acetylmannosamine showed that there were differences in the relative degrees of incorporation of the two radioactive precursors into the various glycoproteins. The greatest incorporation of [¹⁴C]fucose relative to that of N- [³H]acetylmannosamine occurred in some of the glycoproteins of smaller mol. wt.