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Methylarginines in free form were identified in bovine brain. Three compounds were isolated from the basic aliphatic amino acid fraction of bovine brain with several ion-exchange chromatographies. They showed the same Rf values in paper and thin-layer chromatographies as those of authentic NG-monomethylarginine, NG,NG-dimethylarginine, and NG,N'G-dimethylarginine. The migration distance of the isolated compounds in high-voltage paper electrophoresis and the retention times in ion-exchange HPLC were also identical to those of the above authentic methylarginines. We concluded that these three compounds are the methyl derivatives of arginine described above. The amount of these three compounds isolated from 1,090 g of bovine brain was 0.3 mumol of NG-monomethylarginine, 0.1 mumol of NG,NG-dimethylarginine, and 0.5 mumol of NG,N'G-dimethylarginine. The occurrence of these free methylarginines may have an important role in regulating the signal transduction through the nitric oxide system.

—3-Methoxy-4-hydroxyphenylglycol (MHPG) formed a sulphate conjugate when incubated with ATP, Mg2+ ions, Na235SO4 and the high-speed supernatant preparations of rabbit or rat brain. The same reactions could be catalysed by similar enzyme preparations from liver. The sulphated product was separated and identified by paper chromatography. On acid hydrolysis, it released both Na235SO4 and the free glycol. The measurement of this labelled sulphate was used as a specific assay procedure for determining the overall sulphoconjugatory process. The pH optimum of the reaction is 7.8. For rabbit brain, the Km for Na2SO4 determined for the activating system is 3.6 × 10−4m, and that for MHPG for the sulphotransferase reaction is 1.05 × 10−4m. The specific enzyme activity, expressed as nmol 35SO4 incorporated/h/mg protein for a 30-min assay is as follows: rat brain, 2.8; rabbit brain, 1.6; rat liver, 33.4and rabbit liver, 15.0. Dithiothreitol at 3 mm concentration had no significant effect on the sulphation of MHPG in all these preparations.

—The uptake of l[14C]glutamine by a crude isolated nerve ending fraction of rat brain was found to be linear with time for at least 5 min, profoundly temperature-dependent, apparently half-saturated at a substrate concentration of 0·26 mm, partially inhibited by dinitrophenol and ouabain and elevated [K+], weakly Na+-dependent, poorly inhibited by drugs which block uptake of biogenic amines and more strongly inhibited by glutamic acid (IC50= 0·5mm) than by aspartic acid, GABA, glycine or methionine. The [14C]glutamine taken up appeared to be associated with nerve endings and was released by membrane-disruption; about 20 per cent was associated with free mitochondria. Glutamine, δ-aminolevulinic acid and several other amino acids were poor inhibitors of [3H]GABA-uptake; δ-aminolevulinic acid was a poor inhibitor of [3H]glutamine-uptake, whereas glutamine was a moderately effective competitive inhibitor (Ki= 1 mm). [14C]glutamine and [3H]GABA were released from brain slices by electrical stimulation or 50 mm K+, while labeled δ-aminolevulinic acid, leucine, urea, amphetamine and tyramine were poorly released. [14C]glutamine was not released by unlabeled glutamate or several aromatic amines. We conclude that the neuropsychiatric features of porphyria are not likely due to a ‘false transmitter’ role for δ-aminolevulinic acid although such a role for glutamine in hepatic encephalopathy or other neuropsychiatric diseases should be considered.

—Effects of acute or chronic administration of ethanol and its withdrawl on the steady-state levels and turnover rates of certain neurotransmitters have been investigated in mice. The influence of long-term administration of ethanol on the activities of enzymes involved in the metabolism of these transmitters has also been studied. Acute administration of ethanol or acetaldehyde or chronic administration of ethanol resulted in a decrease in the cerebral contents of acetylcholine, acetylCoA and CoA. Brain levels of 5-hydroxytryptamine, norepinephrine and choline remained unchanged after acute administration of ethanol. However, chronic administration of ethanol resulted in a decrease in the norepinephrine content without significantly affecting 5-hydroxytryptamine or choline contents. Cerebral levels of γ-aminobutyric acid increased with both acute or chronic administration of ethanol. The total incorporation of [3H]choline into acetylcholine in brain was depressed upon acute administration of ethanol. After withdrawal of ethanol for one day cerebral levels of norepinephrine returned to normal; however, γ-aminobutyric acid and acetylcholine returned to normal levels at 2 and 4 days after ethanol withdrawal, respectively. Pretreatment of mice with pyrazole, an inhibitor of alcohol dehydrogenase, prevented the ethanol-induced decrease in cerebral acetylcholine levels. The activities of cerebral choline acetyltransferase and glutamic decarboxylase were decreased after 2 weeks of chronic ethanol administration. However, the activities of acetyl cholinesterase and GABA-transaminase remained unaffected after 2 weeks of ethanol treatment

