MANIFESTATION OF METABOLIC COMPARTMENTATION DURING THE MATURATION OF THE RAT BRAIN

—(1) The fate of [U-¹⁴C]leucine was studied in rat brain in vivo from birth to five weeks of age. The major route of leucine metabolism at all ages was conversion into protein. The rate of protein synthesis was low in the newborn; it reached a peak at about 15 days and slowed down moderately later. Incorporation into brain lipids was relatively low under the experimental conditions (less than 2 per cent of the total tissue ¹⁴C). (2) The conversion of leucine-carbon into amino acids associated with the tricarboxylic acid cycle was low in the first 9 days after birth (less than 4 per cent of the acid-soluble ¹⁴C at 10 min after injection) and increased rapidly until 15 days when the level characteristic of the adult was approached (about 20 per cent of the acid-soluble ¹⁴C). The results indicated that the oxidation of acetyl-CoA derived from leucine reached the adult level at an earlier age than that derived from glucose. (3) The glutamine/glutamate specific radioactivity ratio was 0·3 in the brain of newborn animals and increased progressively; it was 1·3 and 2·4 at 15 and 35 days of age respectively. The specific radioactivity of aspartate and of GABA relative to that of glutamate was less than 1 throughout the experimental period. (4) The factors involved in the development of metabolic compartmentation in brain were analysed. It is proposed that although the experimental results show that a 'small’compartment becomes functionally manifested with maturation the primary cause is the development of the‘large’metabolic compartment. (5) Morphological correlates of the metabolic compartments in brain tissue are suggested and it is concluded that the manifestation of metabolic compartmentation is related to maturational changes in glia-neuronal relations rather than to developmental processes affecting the individual components only.     

KINETICS OF COMPETITIVE INHIBITION OF NEUTRAL AMINO ACID TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER

The transport of tryptophan across the blood-brain barrier is used as a specific example of a general approach by which rates of amino acid influx into brain may be predicted from existing concentrations of amino acids in plasma. The kinetics of inhibition of [¹⁴C]tryptophan transport by four natural neutral amino acids (phenylalanine, leucine, methionine, and valine) and one synthetic amino acid (α-methyl tyrosine) is studied with a tissue-sampling, single injection technique in the barbiturate-anesthetized rat. The equality of the K₁ (determined from cross-inhibition studies) and the K_m (determined from auto-inhibition data) for neutral amino acid transport indicate that these amino acids compete for a single transport site in accordance with the kinetics of competitive inhibition. Based on equations derived for competitive inhibition, apparent K_m values are computed for the essential neutral amino acids from known data on amino acid transport K_m and plasma concentrations. The apparent K_m values make possible predictions of the in vivo rates of amino acid influx into brain based on given plasma amino acid concentrations. Finally, a method is presented for determining transport constants from saturation data obtained with single injection techniques.     

THE EFFECTS OF TEN PHENOLIC COMPOUNDS ON HYPOCOTYL GROWTH AND MITOCHONDRIAL METABOLISM OF MUNG BEAN

Ten phenolic compounds were examined for their effect on mung bean (Phaseolus aureus L.) hypocotyl growth and on respiration and coupling parameters of isolated mung bean hypocotyl mitochondria. Three compounds—tannic, gentisic, and p-coumaric acids—inhibited hypocotyl growth and when incubated with isolated hypocotyl mitochondria released respiratory control, inhibited respiration, and prevented substrate-supported Ca²⁺ and PO₄ transport. Vanillic acid also inhibited hypocotyl growth and reduced mitochondrial Ca²⁺ uptake but did not affect respiration or respiratory control of isolated mitochondria. This is the first compound reported to selectively inhibit Ca²⁺ uptake in plant mitochondria. Two other phenolic compounds—α, 3,5-resorcylic and protocatechuic acids—showed no significant effect on hypocotyl growth and did not affect mitochondrial oxidative phosphorylation either separately or in various combinations. Four phenolic compounds—ferulic, caffeic, p-hydroxybenzoic, and syringic acids—showed a significant reduction in mung bean hypocotyl growth but did not inhibit any of the mitochondrial processes examined. The results show that phenolic compounds which alter respiration or coupling responses in isolated mitochondria also inhibit hypocotyl growth and may reflect a mechanism of action for these natural growth inhibitors.     

