IN VlTRO BINDING OF PHENYLALANYL-tRNA TO NEONATAL AND ADULT MOUSE BRAIN RIBOSOMES

The ability of brain ribosomes, isolated from mice of various ages, to bind phenylalanyl-tRNA was measured under various reaction conditions. In the presence of template RNA (polyuridylic acid) the binding could be measured by both enzymic and non-enzymic assays. In general, the binding requirements for the brain system were similar to those previously described for microbial and eukaryotic systems. Although previous studies have shown that ribosomes obtained from increasingly older mow brain tissue were less active in polyphenylalanine synthesis, no significant differences in phenylalanyl-tRNA binding to polysome complexes could be detected. The binding of phenylalanyl-tRNA by ribosomes isolated from both neonatal and mature mouse brain tissue was similar with regard to GTP and polyuridylic acid dependence, magnesium ion concentration and reaction kinetics. Similar binding of phenylalanyl-tRNA by young and mature brain ribosomes was also measured with ribonucleoprotein particles previously stripped with puromycin. The results are discussed in light of the rapid alteration of macromolecular synthesis during postnatal brain development and the possible role of the interaction between ribosomes and tRNA.     

RIBOSOMAL ACTIVITY IN PRENATAL MOUSE BRAIN

Abstract— Regulation of protein synthesis is important for the proper growth and development of the brain. Our previous work on the regulation of protein synthetic activity in fetal mouse brain cell suspensions showed that the rate of protein synthesis decreased during the prenatal period. In the present study, ribosomal activity of cell-free homogenates and purified ribosomes obtained from fetal neural tissue was measured. The post-mitochondrial supernatant (PMS) fraction actively incorporated amino acids into polypeptides using either endogenous mRNA or polyuridylic acid as template. The protein synthetic activity was dependent upon the age of the fetus. Ribosomes purified from this fraction were also active in protein synthesis. Incorporation of phenylalanine was linear for 20 min, and dependent upon the concentration of ribosomes and the pH 5 enzyme fraction. The age dependent decrease in protein synthetic activity observed with the post-mitochondrial supernatant fractions was not found when these purified ribosomes were employed. Ribosomes obtained from fetal, newborn or adult neural tissue were compared and found equally active in their protein synthetic capacity.     

CHARACTERISTICS AND PRODUCTS OF A CELL-FREE POLYPEPTIDE SYNTHESIZING SYSTEM FROM NEONATAL AND ADULT MOUSE BRAIN

—The regulation of protein synthesis by ribosomes isolated from mouse brain tissue was studied using a cell-free polyphenylalanine synthesizing system. Polypeptide synthesis was followed by assaying translocation and analysing the reaction products by BD-cellulose chromatography. The brain ribosomal activity could be divided by these methods into two distinct steps : binding of aminoacyl-tRNA to the ribosome and active translocation leading to subsequent polyphenylalanine synthesis. In comparison to initial binding of aminoacyl-tRNA, translocation in the cell-free system increased the incorporation of labelled phenylalanine by 10-fold. An analysis of the reaction products clearly showed active ribosomal synthesis of oligophenylalanine from [³H]phe-tRNA. Ribosomes isolated from neonatal brain tissue were 2–4 times as active as those obtained from adult brain tissue in polypeptide synthesis. In addition, polypeptides synthesized on the more active ribosomes from neonates tended to be of greater chain length than those from adult. Therefore, the maturation-dependent decrease in ribosomal protein synthetic activity during neural development was shown to be directly associated with the ribosome particles.     

ACTIVITY OF CYCLIC AMP-DEPENDENT MICROSOMAL PROTEIN KINASE AND PHOSPHORYLATION OF RIBOSOMAL PROTEIN IN RAT BRAIN DURING POSTNATAL DEVELOPMENT

Abstract— Microsomes from rat brain exhibited protein kinase activity which was stimulated by cyclic AMP when assayed in the presence of exogenous protein substrate, such as thymus histone. In the absence of exogenous substrate some phosphorylation of microsomal protein occurred, but no stimulation by cyclic AMP could be discerned, probably because of limitations of substrate. The maximal activity of microsomal protein kinase observed in the presence of saturating concentrations of histone and the optimal concentration (5 μ m ) of cyclic AMP remained essentially unchanged from birth to early adulthood, but the magnitude of the stimulation by cyclic AMP was significantly higher at birth than at 30 days of age. Brain ribosomal proteins could be phosphorylated by the cyclic AMP-dependent brain protein kinase. Their total capacity for acceptance of phosphate by means of this phosphorylation reaction remained unchanged throughout the postnatal development of the brain. Our results are consistent with the possibility that phosphorylation of ribosomal protein mediated by cyclic AMP-dependent protein kinase may play a a role in the postnatal regulation of cerebral protein synthesis, as a result of the changes in the levels of cyclic AMP known to occur in brain during postnatal maturation.     