—[14C]-Glutamate and [14C]-glutamine were incorporated into calf brain tRNA in the presence of homologous aminoacyl-tRNA synthetases. When the tRNAs were then deaminoacylated and chromatographed, a number of radioactive products were found in addition to the original amino acids. One of the products of glutamate transformation was identified to be glutamine. Formation of the radioactive products of glutamate in the presence and absence of tRNA indicated that glutamine was produced from glutamate at the level of the free amino acid followed by the incorporation of both substances into tRNA. Examination of the products of deaminoacylation of glutaminyl-tRNA showed that glutamine underwent structural alterations at the level of the aminoacyl-tRNAs to give rise to a cyclic derivative of glutarimide. This reaction was specific for glutamine, and constituted approximately 15 per cent of the total radioactivity in the deaminoacylation products of glutaminyl-tRNA.

— 1 Slices of mouse brain were incubated with [U-14C]alanine, valine, leucine, phenylalanine, proline, histidine, lysine, arginine or aspartic acid, and the extent of metabolism was estimated by analyses utilizing paper chromatography of the tissue extracts and with an amino acid analyser. 2 The metabolism of Ala and Asp was high; of Leu and Pro, moderate; and of Lys, Arg and Phe, low; the metabolism of Val and His was not significant. The time-course of metabolism in most cases showed varying rates, indicating heterogeneous metabolic compartments for the amino acids. 3 Production of CO2 was high from Asp, moderate from Ala, and low from Leu; the other amino acids were not oxidized to CO2 to any significant extent. A large portion of the metabolized label was trapped in the form of Glu or Asp. 4 Metabolism increased with increasing concentration of amino acid to some extent and was largely inhibited by omission of glucose, by anaerobic conditions, or by cyanide. Although these conditions also inhibit uptake, the time-course and extent of inhibition uptake and metabolism were different. 5 With Asp, Ala and Phe, metabolism was lowest in slices from pons-medulla; the brain area exhibiting the highest metabolism differed for each amino acid. The metabolism of Asp was lower in brain samples from newborn than in those from adults; the metabolism of Leu was higher in slices from newborn brain. 6 The results indicate that the majority of the amino acids can be metabolized in brain tissue and that the metabolic rates are influenced by a number of factors, among them the level of amino acids and the level of available energy.

— Radioactive glutamate, glutamine, aspartate and asparagine were incorporated into calf brain tRNA in the presence of homologous aminoacyl-tRNA synthetases. When the aminoacyl-tRNAs were deaminoacylated and the products chromatographed in the phenol solvent, the glutaminyl-and asparaginyl-tRNAs showed two products GnP2 and AnP2 respectively, in- addition to the original amino acids. These new substances moved close to the solvent front in contrast to glutamine and asparagine which had much lower RF values. Attachment of the amino acids to tRNA appeared to be a prerequisite for the formation of these substances, since they were not found in the reaction mixture used for aminoacylation in the absence of incubation or on omission of tRNA or when tRNA was degraded by RNase. Application of the deaminoacylation procedure to pure amino acids also failed to lead to their formation. In an other series of experiments, the dicarboxylic aminoacyl-tRNAs were hydrolysed with pancreatic RNase and then analysed by high voltage paper electrophoresis. Again, the glutaminyl- and asparaginyl-tRNAs showed two new components, GnE3 and AnE3, in addition to the expected glutaminyl- and asparaginyladenosines. GnE3 and AnE3 exhibited much faster electrophoretic mobilities in the direction of the cathode than the adenosine derivatives of the original amino acids and thus appeared to be more positively charged. The presence of these new compounds in the products of deaminoacylation or in R Nase hydrolysates was specific to glutaminyl- and asparaginyl-tRNAs and did not occur in the case of either glutamyl or aspartyl-tRNAs, indicating that the amide group was probably involved in the transformation reaction.