THE ACCUMULATION OF ARACHIDONATE AND DOCOSAHEXAENOATE IN THE DEVELOPING RAT BRAIN

—Fatty acids typical of grey matter lipids (C20:4 and C22:6) and of myelin lipids (C20:1 and C24:1) were estimated in developing rat brains. The polyenoic fatty acids (C20:4 and C22:6) are synthesized from the essential fatty acids (C18:2 and C18:3). The results showed that more than 50 per cent of the adult content of the brain polyenoic acids were laid down by day 15. In contrast, the fatty acids characteristic of myelin lipids did not appear in significant quantities until after this time. These findings distinguish biochemically the different periods of brain development associated firstly with cell division (formation of neurons and glial cells) and secondly with myelination. It is of special interest that the period of cell proliferation is accompanied by the appearance in brain lipids of long-chain polyenoic acids derived from the essential fatty acids.     

BIOCHEMTCAL CHANGES IN THE BRAIN OF EXPERIMENTAL ANIMALS IN RESPONSE TO ELEVATED PLASMA HOMOCYSTINE AND METHIONINE

The effects of high plasma concentrations of homocystine and methionine on the free amino acids of brain have been examined. Incorporation of the label from [³⁵S]methionine into the free amino acid pools of rabbit brain was enhanced in response to high plasma homocystine or high plasma homocystine and mcthionine. Under comparable conditions a marked decrease in the incorporation of the label from [¹⁴C]glycine into the free pool was observed. The corresponding incorporation of ³⁵S and ¹⁴C into brain proteins parallelled the results obtained with incorporation into the free pools of amino acids. Amino acid analyses of the free amino acid pools of rabbit brain revealed a general decrease in the concentration of amino acids in response to high plasma homocystine or high plasma homocystine and methionine. Inhibition of protein synthesis which occurs under the above experimental conditions is a general phenomenon. myelin and other brain fractions being equally affected. The decrease in concentration of brain amino acids also results in a diminution in concentration of the neurotransmitters GABA, dopamine and noradrenaline. The possible relationship of the observed changes to homocystinuria is discussed.     

THE STIGMATIC SECRETION OF THE SWEETPOTATO

The stigmatic exudate of sweetpotato, when removed with organic solvents, consisted chiefly of lipid and phenolic compounds. Only traces of sugar were obtained. The esterified lipids were similar in chain length to capric and lauric acids. The two prinicpal phenolic compounds have UV absorption peaks, and bathochromic shifts on ionization with NaOH similar to those of esters of caffeic acid. On acid hydrolysis, caffeic acid was obtained from these and from three minor phenolic compounds. From two of the minor phenolic compounds, glucose was released by hydrolysis. The phenolic content of stigmas increased up to 20–24 hr before anthesis, and then gradually decreased. Neither compatible nor incompatible pollination affects the amount or composition of the stigmatic exudate.     

BIOSYNTHESIS OF FREE AMINO ACIDS IN THE BRAIN OF JIMPY MICE

—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.     

EFFECT OF THE ADMINISTRATION OF L-5-HYDROXYTRYPTOPHAN AND A MONOAMINE OXIDASE INHIBITOR ON GLUCOSE METABOLISM IN RAT BRAIN

The effects of the presence of large amounts of 5-HT and of its precursor 5-HTP in brain on cerebral utilization of glucose were studied. [U-¹⁴C]Glucose was injected to fed rats that had previously been treated with L-5-HTP, L-5-HTP and an inhibitor—N-[β-(2-chlorophenoxy)-ethyl]-cyclopropylamine hydrochloride (Lilly-51641)-of MAO, or Lilly-51641 alone. Such treatment increased the concentrations of 5-HTP and 5-HT in the brain. After treatment with 5-HTP and Lilly-51641, and to a lesser extent with Lilly-51641 alone, the concentration of glucose in plasma was increased. However, the uptake of glucose by the brain did not appear to be proportionately increased, and this suggested an impairment in this mechanism. After the administration of Lilly-51641 alone and more especially of Lilly-51641 plus 5-HTP, the concentration of glucose in the brain was increased. This increase was thought to be due to an impairment of glucose utilization, because the flux of ¹⁴C from glucose to amino acids in the brain was reduced. The concentrations of most major amino acids in the brain were not greatly affected by these treatments. GABA and alanine concentrations in the brain were modestly increased after treatment with 5-HTP alone or in combination with Lilly-51641. The present results suggest that the metabolism of glucose to amino acids in the brain is altered when the concentration of 5-HTP, or more especially that of 5-HT, in the brain is increased.     