CELL-FREE PROTEIN SYNTHESIS BY MOUSE BRAIN DURING EARLY DEVELOPMENT

—Cell-free homogenates were employed to study the nature of the mechanism that is responsible for the rapid decrement in protein synthesis during early neural development. There was a progressive loss of polypeptide synthesis in post-mitochondrial fractions that were isolated from increasingly older tissue. By the time the animals were approximately 17 days old, the rate of amino acid incorporation had decreased to the rate that was measured in adult brain preparations. This decrement in synthetic activity was similar to that previously measured in developing intact brain cells. The loss in protein synthesis was demonstrated to be independent of cellular membrane permeability and under the influence of intracellular control mechanisms. Although the nature of the control mechanism is still not clear, a lack of template RNA to direct protein synthesis was not the limiting factor in the decreased synthesis of the older brain preparations.     

NUCLEAR-CYTOSOL INTERACTIONS THAT FACILITATE RELEASE OF RNA FROM MOUSE BRAIN NUCLEI

Abstract— Mouse brain nuclei were incubated in vitro under conditions that primarily lead to the synthesis of radioactive polydisperse and messengerlike nuclear RNA. After incubation the effects of Mg² concentrations, nucleoside triphosphate levels and brain cytosol were examined with regard to their ability to influence the release of RNA from brain nuclei. The presence of 8 mM -MgCl₂ and a total of 0.3 mM-nuclcoside triphosphates during the labelling procedure allowed only a minimal amount of RNA to be released. However, when the MgCl₂ was decreased to 2 mM and the nucleoside triphosphates were increased to 1 mM, a stimulation of RNA release was observed. The addition of unfractionated brain cytosol under these conditions resulted in an inhibition of RNA release.
G-100 Sephadex filtration removed detectable RNase activity from the cytosol preparations and allowed the identification of fractions that were able to facilitate nuclear RNA release by 3-fold. The fractions that stimulated release did not have detectable levels of RNase, protease or DNA-dependenl RNA polymerase. Under conditions that provided maximum nuclear RNA release by both labelled mouse brain and neuroblastoma nuclei, no release of DNA could be measured. The cytosol fractions that facilitated RNA release did not have a high affinity for nuclear RNA or an ability to stimulate nuclear RNA synthesis. However, other components in the cytosol were shown to stimulate RNA metabolism in isolated mouse brain nuclei and to have a relatively high binding affinity to nuclear RNA. Further purification of the RNA release components in the brain cytosol by DEAF. Sephadex chromatography allowed an increase in specific activity of at least 40-fold. The thermal lability, effective filtration size, and solubility in phenol suggested that the cytosol factors that facilitiated nuclear RNA release were associated with cellular proteins.     

SELECTIVE RETENTION AND FILTRATION OF BRAIN NUCLEIC ACIDS IN AGAROSE GELS

Abstract— Total nucleic acids of rat brain have been separated by agarose gel chromatography at 2 m -NaCl into DNA. transfer RNA plus low molecular weight RNA. and high molecular weight RNA fractions. The DNA fraction contained less than 1 per cent RNA by weight judged by either short-term or long-term labelling with ortho[³²P]phosphate. The high molecular weight RNA fraction contained 28 s and 18 s ribosomal RNAs and a heterogeneous population of 20-60 s RNAs, apparent after short-term labelling and characterized by a high content of nearest-neighbour-labelled uridylic acid. The rapidly sedimenting (>30 s ) portion of these RNAs could be largely separated from ribosomal RNAs by gel filtration using 4% agarose. The ribosomal RNAs could be fully resolved into 28 s and 18 s components by agarose gel chromatography at 0.5 m -0.6 m -NaCl, as shown by analysis of their sedimentation and nucleotide composition.     

QUANTITATIVE AND QUALITATIVE CHANGES IN RIBONUCLEIC ACIDS OF RAT BRAIN DEPENDENT ON AGE AND TRAINING EXPERIMENTS

—Investigations of rat brain RNA were carried out by phenol extraction of the whole brain and chromatographic fractionation into ribosomal RNA and transfer RNA. (1) The amounts of both RNA species increase in the course of the animal's development reaching a maximum at about the tenth week of life. The ratio of both species remains constant throughout the growth to the twentieth week. After the rats had been trained how to reach their forage by balancing on a stressed rope, the rRNA content was found to be significantly higher, whereas the tRNA content was unchanged. (2) The portion of ribosomes bound in polysome complexes decreases with increasing age of rats. Conditioning of the animals brings about again an increase in polysome content. It is supposed that this reflects an enhanced synthesis of specific proteins in young developing rats and in the course of conditioning. (3) In young rats a second valine specific tRNA could be found as a minor component in addition to the major valyl-tRNA. This additional component disappears as the animals advance to an age of 3 weeks and it could not be detected in the brains of rats after training experiments. In tRNAs specific for the amino acids leucine, lysine and phenylalanine no kind of deviation could be stated.     