— Glutamyl-, glutaminyl-, aspartyl- and asparaginyl-tRNAs were separated into different isoacceptor species by reverse phase column chromatography. RNase hydrolysates of any of the isoacceptor [14C]aminoacyl-tRNAs for a given amino acid gave radioactivity profiles, on paper electrophoresis, very similar to unfractionated tRNA. This suggested a lack of tRNA specificity for the transformation reaction involving the aminoacyl moieties of asparaginyl and glutaminyl-tRNAs. GnP2 and AnP2 detected in the products of deaminoacylation of glutaminyl and asparaginyl-tRNA showed a number of properties in common with GnE3 and AnE3 present in the RNase hydrolysates of the same tRNAs. Thus, GnP2 and GnE3 chromatographed in the same position in the phenol: water solvent and both yielded glutamate on acid hydrolysis and a mixture of glutamine and isoglutamine on alkaline hydrolysis. Similarly, AnP2 and AnE3 had the same RF value in phenol :water chromatography and gave aspartate or a mixture of asparagine and isoasparagine when hydrolyzed with acid or alkali. On the basis of these results and other evidence, GnP2 and GnE3 were assigned the structure, α-aminoglutarimide; AnP2 and AnE3 were identified as α-aminosuccinimide. These cyclic compounds are presumed to be formed by nucleophilic attack of the amide nitrogen of asparagine or glutamine on the carbon of the aminoacyl ester carbonyl group. The cyclization-deesterification appeared to be facilitated by RNase hydrolysis of aminoacyl-tRNA indicating that the aminoacyl-tRNA is probably more resistant to this reaction than aminoacyladenosine. Neither the imides nor the amino acid isoamides were detected in the reaction mixture in which aminoacylation of tRNA was performed, suggesting that a mechanism may exist for inhibition of the cyclization reaction under conditions of active aminoacylation.

—An enzyme from rat brain catalysing the synthesis of the histidine-containing dipeptides carnosine and homocarnosine (l.-histidine: β-alanine ligase (AMP) [EC 6.3.2.11]) was purified about 30-40-fold from a 100,000 g supernatant. Assays were conducted by measuring the incorporation of L-[14C]histidine into carnosine and homocarnosine isolated by paper electrophoresis from the incubation mixture. The ratios of specific activities for the formation of carnosine and homocarnosine were not significantly different for the various purification steps. This was taken as evidence of one enzyme synthesizing both dipeptides. In studying the properties of this enzyme, a pH optimum of 7.4 was shown for carnosine synthesis. The concentrations of amino acid substrates giving maximal synthesis of both dipeptides were in the physiological range found for rat brain. An apparent requirement for ATP, Mg2+, and DPN was seen for dipeptide synthesis. A substrate dependent, enzymecatalysed 32PPi-ATP exchange reaction was observed, suggesting the formation of an aminoacyl-AMP intermediate. Certain other nucleoside triphosphates could substitute for the ATP; this effect showed a specificity toward the dipeptide being synthesized. The apparent requirement for DPN was quite specific, with a number of related compounds having no effect. The stoichiometry of enzyme-catalysed carnosine synthesis was studied. A one to one relationship between carnosine formed and ATP hydrolysed was demonstrated. However, the ratio between carnosine synthesized and DPN hydrolysed was about 6 to 1, indicating a catalytic role for the DPN. The breakdown of DPN did not occur with enzyme alone but was dependent on the presence of substrate.

Kynurenine formamidase (aryl-formylamine amidohydrolase, EC 3.5.1.9) was found to be present in rat brain and was partially purified and characterized. The partially purified enzyme catalysed the hydrolysis of 5-hydroxyformyl-dl-kynurenine to 5-hydroxy-dl-kynurenine and that of formyl-l-kynurenine to l-kynurenine at similar rates. The apparent Km values of the enzyme for 5-hydroxyformyl-dl-kynurenine and formyl-l-kynurenine were 4.0 ± 10−4 and 1.8 ± 10−4m, respectively. The enzyme was active over a wide pH range (5.5–8.5). The activity was inhibited by low concentrations of Ag+ and Hg2+. The physiological significance of the enzyme is discussed.

— A simple, rapid, sensitive and economical assay technique has been developed for any enzyme whose substrate and product differ by charge. It is based on a combination of rapid vacuum filtration with an ion exchange resin. The technique has been successfully applied to brain glutamate decarboxylase.

— The formation of N-acetyl-aspartyl-peptides in mouse brain homogenates is described. The formation is completely dependent on histamine and on an ATP-regenerating system, and partially dependent on the addition of N-acetyl-aspartate and certain other amino acids. The N-acetyl-aspartyl-peptide formation is probably non-ribosomal and is apparently not due to hydrolysis of preformed proteins.