A COMPARATIVE STUDY OF THE METABOLISM OF DE NOVO SYNTHESIZED FATTY ACIDS FROM ACETATE AND GLUCOSE, AND EXOGENOUS FATTY ACIDS, IN SLICES OF RABBIT CEREBRAL CORTEX DURING DEVELOPMENT

Slices of rabbit cerebral cortex, from the foetal stage to the adult have been used to compare lipid synthesis from fatty acids synthesized de novo from [U-¹⁴C]glucose and [1-¹⁴C]acetate, with lipid synthesis from exogenous albumin-bound [1-¹⁴C]palmitate. Incorporation into cellular lipid has been determined in terms of DNA, protein, wet wt. of tissue and wet weight of whole brain. On a wet wt. basis, maximum incorporation of glucose carbon into lipid occurred in the foetal brain while lipid synthesis from acetate and palmitate was maximum at 4–14 days after birth. Glucose and acetate were incorporated into a diversity of lipids (with increasing amounts of phosphatidylcholine synthesized during maturation), while palmitate was incorporated into the free fatty acid and triglyceride fractions. A greater proportion of acetate was incorporated into fatty acids of chain-length longer than C16 compared with the incorporation of palmitate. However, on a molar basis de novo synthesized and exogenous palmitate were elongated, desaturated and incorporated into phospholipids at a similar rate, while exogenous palmitate was incorporated to a greater extent than de nova synthesized fatty acid into the triglyceride fraction. This difference in metabolism may be due to the different size of the non-esterified fatty acid pool in the two situations. At the period of their most active formation, the very long-chain fatty acids may be synthesized from a pool of the C18 series of fatty acids (saturated and monoenoic) not in equilibrium with the bulk of C₁₈ acids in cerebral lipids. This could be a pool of acyl groups derived from ethanolamine phospholipids.     

THE INFLUENCE OF PORTOCAVAL ANASTOMOSIS ON THE METABOLISM OF LABELLED OCTANOATE, BUTYRATE AND LEUCINE IN RAT BRAIN

Rats were given a portocaval anastomosis and 3 weeks later, when the only ultrastructural change in the CNS is watery swelling of astrocytes, several aspects of brain metabolism were studied. The uptake of leucine by the brain, its incorporation into protein and its oxidation were followed after the simultaneous injection of a mixture of L-[1¹⁴C]leucine and L-[4,5-³H]leucine. The concentration of leucine in blood was lowered in the operated animals whereas in brain it was increased. The specific radioactivity of leucine in the brain was comparable to values in control animals and there was no evidence of a decrease in incorporation of [1-¹⁴C]leucine into brain proteins over the short experimental time period studied. The only difference from the controls in the oxidation of [4,5-³H]leucine was a greater accumulation in glutamine. The amount of glutamine in the brains of the operated animals had increased 4-fold at the time of the metabolic studies. From dual-labelled experiments in which a mixture containing [1-¹⁴C]butyrate and L-[4,5-³H]leucine was injected intravenously, it was shown that, in both control and operated animals, the pools of brain glutamate and glutamine labelled from butyrate were metabolically distinct from those labelled from leucine. The total radioactivity appearing in brain from [1-¹⁴C]butyrate was markedly reduced in operated animals, but the radioactivity from L-[4,5-³H]leucine was not. The metabolism of [1-¹⁴C]octanoate was compared with that of [1-¹⁴C]butyrate. In control animals the labelling of metabolites was almost identical with either precursor. In operated animals there was no reduction in the uptake of [1-¹⁴C]octanoate into the brain. There was evidence that the size of the glutamine pool labelled, relative to glutamate, was increased but that it had a slower fractional turnover coefficient. A link between astroglial changes and an impairment to the carrier mechanism for transport of short chain monocarboxylic acids across the blood-brain barrier is suggested.     