MEASUREMENTS OF RATES OF PROTEIN SYNTHESIS IN RAT BRAIN SLICES

The use of tracer concentrations of labelled amino acids to measure incorporation in incubated slices of brain results in wide fluctuations with time in the specific activity of the precursor. Using concentrations of about 1 mm of labelled amino acid facilitates the accurate measurement of rates of synthesis. These higher precursor levels in the medium decrease the fluctuations in free amino acid specific activity due to dilution by endogenous amino acid and the production of amino acid by protein degradation, and decrease the lag in incorporation due to transport phenomena. Concentrations of 1 mm amino acid in the medium did not inhibit protein synthesis; with valine, leucine, phenylalanine, lysine and histidine, incorporation rates were similar when measured at trace concentrations and at 1 mm medium levels. The source of amino acid for protein synthesis appears to be intracellular. No evidence could be found for the preferential use of extracellular medium amino acid. The rate of incorporation of amino acids in incubated slices of rat brain was 0.087 per cent of the protein amino acid/h.     

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.     

CHARACTERIZATION OF BRAIN RIBONUCLEOPROTEIN PARTICLES

Abstract— Brain RNP particles were characterized to determine whether they play a role in the regulation of brain protein synthesis. RNP particles were isolated from the postribosomal supernatant of cerebral hemispheres of young rabbits, employing conditions which minimize adventitious protein-RNA interactions. Brain RNP particles consist of a different set of proteins compared to proteins associated with either 40 and 60s ribosomal subunits or polysomal mRNA. Poly(A+)mRNA from brain RNP particles stimulates the incorporation of [³⁵S]methionine in a wheat embryo cell-free system and codes for a different set of proteins compared to poly(A+)mRNA isolated from polysomes (with some overlap; i.e. mRNA coding for brain-specific S100 protein is present in both RNP particles and polysomes).
Addition of total brain RNP particles to a cell-free wheat embryo system inhibits the endogenous incorporation of [³⁵S]methionine. Total RNP particles were fractionated by sucrose density gradient centrifugation into a'light'and a'heavy'fraction. The light RNP fraction inhibited while the heavy RNP fraction stimulated protein synthesis in the wheat embryo cell-free system. Analysis of the protein composition of fractionated RNP particles revealed that the light and heavy RNP particles contained different sets of proteins. Together these results suggested that one class of brain RNP particles may contain a translational inhibitor and may be involved in the regulation of protein synthesis in the brain.     

Binding and translation of turnip-yellow-mosaic-virus ribonucleic acid by skeletal-muscle ribosomes from normal and diabetic rats

Ribosomes from skeletal muscle of diabetic rats were less active than normal ribosomes in protein synthesis directed by turnip-yellow-mosaic-virus RNA. The proportion of ribosomes from muscle of diabetic rats capable of binding turnip-yellow-mosaic-virus RNA was greater than normal, but there was no difference in the equilibrium constants for the binding reaction. The turnip-yellow-mosaic-virus RNA was bound preferentially to the small (40S) ribosomal subunit, whereas the decrease due to diabetes in its translation was associated with the large (60S) subunit. Thus the diminished capacity of ribosomes from muscle of diabetic rats to translate turnip-yellow-mosaic-virus RNA was not the result of decreased binding of the template.     