—The accumulation of [3H]choline into synaptosome-enriched homogenates of rat corpus striatum, cerebral cortex and cerebellum was studied at [3H]choline concentrations varying from 0.5 to 100 μm. The accumulation of [3H]choline in these brain regions was saturable. Kinetic analysis of the accumulation of the radiolabel was performed by double-reciprocal plots and by least squares iterative fitting of a substrate-velocity curve to the data. With both of these techniques, the data were best satisfied by two transport components, a high affinity uptake system with Km. values of 1.4 μM (corpus striatum), and 3.1 μM (ceμ(cerebral cortex) and a low affinity uptake system with respective Km. values of 93 and 33 μM for these two brain regions. In the cerebellum choline was accumulated only by the low affinity system. When striatal homogenates were fractionated further into synaptosomes and mitochondria and incubated with varying concentrations of [3H]choline, the high affinity component of choline uptake was localized to the synaptosomal fraction. The high affinity uptake system required sodium, was sensitive to various metabolic inhibitors and was associated with considerable formation of [3H]acetylcholine. The low affinity uptake system was much less dependent on sodium, and was not associated with a marked degree of [3H]acetylcholine formation. Hemicholinium-3 and acetylcholine were potent inhibitors of the high affinity uptake system. A variety of evidence suggests that the high affinity transport represents a selective accumulation of choline by cholinergic neurons, while the low affinity uptake system has some less specific function.

— The aminotransferase activity of homogenates of brains from adult and neonatal rats has been investigated. Aminotransferase activity was demonstrated wtih 15 of 22 amino acids incubated with seven keto acids. The basic amino acids exhibited little or no activity. 1 The greatest activity was obtained when glutamate or aspartate was incubated with α-ketoglutarate or oxaloacetate. Significant activity was also observed when the neutral aliphatic and aromatic amino acids were incubated with these two keto acids. 2 Activity with pyruvate was obtained principally upon incubation with glutamate and alanine. Most of the other amino acids that underwent transamination with α-ketoglutarate also did so with pyruvate, although at a lower rate. 3 When phenylpyruvate was added to the medium, glutamate, phenylalanine and tyrosine transaminated most actively. 4 Incubations with 11 amino acids and glyoxylic acid demonstrated aminotransferase activity, with glutamate and ornithine being the most active substrates. 5 α-Ketoisocaproate and α-ketoisovalerate accepted amino groups primarily from the branched-chain amino acids. Except for glutamate, activity with other amino acids was low or not detectable. 6 A comparison of aminotransferase activity in the newborn brain with that in the adult brain showed that the greatest change in activity occurred for glutamate with pyruvate or for alanine with α-ketoglutarate, these activities increasing about 10-fold from birth to adulthood; during this time activities with most other amino acids increased two- to threefold. Amino transfers from the branched-chain amino acids showed no increase with maturation, and some reactions, such as that with methionine and a number of keto acids, decreased from birth to adulthood. 7 Our results correspond in general to previous studies of aminotransferase activity in brain and in liver. However, our study also indicates a possible second aminotransferase acting on the branched-chain amino acids, the presence of aminotransferase activity for methionine and asparagine, and relatively high aminotransferase activity for glutamine or ornithine when incubated with glyoxylic acid rather than other keto acids. Moreover, phenylpyruvate and glyoxylate are active in amino transfers and may serve as substrates for a number of aminotransferases.

— Putreanine was proven to be formed from spermidine in rat brain by intra-ventricular injection of radioactive spermidine. The ability to form putreanine from spermidine was also found in rat liver. The turnover rate of putreanine was slower in rat brain than in rat liver.

Significant quantities of corticosterone were associated with macromolecules of the brain cytosol following intrathecal administration of [3H]corticosterone to adrenalectomized rats. Fifteen times more steroid was found associated with proteins from adrenalectomized rats than from control animals or adrenalectomized animals pretreated with corticosterone. Pretreatment of adrenalectomized rats with 11-dehydrocorticosterone, Cortisol and cortisone decreased the amount of [3H]corticosterone found associated with protein, whereas progesterone, oestradiol and testosterone did not interfere with the association of [3H]corticosterone with macromolecules of the cytosol. Further evidence for protein-steroid interaction was obtained by incubating [3H]corticosterone (B), [3H]cortisol (F), or 11-[3H]deoxycortisol (S) with brain cytosols. The degree of binding was in the order B > F > S.

— The de novo biosynthesis of purine nucleotides in rat brain was markedly and rapidly inhibited by intracerebral injections of azaserine. After 48 hr the animals became paraplegic. The biosynthesis of purine and pyrimidine nucleotides, RNA and protein was investigated by radioactive tracer studies over a 4-day period in an attempt to correlate the azaserine-induced, metabolic lesions found in rat brain and the manifested, peripheral, neurological disturbance.

