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 BY CEREBRAL POLYSOMES PRETREATED WITH PUROMYCIN

Abstract— Polysomes prepared from rat cerebral microsomes, following preincubation with a high concentration of puromycin (2.5 mM) in the presence of rat liver soluble enzymes, were very similar to normal polysomes in yield, A 260_nm:A 280_nm ratio and in absorbance profile on sucrose density gradients. However, the capacity for amino acid incorporation was inhibited by more than 50 per cent by puromycin treatment. The extent of inhibition far exceeded what could be expected from the amount of residual puromycin bound to polysomes, suggesting that some essential step in polypeptide synthesis was damaged. An examination of the labelled polypeptides, using sucrose density gradient centrifugation, showed that most of the new chains synthesized by puromycin-polysomes were released into solution. However, small amounts of polypeptides of high specific radioactivity were distributed among the polysomal aggregates. In contrast to normal polysomes, the specific radioactivity of puromycin polysomes was the highest in aggregates of six or more ribosomes and declined sharply at the levels of trimers and dimers. It is suggested that cerebral polysomes pretreated with puromycin become defective in the termination mechanism with the consequence that even though they are capable of moving at least short distances on the messenger RNA and of releasing the polypeptide chains formed, a concomittant release of monomeric ribosomes is obstructed. This may result in the‘clogging’of the terminus of the mRNA, thus blocking further polypeptide synthesis.     

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.     

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.     

ISOLATION OF PURIFIED BASIC PROTEIN FROM HUMAN BRAIN

—A simplified method for the isolation of an encephalitogenic basic protein from myelin is described. The basic polypeptide is easily extracted from the interfacial protein which separates when chloroform-methanol extracts of myelin are treated with a synthetic upper phase containing citrate. Evidence is presented for the purity and identity of the protein. The disc polyacrylamide gel electrophoretic pattern of this protein was consistent with a high degree of homogeneity.     

ISOLATION AND CHARACTERIZATION OF GLUCOCEREBROSIDES FROM ADULT RAT BRAIN1

Abstract— By chromatography on borate-coated silicic acid, glucocerebrosides, galactocerebrosides, sulfatides and sphingomyelins from brain tissue could be efficiently separated. Adult rat brain was found to contain 54.1 ± 1.5 nmol of glucocerebrosides per gram fresh weight. Ninety percent of the glucocere-broside fatty acids were palmitate, stearate and oleate; fatty acids with chain lengths above C₂₀ were virtually absent. No hydroxy fatty acids were found. The long chain bases of adult rat brain glucocerebrosides consisted of 74.6% C₁₈-sphingosine, 24.4% C₁₈-sphinganine and 1.1% C₂₀-sphingosine. These results are compared to those obtained from glucocerebrosides from immature rat brains (Abe & Norton , 1974) and discussed in respect to changes occurring during brain development.     

ACTH-SENSITIVE PROTEIN KINASE FROM RAT BRAIN MEMBRANES

—The protein kinase which in rat brain synaptosomal plasma membranes is responsible for the phosphorylation of a protein band B-50 (MW 48, 000) was inhibited by the behaviorally active peptide ACTH_1–24 and not stimulated by cAMP. Treatment with 0.5% Triton X-100 in 75 mM-KCl solubilized 15% of the total B-50 protein kinase activity and preserved the sensitivity of the enzyme to ACTH_1–24. The rate of endogenous phosphorylation of protein band B-50 was different in intact SPM, solubilized fraction and residue. cAMP stimulated the endogenous phosphorylation of the solubilized fraction in a rather general manner. The solubilized membrane material also phosphorylated B-50 proteins which were previously extracted from membranes. Column chromatography of the solubilized material over DEAE-cellulose pointed to the presence of multiple protein kinase activities from rat brain synaptosomal plasma membranes, one of which was the ACTH-sensitive B-50 protein kinase.     

CHANGES IN POLYSOMES OF THE DEVELOPING RAT BRAIN

Abstract— Rat brain polysomes were prepared from a deoxycholate-treated postmito-chondrial supernatant in the presence of 2% bentonite and 1 mg/ml of yeast RNA to prevent partial degradation during preparation.