DEVELOPMENT OF LIPOGENESIS IN RAT BRAIN CORTEX: THE DIFFERENTIAL INCORPORATION OF GLUCOSE AND ACETATE INTO BRAIN LIPIDS IN VITRO

—The oxidation to CO₂ and the incorporation of [U-¹⁴C]glucose and [U-¹⁴C]acetate into lipids by cortex slices from rat brain during the postnatal period were investigated. The oxidation of [U-¹⁴C]glucose was low in 2-day-old rat brain, and increased by about two-fold during the 2nd and 3rd postnatal weeks. The oxidation of [U-¹⁴C]acetate was increased markedly in the second postnatal week, but decreased to rates observed in 2-day-old rat brain at the time of weaning. Both labeled substrates were readily incorporated into non-saponifiable lipids and fatty acids by brain slices from 2-day-old rat. Their rates of incorporation and the days on which maximum rates occurred were different, however, maximum incorporation of [U-¹⁴C]glucose and [U-¹⁴]acetate into lipid fractions being observed on about the 7th and 12th postanatal days, respectively. The metabolic compartmentation in the utilization of these substrates for lipogenesis is suggested. The activities of glucose-6-phosphate dehydrogenase, cytosolic NADP-malate dehydrogenase, cytosolic NADP-isocitrate dehydrogenase, ATP-citrate lyase and acetyl CoA carboxylase were measured in rat brain during the postnatal period. All enzymes followed somewhat different courses of development; the activity of acetyl CoA carboxylase was, however, the lowest among other key enzymes in the biosynthetic pathway, and its developmental pattern paralleled closely the fatty acid synthesis from [U-¹⁴C]glucose. It is suggested that acetyl CoA carboxylase is a rate-limiting step in the synthesis de novo of fatty acids in developing rat brain.     

THE EFFECT OF MORPHINE ON THE TRANSPORT AND METABOLISM OF INTRACISTERNALLY-INJECTED LEUCINE IN THE RAT

—Intracisternally injected l or d-[¹⁴C]leucine was retained longer in the brains of morphine-treated rats than in saline-injected control animals. This resulted in higher levels of the labelled leucine and of labelled metabolites of the l-isomer in free pools of brain tissue. However, the absolute levels of brain amino acids and the relative distribution of radioactivity among l-leucine metabolites in brain were unaffected by treatment with morphine, indicating that no disturbance of leucine oxidation through the citric acid cycle was produced by the drug. The inhibition of protein synthesis caused by acute administration of morphine was calculated to be greater than previously reported since morphine treatment increased the specific radioactivity of the free pool of leucine in brain following the intracisternal injection of the labelled amino acid. Possible mechanisms responsible for these morphine effects are discussed.     

METABOLISM OF GLUCOSE, AMINO ACIDS, AND SOME RELATED METABOLITES IN THE BRAIN OF TOADS (BUFO BOREAS) ADAPTED TO FRESH WATER OR HYPEROSMOTIC ENVIRONMENTS