—(1) Phenylalanine, proline and presumably tyrosine are precursors of the small glutamate pool in brain. This follows from the finding that with these precursors the specific radioactivity of glutamine is higher than the specific radioactivity of glutamate. (2) Glucose is not as efficient a precursor of glutamate and related amino acids in the brain of 10-day-old mice as it is in the adult brain. (3) Acetate, phenylalanine, tyrosine and proline are incorporated to about the same extent in glutamate, aspartate and glutamine in the brains of 10-day-old and adult mice. (4) The results suggest that the brain of the immature animal uses substrates other than glucose, relative to glucose better than the brain of adult animals.

— Labelled fucose administered to rats in vivo was rapidly incorporated into brain glycoproteins, but not into any other brain constituents, including glycolipids and acid mucopolysaccharides. Maximum incorporation of tritium-labelled fucose into brain glyco-proteins occurred 3–6 h after intraperitoneal injection in young or adult rats, and the half-time for the turnover of glycoprotein-fucose in young rats was approximately 2 weeks. Within 3 h after the administration of either [1-3H]fucose or fucose generally labelled with tritium, 75 per cent of the total acid-soluble radioactivity in plasma and brain was found to be volatile, and by 24 h after injection more than 90 per cent of the acid-soluble radioactivity was volatile. The tritium in labelled fiicose appears to undergo arapid exchange reaction with hydrogen atoms in body water, although the tritium in fucose glycosidically- linked to glycoproteins is biologically stable. The rapid disappearance of labelled free fucose from the plasma and tissues of the rat precludes the possibility of any significant degree of reutilization of labelled precursor, and provides support for other data indicating that the turnover of fucose in brain glycoproteins is relatively slow in comparison to that of hexosamine and sialic acid. Activities of α-L-fucosidase in rat brain, with pH optima at 40 and 6.0, had essentially the same Km (4 × 10−4 M and 3.2 × 10−4 M, respectively) with p-nitrophenyl-α-L-fucopyranoside as substrate. Activities of both were competitively inhibited by L-fucose. However, the Kt measured at pH 4 (1.9 × 10−2) was almost ten times greater than that measured at pH 6 (1.5 × 10−4).

— Slices from various regions of rat brain, incubated at 25°C, rapidly accumulate [3H]GABA from the surrounding medium until after 60min tissue:medium ratios as high as 300 may be achieved. Kinetic analysis has demonstrated two distinct uptake systems for GABA in all the brain regions examined. One system has a relatively high substrate affinity (Km= 1.2 ± 10-5 M) while the other has a lower affinity (Km= 4 ± 10-4 M). Studies at low GABA concentration (5 ± 10-8 M), as well as estimates of maximum velocities, have shown that the distribution of the high affinity uptake system is heterogeneous. Cortex, hypothala mus, midbrain and hippocampus have relatively high uptake rates while the striatum, cerebellum and pons and medulla have a lower uptake rate. Maximum velocities for the low affinity uptake system show much less regional variation. Lithium, either added to the incubation medium or fed to rats, had no effect on the uptake of GABA by cortical slices.

— Further investigations have been made into ergothioneine in the brains of several mammalian species, and the distribution of ergothioneine in the brain of the ox is described. It has not been possible to confirm many of the findings of earlier workers and the results do not appear to support their conclusion that ergothioneine is identical with the cerebellar factor.

— 1 The in vivo metabolism of glutamate in rat neuron cell bodies and neuropil was studied after intraventricular injection of (U-14C)glutamic acid followed by separation of the tissue into neuronal and neuropil fractions. 2 The losses of amino acid and of radioactivity during the fractionation were equivalent. Recoveries were: glutamate, 32; glutamine, 15; aspartate, 25; GABA, 41; alanine, 30 per cent. In the washed cell fractions glutamine was 45 per cent and alanine 132 per cent higher in the neuronal fraction, glutamate was 62, GABA 77 and aspartate 95 per cent of neuropil levels. This contrasted with results obtained previously for in vitro incorporation. Calculation from these results indicated that 28 per cent of the original cell suspension was neuronal, 72 per cent neuropil. In the final cell preparations, 29 per cent of the neuron cell bodies and 26 per cent of the neuropil were recovered. 3 Specific activity of glutamate in the neuronal fraction 15 min after injection was higher than in the original suspension, but had declined to 30 per cent of its initial value by 2 h. In the neuropil, specific activity of glutamate was below that of the cell suspension at 15 min, but at later times rose above it by up to 40 per cent. 4 Radioactivity was detected in aspartate and glutamine 15 min after injection and GABA by 60 min after injection. In the original cell suspension the specific activity of glutamine was higher than that of glutamate at all times (the Waelsch effect) but aspartate and GABA were lower than glutamate. 5 In the neuronal fraction the specific activity of glutamine was below that of glutamate at all times, indicating a precursor-product relationship. In the neuropil fraction, glutamine specific activity remained above glutamate for the first hour. 6 These results are discussed in relation to the interpretation of the Waelsch effect in terms of metabolic compartmentation.