• 1 The polysomal preparations had an absorption maximum at 260 mμ and an absorption minimum at 235 mμ. The ratio of absorption maximum to minimum and the RNA to protein ratio were 1·58 and 1·06 respectively in 6-day-old rat brain polysomes. The sedimentation patterns showed six distinct peaks with sedimentation coefficients of 235S, 185S, 173S, 135S, 100S and 80S, indicating that these preparations have the characteristics of pure heavy polysomes.

• 2 The rate of [¹⁴C]phenylalanine incorporation into brain polysomal protein was maximal at approximately 10 days of age and decreased thereafter. A similar progressive reduction with increasing age was found in the stimulation of phenylalanine incorporation by the addition of 60 μg/tube of polyuridylic acid. However, the incorporation of phenylalanine into young rat brain polysomes was usually greater even with the addition of polyuridylic acid than in the older animals.

• 3 The comparative studies on sucrose density gradient centrifugation of polysomes between young and adult rat brains showed a considerable decrease of heavy polysomes in the older animals.

• 4 The effect of various factors on the stability of brain polysomes from both ages has been studied. The rates of RNA, protein and acid-soluble phosphorus release from polysomes of the adult rat brains were usually greater in the presence of high salt concentration, ethylenediaminetetra-acetic acid and urea than those from the corresponding preparations of younger animals. On the basis of evidence obtained from the above results it suggested that the adult brain polysomes were more unstable than those of younger animals.

• 5 The amount of polysomal RNA linearly increased up to the first 20 days after birth and then levelled off. The ratio of G + C/A + U of polysomal RNA was less in the young rat brains, falling to 1·30 as compared to 1·50 in older animals. The differences were statistically significant at less than a 1% level of confidence.

• 6 Polysomal preparations also contained RNase, phosphomonoesterase, phospho-diesterase and 5′-nucleotidase activities which cannot be washed off. The specific activities of these enzymes were generally higher in young rat brains than those in the adult.

     

DNA-DEPENDENT RNA POLYMERASES FROM RAT BRAIN, AND RNA SYNTHESIS IN GLIAL AND NEURONAL CELL-ENRICHED FRACTIONS, AFTER PROTEIN RESTRICTION

Abstract— An assay for the dopaminergic agonist, apomorphine, is described. The assay is capable of detecting as little as 3 pmol of apomorphine. The basis of the assay is the O-methylation of apomorphine utilizing S-[methyl-³H]adenosyl-L-methionine and a partially purified catechol-O-methyl transferase to form [methyl-³H]apocodeine. The radiolabeled apocodeine is purified by solvent partition. Following systemic administration of apomorphine, the compound is uniformly distributed throughout the brain of the rat: there is no selective accumulation of apomorphine in dopaminergic regions of the brain. The level of apomorphine found in the substantia nigra is as great as the level found in the caudate-putamen. This latter observation, in addition to a variety of other evidence, raises the possibility that some of the in vivo effects of apomorphine may occur via an action of the drug within the substantia nigra, rather than by an action within the caudate-putamen.     

THE SYNTHESIS OF GLUTAMATE BY RAT BRAIN MITOCHONDRIA

The synthesis of glutamate from 2-oxoglutarate generated by the citric acid cycle and ammonium acetate has been studied in brain mitochondria of synaptic or non synaptic origin. Non synaptic brain mitochondria synthesise glutamate at twice the rate (1.3 nmol. min^?1. mg protein^?1) of synaptic mitochondria (0.65 nmol. min^?1. mg protein^?1) when pyruvate is the precursor for 2-oxoglutarate, but at a similar rate (0.9 and 0.7 nmol. min^?1, mg protein^?1) when 3 hydroxybutyrate is the precursor. Glutamate synthesis from ammonium acetate and extramitochondrially addcd 2-oxoglutarate (5 mM) by both synaptic and nonsynaptic mitochondria was 5-fold higher (5-6nmol. min^?1. mg protein^?1) than glutamate synthesis from endogenously produced 2-oxoglutarate. In the uncoupled state (or un-coupler + oligomycin) the rate was reduced by half. (2.5-3 nmol. min^?1. mg protein^?1) as compared to mitochondria synthesising glutamate in states 3 or 4 (± oligomycin). The changes in brain mitochondrial nicotinamide nucleotide redox state have been monitored by fluorimetric, spectrophotometric and enzymatic techniques during glutamate synthesis and compared with liver mitochondria under similar conditions. On the instigation of glutamate synthesis by NH⁺₄ addition a significant NAD(P)H oxidation occurs with liver mitochondria but no detectable change occurs with brain mitochondria. Leucine (2 mM) causes a doubling of glutamate synthesis by both synaptic and non synaptic brain mitochondria with no detectable change in the NAD(P)H redox state. The results are discussed with respect to the control of glutamate synthesis by mitochondrial redox potential and the possible intramitochondrial compartmentation of this process.     