TRANSFER RNA AND THE REGULATION OF PROTEIN SYNTHESIS IN RAT CEREBRAL CORTEX DURING NEURAL DEVELOPMENT

Protein synthesis was measured in ribosomal systems derived from the cerebral cortex of 5-and 35-day-old rats. Under optimal conditions incorporation of radioactive leucine per mg ribosomal protein was four times higher with ribosomes from the younger animals than with ribosomes from the 35-day-old rats. This suggests that a decrease in the rate of protein synthesis occurs during neural development. Both ribosomes and the pH enzyme fraction from the cerebral cortex of 35-day-old rats had lower activities than preparations from the younger rats. Cerebral cortical ribosomes from 35-day-old animals had a lower polyribosome content than similar preparations from 5-day-old rats. A three-fold higher requirement for the pH 5 enzyme fraction was observed with the ribosomal system from 5-day-old rats, an observation which correlated with the yields of pH 5 enzyme and ribosomal protein from the younger tissue. The nature of the changes in the composition of the pH 5 enzyme fraction was investigated. Methylated albumin kiesselguhr (MAK) and Sephadex G-75 column chromatography showed that RNA from the pH 5 enzyme fraction was heterogeneous, containing tRNA, rRNA, and a small molecular weight RNA. This latter RNA, perhaps a degradation product of rRNA, comprised the greatest portion of RNA from the pH 5 enzyme fraction of cerebral cortex. The data obtained with MAK chromatography were used to estimate the total tRNA content of the cerebral cortex, with no age-related differences being observed. Since evidence of RNA degradation was seen, tRNA was also isolated by phenol extraction of whole cerebral cortex in the presence of bentonite. Purification of tRNA by NaCl and isopropanol fractionation gave preparations with no detectable rRNA or small molecular weight RNA. With this purification method, the tRNA yield was greater than estimated by the MAK method, demonstrating that losses of tRNA occurred during the cell fractionation steps. With the purification method 1.6 times more tRNA was obtained from the cerebral cortex of 5-day-old animals than from the older tissue. This higher level of tRNA in the younger, more active tissue appeared to involve all tRNA species, since in vitro aminoacyiation studies revealed nearly identical acceptance values for 18 individual amino acids. These results suggest that the rate of protein synthesis in cerebral cortex is regulated in part by the total amount of tRNA present to translate the higher level of polysome-bound mRNA.     

Regulation of Prenatal and Postnatal Protein Synthesis in Mouse Brain

Abstract: Regulation of protein synthesis during prenatal and postnatal brain development was examined using postmitochondrial supernatant (PMS) fractions and isolated ribosome-pH 5 enzyme systems from fetal, neonatal, and adult neural tissue. The rate of polyuridylic acid (poly-U)-dependent protein synthetic activity was inversely proportional to the endogenous rate of protein synthesis in either the PMS fractions or ribosomal preparations. A careful analysis of the kinetics of the poly-U-dependent polypeptide synthesis revealed that there was a lag in the time at which certain of the PMS preparations could begin to utilize the poly-U template as sole source of mRNA. The lag period was dependent upon the developmental age of the neural tissue used and the Mg²⁺ concentration of the protein synthesis reaction. Since previous work reported that the observed developmental decrease in the rate of polypeptide synthesis utilizing a poly-U template could not be measured in a purified ribosomal-pH 5 enzyme system, ribosomes were obtained by several isolation techniques to determine if the purification procedure might have affected the ribosomes in some manner by removing a specific protein(s) involved in ribosome-cytosol interactions. At 6 mM-Mg²⁺ the rate of poly-U-dependent protein synthesis was inversely proportional to the rate of endogenous synthesis and depended upon the method used to isolate the ribosomes: microsomes ∼Triton X-100-treated < DOC-treated < KCl-treated. However, there was no age-dependent effect with any of the ribosomal preparations. The data suggest that there is a developmental modulating effect of ribosomal activity in PMS preparations which is not found in association with the isolated ribosome-pH 5 enzyme protein synthesizing system.     

THE EFFECTS OF PHENYLALANINE ON AMINO ACID METABOLISM AND PROTEIN SYNTHESIS IN BRAIN CELLS IN VITRO1

Incubation of brain cell suspensions with 14 mM-phenylalanine resulted in rapid alterations of amino acid metabolism and protein synthesis. Both thc rate of uptake and the final intracellular concentration of several radioactively-labelled amino acids were decreased by high concentrations oi phenylalanine. By prelabelling cells with radioactive amino acids, phenylalanine was also shown to effect a rapid loss of the labelled amino acids from brain cells. Amino acid analysis after the incubation of the cells with phenylalanine indicated that several amino acids were decreased in their intracellular concentrations with effects similar to those measured with radioisotopic experiments (large neutral > small and large basic > small neutral > acidic amino acids). Although amino acid uptake and efflux were altered by the presence of 14 mwphenylalanine, little or no alteration was detected in the resulting specific activity of the intracellular amino acids. High levels of phenylalanine did not significantly altcr cellular catabolism of either alanine, lysine, leucine or isoleucine. As determined by the isolation of labcllcd aminoacyl-tRNA from cells incubated with and without phenylalanine, there was little or no alteration in the level of this precursor for radioactive alanine and lysine. There was, however, a detectable decrease in thc labelling of aminoacyl-tRNA for leucine and isoleucine. Only aftcr correcting for the changes of the specific activity of the precursors and thcir availability to translational events, could the effects of phenylalanine on protein synthesis be established. An inhibition of the incorporation into protein for each amino acid was approximately 20%.     

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.     

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.