— 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.

—Using either tryptophan or 5-hydroxytryptophan as the precursor, and examining the metabolites in whole rat brain and in brain regions of dog, the pattern of metabolites resembled that found under physiological conditions only after tryptophan administration. From these and other observations on the cerebral 5-hydroxyindoles the main conclusions are firstly, that there are regional differences within brain in storage, turnover or metabolic fate of 5-HT. Secondly, that the normal pathway appears to be well localized biochemically with linking of its succeeding steps, and thirdly, that turnover through the system is normally controlled by intracerebral tryptophan 5-hydroxylase in both rats and dogs although there are differences between the species in the cerebral metabolism of 5-HT.

— Some metabolic and biochemical effects of fluorocitrate were studied in vivo in rat brain and cat spinal cord. During the preconvulsant and convulsant phases of fluorocitrate poisoning the contents of free glutamate, glutamine and aspartate declined progressively, while that of alanine increased. Incorporation of 14C from [U-14C]glucose into these amino acids also decreased, although somewhat more gradually. GABA exhibited a biphasic change, its content rising after an initial decrease while its relative specific activity rose initially and subsequently diminished. Incorporation of 14C from [U-14C]glucose and [U-14C]lysine into neural protein declined sharply. The citric acid content rose markedly in rat brain and cat spinal cord. In rat brain the glycogen content declined but ATP and ammonia contents were unchanged. The significance of these results with respect to energy metabolism and the possible mechanism of the convulsions during fluorocitrate poisoning is discussed.

— Adenosine metabolism in the homogenate of brain mainly undergoes deamination to inosine and hypoxanthine, while uniformly labelled [14C]adenosine injected into the carotid artery or [8-14C]adenosine incubated with brain slices was mostly phosphorylated to [14C]adenine nucleotides in brain cells. Adenosine kinase has now been partially purified from homogenates of guinea pig brain. The kinase preparation was free of adenosine deaminase, almost free of adenosine triphosphatase and had a Km of the order of 2 × 10-5M for adenosine. Kinetic studies with brain slices showed that adenosine reached the cells by diffusion and that the diffusion was facilitated by subsequent phosphorylation to adenine nucleotides. From the following experimental results, it is concluded that the phosphorylation is catalysed by adenosine kinase quantitatively. (1) During the uptake and phosphorylation of adenosine by brain slices, the nucleoside did not split to adenine and ribose moieties. (2) The rate of formation of adenine nucleotides in the slices was a hyperbolic function of the concentration of adenosine in the medium, showing an apparent Km foradenosine of the order of 2 × 10-5 M. (3) Some analogues of adenosine inhibited both the facilitated diffusion of adenosine and the kinase activity, but ouabain (0.005 mM) did not inhibit either.

SUMMARY A study was conducted on the in vivo incorporation of l-[14C]-serine into the lipids and proteins of the various subcellular fractions of the developing rat brain before and during the stage of active myelination. The total radioactivity in the various fractions at 12 days of age was higher than that at 3 days, while the radioactive specific activity was reversed. The specific activities of the proteins and lipids were higher at 3 days of age with the exception of the subcellular fraction containing myelin. At both ages the lipids of the various cellular fractions had similar specific activities, a finding that suggests a common source for lipid biosynthesis. Incorporation of radioactivity into the various phospholipids was in the following order: phosphatidyl serine > phosphatidyl ethanolamine > phosphatidal serine > sphingomyelin and phosphatidyl choline. Of all the phospholipids, the plasmalogens increased most in total radioactivity during the period when meylination was most active. Serine-containing phospholipids appear to be most tightly bound to proteins. The brain mitochrondrial fraction contained most of the phosphatidyl serine decarboxylase activity with some activity in the nuclei. Biosynthesis of phosphatdyil ethanolamine through decarboxylation of phosphatidyl serine could take place in rat brain. Four unidentified radioactive metabolites were found in the acid-soluble fraction in addition to l-[14C]serine.

— The distribution of tryptophan hydroxylase in the cat brain was investigated and found to parallel roughly the distribution of serotonin. The most active areas are the caudate nucleus, septal area, anterior perforating substance, hypothalamus, amygdala and various areas of the midbrain.