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.     

QUANTITATIVE ISOLATION AND PROPERTIES OF NEARLY HOMOGENEOUS POPULATIONS OF UNDEGRADED FREE AND BOUND POLYSOMES FROM RAT BRAIN1

Abstract— A procedure is described for the preparation of free and bound polysomes from whole homogenate of rat brain tissue. Brain is homogenized in a sucrose-polysome buffer medium high in KCl (250 mm). After a 12-min centrifugation at 135,000 g, the free polysomes in the supernatant are decanted and saved, while the membrane bound polysomes in the pellet are resuspended in homogenizing medium, homogenized in the presence of detergent (Triton X-100), centrifuged for 5min at 1470 g to remove nuclei, decanted, treated with deoxycholate and centrifuged for 10 min at 24,000 g to remove deoxycholate-insoluble material. Polysomes in the two supernatants are harvested by centrifugation through sucrose gradients prepared in high KCl polysome buffer, and with or without cell sap. Free and bound polysomes prepared in this manner are undegraded, equally active in cell-free protein synthesis, and largely free of the usual contaminants. Cross-contamination is minimal (>10%). The recovery of polysomes is at least 95%. The distribution of ribosomes and polysomes in rat brain is 58% free and 42% membrane-bound. The distribution of rat brain RNA is 65% ribosomal and 35% non-ribosomal. Conditions are described for the visualization and analysis of the entire complement of free and bound ribosomes. The size fractionation procedure is rapid and reproducible, requires much less ultracentrifugation than the density-gradient technique, and provides a nearly quantitative means of isolating undegraded free and bound polysomes of rat brain tissue.     

NUCLEAR PROTEIN PATTERNS IN DEVELOPING AND ADULT BRAIN AND IN ETHYLNITROSOUREA-INDUCED NEUROECTODERMAL TUMOURS OF THE RAT1,2

In a comparative study, the patterns of histones and non-histone proteins were analysed in the chromatin of foetal (18th day of gestation), 10-day-old, and adult BD IX-rat brain, as well as in the chromatin of two ethylnitrosourea-induced neuroectodermal tumours (TV1A1 and GV1A1) and the corresponding malignant cell culture lines TV1C1 and GV1C1. Separation of nuclear proteins at high resolution was obtained by electrophoresis in 15% and 10% polyacrylamide gels containing urea (2·5 m or 6·25 m ). In spite of an overall similarity, significant quantitative and qualitative differences were observed between the respective non-histone proteins banding patterns of normal brain and the neoplastic cells analysed. The non-histone protein banding patterns of brain (∼40 different bands) at different stages of development revealed both quantitative differences and the presence of particular bands characteristic of foetal or adult brain, respectively. Both the‘foetal’and‘adult’non-histone protein bands also appeared in the electrophoretograms of the neoplastic neuroectodermal cells.     