The levels and specific radioactivities (SA) of glucose, lactate, pyruvate, α-oxoglutarate and seven amino acids in the brain of toads adapted to fresh water or to an hyperosmotic environment were analysed at various times (5 min–4 h) after an injection of [U-¹⁴C]glucose into the bloodstream. The concentrations and SA of glucose, lactate and five amino acids in blood plasma also were measured. In addition, the SA of glutamine, glutamate, aspartate and GABA in brain were determined 30 min after an injection of [1,5-¹⁴C]citrate into the cisterna magna. The flow of labelled carbon atoms from glucose to amino acids and related metabolites in the toad brain was qualitatively similar to that in the mammalian brain, but quantitatively less than one-tenth of the rate in the brain of rats. Hyperosmotic adaptation induced a large increase in the levels of glucose and amino acids in the brain without affecting the rate of glucose utilization. The SA of several amino acids relative to the SA of glucose were initially lower in hyperosmotically-adapted toads than in toads adapted to fresh water, presumably because of a greater dilution of isotope by the larger amino acid pools in the hyperosmotically-adapted toads. The rates of synthesis of alanine and glutamine from pyruvate and glutamate, respectively, appeared to increase with hyperosmotic adaptation, but the rate of GABA synthesis from glutamate was unaltered. The SA of α-oxoglutarate and glutamate were similar at all time periods in both groups of toads, an indication that these compounds were interconverted much more rapidly than the rate at which α-oxoglutarate was formed from isocitrate. The SA of lactate in comparison to that of glucose varied but was always considerably lower, even at 4 h after the [¹⁴C]glucose injection. After[U-¹⁴C]glucose, glutamine had a SA lower than that of glutamate, whereas after the injection of [1⁴C]citrate, glutamine was formed with a SA much higher than that of glutamate. Hence, glutamate in the toad brain exhibited metabolic compartmentation similar to that in rat brain.     

DECREASED METABOLISM IN VIVO OF GLUCOSE INTO AMINO ACIDS OF THE BRAIN OF THIAMINE-DEFICIENT RATS AFTER TREATMENT WITH PYRITHIAMINE

Abstract— Thiamine deficiency produced by administration of pyrithiamine to rats maintained on a thiamine-deficient diet resulted in a marked disturbance in amino acid and glucose levels of the brain. In the two pyrithiamine-treated groups of rats (Expt. A and Expt. B) there was a significant decrease in the levels of glutamate (23%, 9%) and aspartate (42%, 57%), and an increase in the levels of glycine (26%, 27%) in the brain, irrespective of whether the animals showed signs of paralysis (Expt. A) or not (Expt. B). as a result of thiamine deficiency. A significant decrease in the levels of γ-aminobutyrate (22%) and serine (28%) in the brain was also observed in those pyrithiamine-treated rats which showed signs of paralysis (Expt. A). Threonine content increased by 57% in Expt. A and 40% in Expt. B in the brain of pyrithiamine-treated rats, but these changes were not statistically significant. The utilization of [U-¹⁴C]glucose into amino acids decreased and accumulation of glucose and [U-¹⁴C]glucose increased significantly in the brain after injection of [U-¹⁴C]glucose to pyrithiamine-treated rats which showed abnormal neurological symptoms (Expt. A). The decrease in ¹⁴C-content of amino acids was due to decreased conversion of [U-¹⁴C]glucose into alanine, glutamate, glutamine, aspartate and γ-aminobutyrate. The flux of [¹⁴C]glutamate into glutamine and γ-aminobutyrate also decreased significantly only in the brain of animals paralysed on treatment with pyrithiamine. The decrease in the labelling of, amino acids was attributed to a decrease in the activities of pyruvate dehydrogenase and α-oxoglutarate dehydrogenase in the brain of pyrithiamine-treated rats. The measurement of specific radioactivity of glucose, glucose-6-phosphate and lactate also indicated a decrease in the activities of glycolytic enzymes in the brain of pyrithiamine-treated animals in Expt. A only. It was suggested that an alteration in the rate of oxidation in vivo of pyruvate in the brain of thiamine-deficient rats is controlled by the glycolytic enzymes, probably at the hexokinase level. The lack of neurotoxic effect and absence of significant decrease in the metabolism of [U-¹⁴C]glucose in the brain of pyrithiamine-treated animals in Expt. B were probably due to the fact that animals in Expt. B were older and weighed more than those in Expt. A, both at the start and the termination of the experiments.     

EFFECT OF PHENYL AND PHENOLIC ACIDS ON MEVALONATE-5-PHOSPHATE KINASE AND MEVALONATE-5-PYROPHOSPHATE DECARBOXYLASE OF THE RAT BRAIN

Abstract— Phenyl and phenolic acids are known to inhibit metabolism of mevalonate in rat brain. The site of inhibition has been found to be mevalonate-5-pyrophosphate decarboxylase. Phenolic acids also inhibited mevalonate-5-phosphate kinase on preincubation. The kinetics showed that p -coumaric acid and isoferulic acid were competing with substrates, mevalonate-5-phosphate or mevalonate-5-pyre phosphate, whereas others showed an uncompetitive type of inhibition. Chlorophenoxyisobutyrate, a hypocholesterolaemic drug, had no effect on these enzymes. An improved method for the synthesis of mevalonate-5-phosphate and mevalonate-5-pyrophosphate, labeled at carbon-1, is described.     