— Synaptosomes from guinea-pig cerebral cortex contain a fetuin: sialyl glyco-protein: glycosyl transferase; evidence is presented which indicates that both a sialyl transferase; evidence is presented which indicates that both a sialyl transferase and endogenous acceptors were located in the synaptosome ‘ghost’ fractions. Following solubilization of synaptosomes with Triton X-100 and the use of fetuin minus NANA as acceptor, 25 per cent of the transferase was recovered after centrifugation and column chromatography on Sephadex G-100 and G-200 with a 64·0-fold purification. The enzyme had a pH optimum of 6·3, required no divalent metal cation for activity, and exhibited high activity with either fetuin minus sialic acid, prothrombin minus sialic acid, Tamm-Horsfall glycoprotein minus sialic acid, or orosomucoid minus sialic acid as acceptor; neither BSM nor PSM minus NANA functioned as an effective acceptor. The fetuin:sialyl transferase using fetuin minus sialic acid and CMP-sialic acid as substrates a and b, respectively, gave the following kinetic constants when using the Cleland bisubstrate model: Ka= 35μM; Kb= 3 μM; Kia, = 25 μM; Kib= 25μM; and V1= 92 pmoles. min−1.mg−1 of protein. The following divalent cations inhibited the reaction: Ba2+ > Hg2+ > Pb2+ > Cu2+.

—The enzymatic decarboxylation of l-DOPA was measured in isotonic dextrose homogenates of different regions of the human brain by estimating 14CO2 evolved from tracer amounts of d l-DOPA[carboxy1-14C]. Enzyme activity was linear with respect to tissue concentration and time of incubation. The reaction exhibited a pH maximum at 7·0, was completely dependent upon the presence of high concentrations of pyridoxal phosphate, proceeded at the same rate in an atmosphere of air and nitrogen, and produced dopamine in addition to CO2 as a reaction product. The enzyme preparation behaved like an aromatic l-amino acid decarboxylase: it also decarboxylated o-tyrosine and when incubated with 5-hydroxytryptophan, serotonin was isolated as the reaction product; but it was devoid of activity towards d-DOPA[carboxy1-14C]. Within the human brain, l-DOPA decarboxylase was most active in the putamen and caudate nucleus; the pineal gland, hypothalamus, and the reticular formation and dorsal raphe areas of the mesencephalon exhibited considerable activity. Areas of cerebral cortex exhibited very low enzymatic activity and in regions composed predominantly of white matter, l-DOPA decarboxylase activity was not significantly above blank values. The activity of l-DOPA decarboxylase in the human putamen and caudate nucleus tended to decrease with the age of the patients; in comparatively young subjects (46 yr old) the enzyme activity compared favourably with that found, by means of the same assay technique, in the caudate nucleus of the cat.

— In further experiments on the effects of antibiotic agents on protein synthesis in the goldfish brain, doses of intracranially-injected puromycin or acetoxycycloheximide higher than those previously employed did not hasten the onset of inhibition of incorporation of intraperitoneally-injected [3H]leucine into brain protein. The antibiotic-resistant incorporation was not due to the presence of labelled blood protein in the brain. After the intracranial injection of labelled puromycin, the appearance of radioactivity in the acid-soluble fraction of brain was blocked by acetoxycycloheximide. Repeated daily intracranial or intraperitoneal injections of puromycin were lethal, and acetoxycycloheximide was not protective, indicating that peptidyl-puromycin was present in the brain but did not account for the lethality of puromycin. Behavioural experiments argued against but did not totally exclude the possibility that peptidyl-puromycin was responsible for the amnestic effect of puromycin. Puromycin aminonucleoside, O-methyl tyrosine and 5-guanylyl methylenediphosphonate had little or no effect on protein synthesis in brain, but gougerotin was slightly inhibitory.

— We have studied the subcellular distribution of exogenous and endogenous serotinin in slices from the hypothalamus and midbrain of several species. In a procedure which appears to label the endogenous pools, tissue slices were incubated with low concentrations of [3H]5-HT (5 × 10-8 M), for 45 min, when there was apparent equilibrium between [3H]5-HT of tissue and medium. After the tissue slices were homogenized in 0-32 M-sucrose and subjected to differential centrifugation, the distribution of exogenous and endogenous 5-HT in pellets and supernatant fluid was similar. In some experiments, the crude mitochondrial pellets were resuspended in 0-32 M-sucrose, layered on linear, continuous density gradients of sucrose (1 -5-0-32 M), and centrifuged for short times (incomplete equilibrium centrifugation). The subcellular distribution of particulate tritium, total tritium, and particulate endogenous 5-HT was the same in portions of the gradients containing synaptosomes. The peak distribution of [3H]5-HT in sucrose gradients was separable from the peak for [14C]GABA by four to five fractions; potassium (a marker for cytoplasm occluded within synaptosomes) occurred in the regions of the gradients containing most of the labelled compounds. The distribution of monoamine oxidase activity (a mitochondrial marker) overlapped the distribution of [3H]5-HT after a 15 min centrifugation but appeared in denser regions of the gradient after centrifuging for 2 h. Particles containing [3H]5-HT and [I4C]NE were slightly but consistently separable in synaptosomal fractions isolated from the hypothalamus or midbrain of rat, guinea pig and hamster.