IN VITRO BIOSYNTHESIS OF TUBULIN ON TOTAL, FREE AND MEMBRANE-BOUND POLYSOMES FROM THE DEVELOPING RAT BRAIN

Abstract— Incorporation of [³H]leucine into tubulin and total protein was examined using a polysomal system from newborn (1-day-old). young (10-day-old) and adult (3-month-old) rat brains and cerebral cortices. The rate of tubulin biosynthesis (specific radioactivity) was always lower than that of total protein biosynthesis. No significant differences in the specific radioactivities of the synthesized total proteins were found between the newborn and young brain polysomal system, although young cerebral cortical polysomes were less active than newborn cerebral cortical polysomes. The adult brain (or cerebral cortical) polysomes were less active, about 20-30% lower than the young brain (or cerebral cortical) polysomes. The incorporation of [³H]leucine into tubulin showed a progressive decrease in the polysomal systems isolated from the newborn, young and adult rat brains and cerebral cortices. These tendencies were similar in every cell sap taken from newborn, young and adult rat brain homogenates.
In order to examine the relative activities of free and bound polysomes of the developing rat brain in tubulin biosynthesis. double-labelling experiments were carried out. Labelled tubulin was purified by the assembly and disassembly method, followed by SDS gel electrophoresis, or by vinblastine precipitation method, followed by SDS gel electrophoresis; then identification by co-electrophoresis with native brain tubulin, molecular weight determination and demonstration of specific aggregation in the presence of GTP followed. Free and bound polysomes showed approximately similar activities during tubulin biosynthesis. Furthermore, relative activities of tubulin biosynthesis by free and bound polysomes did not significantly change during development.     

FRACTIONATION OF THE RNA COMPONENTS OF RAT BRAIN POLYSOMES

Abstract— The incorporation in vivo of [³H]uridine into the RNA isolated from the free polyribosomes of rat cerebral cortex was studied. Sedimentation in sucrose gradients showed that initially (at times less than 60 min after injection of precursor) the label was associated with a heterodisperse species, while at longer times there was an increased coincidence of label with stable rRNA. Further fractionation was accomplished by means of differential extraction with phenol and analysis on polyacrylamide-agarose gels. Most of the rapidly labelled RNA was concentrated in a fraction obtained at pH 8-3 and 40°C. The base composition of this fraction differed greatly from that of rRNA, preribosomal RNA and DNA. Analysis by electrophoresis on polyacrylamide-agarose gels showed it to be composed of several distinct species in addition to residual 18 and 28S rRNA. Most of the latter was concentrated in a fraction extracted at pH 60 at 0°C.     

RIBOSOMAL SUBUNITS FROM NEONATAL MOUSE BRAIN HIGHLY ACTIVE IN POLYPHENYLALANINE SYNTHESIS

Abstract— A highly active subcellular protein synthesising system is described, in which uncomplexed ribosomes isolated from 5 to 7 day old mouse brain can be reprogrammed with polyuridylic acid. Either purified free polyribosomes or microsomes were used as the starting material for the preparation of uncomplexed ribosomes by treatment with 0.5 m -KCl and puromycin. After reduction of the salt concentration 80S ribosomes were isolated by washing through sucrose. When, subsequently, zonal centrifugation in equivolumetric sucrose gradients containing 0.5 m -KCI was performed, purified ribosomal subunits were obtained. Cross-contamination of subunits was less than 5%. Re-associated ribosomes and recombined isolated ribosomal subunits both showed high activities in vitro. Incorporation levels of 50–60 phenylalanine residues per ribosome could be reached, at a rate of 0.5–2.0 residues/min/ribosome, depending on the activity of the high speed supernatant enzymes added. It was shown by paper chromatography of the cell-free product that only oligophenylalanine formation takes place. It was estimated that 6&70% of the ribosomes present in vitro were actively participating in the protein synthesis process.     

CTP SYNTHETASE ACTIVITY IN NEONATAL AND ADULT RAT BRAIN

—The activity of CTP synthetase (UTP:ammonia ligase (ADP-forming), EC 6.3.4.2) in adult and new-born rat brain was determined by an enzyme assay using [¹⁴C]UTP as a substrate. The activity was age-dependent and showed a distinctive distribution pattern between the cerebral hemispheres and cerebellum. The possible correlations between the activity of CTP synthetase and the rate of RNA or lipid biosynthesis, as well as the regulatory importance of the enzyme in the formation of cytidine nucleotides are discussed.     

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.