METHYLATED AMINO ACID RESIDUES OF PROTEINS OF BRAIN AND OTHER ORGANS

—Methods for the determination of methyl-lysine, methyllarginine and methylhistidine residues of tissue proteins are described. They consist of preliminary purification of basic amino acids, enzymic removal of lysine, arginine and histidine followed by amino acid analysis. Recovery rates and specificities of the method were satisfactory. The contents of methylamino acids in proteins of mammalian organs were determined. The distribution of proteins containing the methylamino acids in human brain showed that the concentrations of methyl-lysine and N^G,N′^G-dimethylarginine were highest in the gray matter of the cerebellar cortex and relatively high in regions rich in gray matter, while those of N^G-mono- and N^G,N′^G-dimethylarginine were highest in the white matter. The following findings suggest that most of the N^G-mono- and N^G,N′^G-dimethylarginine was associated with the myelin basic protein. The distribution of the methylarginine residues of acid-soluble proteins in bovine brains coincided with the cerebroside pattern. The concentrations of the amino acids in acid-soluble proteins of rat brain increased concomitantly with the increase of cerebroside. The methylamino acid content in proteins increased during the purification of the myelin basic protein from the white matter of human and bovine brains. Proteins containing N^G,N^G-dimethyiarginine and di- and trimethyl-lysine are concentrated in cell nuclei. The first amino acid was found mainly in nucleoplasmic proteins and the other two were found in histones. The concentration of 3-methylhistidine residue, highest in muscular proteins, is low in cerebral proteins and is probably derived from proteins of walls of blood vessels in the brain.     

THE INFLUENCE OF INTRAVENTRICULARLY INJECTED AMINO ACID EXCITANTS ON THE LABELLING OF ENDOGENOUS BRAIN AMINO ACIDS FROM [U-14C]ACETATE IN NEMBUTALIZED MICE

Mice were anaesthetized with nembutal and the effects of intraventricularly injected excitant amino acids on [U-¹⁴C]acetate metabolism were investigated. The natural excitant amino acids, l -glutamate and l -aspartate, reduced the incorporation of ¹⁴C from [U-¹⁴C]acetate into glutamine, GAB A and possibly alanine. The synthetic excitant amino acid, N-methyl-d -aspartate caused a reduction in the incorporation of ¹⁴C from intraventricularly injected [U-¹⁴C]acetate into all of the brain amino acids labelled by [U-¹⁴C]acetate within 5 min. It is suggested that these effects may be due to changes in pool sizes of tricarboxylic cycle intermediates, to inhibition of acetyl-CoA formation, or both. Differences in the metabolic effects of the synthetic and natural excitants are interpreted in terms of the uptake of the natural amino acids into glutamine-forming pool(s) of glutamate metabolism.     