— After intracerebral administration of [14C]histidine to mice the major labelled substance found in the brain extracts was histidine itself; small amounts of labelled carnosine and homocarnosine were detected. No other labelled substances were detected on radio- autographs of two-dimensional TLC's of the extracts. In the case of the frog, radioactive histidine, N-acetylhistidine, carnosine and homocarnosine were found in the brain extracts at various times after intracerebral injection of the labelled histidine. With time, approximately 90 per cent of the radioactivity in the extracts was found in the N-acetylhistidine. In neither the mouse nor frog could we find unequivocal evidence for the formation either of histamine or imidazoleacetic acid from intracerebrally administered histidine, but our analytical procedures may have lacked sufficient sensitivity to pick up extremely low activities of histamine and imidazoleacetic acid. Experiments with [14C]histamine administered intracerebrally into mice demonstrated the major pathway of metabolism in brain to be histamine methylhistamine methylimidazoleacetic acid. No detectable label appeared in inlidazoleacetic acid. In the frog intracerebral administration of the labelled histamine led to the formation of methylhistamine and imidazoleacetic acid, but at most only traces of methylimidazoleacetic acid were found. The injection of [14C]imidazoleacetic acid intra- cerebrally into mice and frogs resulted in virtually no loss of the label in the form administered in the frog brain over a period of 4 h and in a slow rate of decrease in the mouse brain. No radioactive metabolites of imidazoleacetic acid were found in either species. The limitations of trying to determine natural functions of substances in brain by following the fate of exogenously administered materials is discussed.

— 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 0.5m-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.

gamma-Amino-beta-[3H]hydroxybutyric acid ([3H]-GABOB) was formed in rat brain from 2-[3H]-hydroxyputrescine that had been chemically synthesized from 2-oxoputrescine and [3H]sodium borohydride. After the injection of 2-[3H]hydroxyputrescine into the lateral ventricle of a rat brain, the rat was killed and then the brain was removed. [3H]GABOB in the brain was identified by a combination of ion-exchange chromatography, high-voltage paper electrophoresis, and recrystallization of the radioactive compound with authentic GABOB.

— 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.

– γ-Glutamyl amides of histamine, serotonin and dopamine were formed from these amines by the transfer of the γ-glutamyl moiety from γ-glutamyl peptides in the presence of γ-glutamyl transpeptidase. [I4C]Histamine was injected intraventricular into rats, and the formation of γ-glutamyl-[14C] histamine in the brain was confirmed by purification and identification with the authentic compound. The radioactivity was highest 30 min after the injection. The possible significance of γ-glutamyl amides in nerve transmission is discussed.

— A method has been developed for the isolation of a previously undescribed fibrous protein from rat brain. The newly isolated material consists of bundles of tightly packed 70-80 A diameter filaments. Based on studies employing degenerated rat optic nerve, it is proposed that these filaments correspond to the well-described astrocyte filaments observed in sectioned preparations of mammalian brain. The purified filaments are stable over a wide temperature range, are not disrupted by colchicine, and exhibit limited solubility in the absence of denaturants or detergents. In neutral SDS-polyacrylamide gel electrophoresis, the filament protein runs as a single band with an apparent molecular weight of 57,000 daltons. This preparation also migrates as a single band in alkaline urea gels, and as a well-resolved doublet on discontinuous SDS-urea gels. In all three electrophoretic systems, the filament subunits co-migrate with rat brain tubulin. Comparative peptide maps of the filament subunits and tubulin indicate a large degree of homology. Our results suggest that microtubules and astrocyte filaments are composed of the same or very similar protein subunits.

—The metabolism of free amino acids: γ-aminobutyric acid (GABA), glutamine, glycine and glutathione has been studied. The labelling of these free amino acids in normal and in myelin-deficient brains of Jimpy mice was followed after intraperitoneal injection of 14C-labelled glucose precursor. The quantitative distribution of these amino acids in the two kinds of mouse brain has been compared. A higher level of GABA and a faster labelling of the amino acids in Jimpy than in normal mouse brain was observed.