THE CELL-FREE SYNTHESIS OF ALKALOID-BINDING PROTEINS IN IMMATURE RAT BRAIN1

Abstract— cell-free amino acid incorporating system from immature rat brain, consisting of ribosomal and soluble fractions, has been investigated for its capacity to incorporate [¹⁴C]amino acids into specific soluble proteins that interact with vinblastine sulfate and colchicine. The soluble ¹⁴C-labeled proteins formed in the cell-free system during incubation were compared with similar soluble proteins from immature rat brain which had been labeled in vivo by the incorporation of ¹⁴C-labeled amino acids. Criteria for the formation of vinblastine-binding, ¹⁴C-labeled proteins were: (1) aggregation of ¹⁴C-labeled soluble protein by one mm -vinblastine sulfate and (2) immunoprecipitation of ¹⁴C-labeled soluble protein by an antiserum against vinblastine sulfate-precipitable material. Criteria for the formation of [³H]colchicine-binding, ¹⁴C-labeled protein were based upon: (1) co-precipitation of the ³H-and ¹⁴C-labeled materials by vinblastine sulfate and (2) the coincidence of ³H- and ¹⁴C-labeled elution peaks from columns of Sephadex G-200, DEAE-Sephadex A-50 and isoelectric focusing. Both in the in vitro and in the in vivo system, ¹⁴C-labeled amino acids were incorporated into soluble proteins of the post-microsomal supernatant fraction. Proteins labeled with ¹⁴C-labeled amino acids in vitro and in vivo yielded comparable and qualitatively identical results by the criteria tested, including the formation of immunoprecipitates. In the in vitro system, ¹⁴C-labeled amino acids were incorporated into protein with a molecular weight of approx 120,000, an isoelectric point of 5.3 and with a chromatographic mobility on Sephadex G-200 which is identical to [³H]colchicine-binding protein. The above experimental results are presumptive evidence for the synthesis of vinblastine-binding and colchicine-binding proteins in the in vitro cell-free system.     

THE INCORPORATION OF RADIOACTIVE AMINO ACIDS INTO BRAIN SUBCELLULAR PROTEINS DURING TRAINING

—Total proteins, free amino acids, tritiated water and subcellular proteins of mouse brain were examined for changes in radioactivity during operant conditioning after subcutaneous administration of labelled amino acids. The conditioning was based on appetitive learning, using sweetened milk as a reward. During training and incorporation for 20-30 min, both [³H]leucine and [1-¹⁴C]leucine underwent a significant increase in catabolism, resulting in a decreased radioactivity in the free amino acids. [2-²H]Methionine underwent a rapid loss of isotope, so that 90% of the radioactivity was in the form of tritiated water at the end of training, and this phenomenon masked any possible effect of training. The brain uptake of [³⁵S]methionine increased during the training, resulting in an increased radioactivity in the proteins. Uptake of [³H]lysine increased slightly during training only after 1 h incorporation and not after 20 or 30 min, as judged from a time course of radioactivity in the free amino acids. Incorporation into nuclear proteins increased selectively during 20 min, and into nuclear and cytosol proteins after 60 min incorporations. It is concluded that changes in the observed rate of incorporation of a precursor into brain subcellular proteins under the influence of behaviour might be the result of changes in precursor catabolism or uptake, or both, and that each amino acid behaves in a different way. Even the same amino acid gives different results depending on the isotope and its position in the amino acid.     

EFFECT OF THYROID HORMONE ON THE BIOCHEMICAL MATURATION OF RAT BRAIN: CONVERSION OF GLUCOSE-CARBON INTO AMINO ACIDS

• 1 The rapid and extensive conversion of glucose-carbon into amino acids is an index of the final coordination of the mechanisms underlying energy metabolism in the adult brain. This phenomenon develops in the rat during a short period extending from 10 to about 19 days after birth. The underlying factors have been analysed.

• 2 The development of the pattern of distribution of glucose-carbon characteristic of the adult brain was markedly influenced by the thyroid state of the animals. The age-curve for the conversion of glucose-carbon into brain amino acids was displaced to the left after treatment with thyroid hormone (T₃) in infancy thus indicating an accelerated maturation. Conversely, neonatal thyroidectomy resulted in a significant retardation in the conversion of glucose-carbon into amino acids.

• 3 The specific radioactivity of glutamate increased five-fold in the brain of normal rats from the 10th to the 19th day of age. The values (as a percentage of those for littermate controls) were 220 in the case of the 10 day-old thyroid treated rats and about 30 for the 19 day-old thyroid deficient animals. At the age of 10 days neither treatment affected the concentration of glutamate which was also only slightly less than the control values in the brain of 19 day-old thyroid deficient animals (–17 per cent).

• 4 Specific pool(s) of glutamate associated with the formation of GABA can be demonstrated in the brain of 19 day-old rats after administration of [U-¹⁴C]glucose as a result of anoxia post mortem. These pools did not develop in the brain of 10 day-old animals. Neonatal thyroidectomy retarded the development of these glutamate pools.

• 5 Evidence is summarized which indicates that the development of the rapid conversion of glucose-carbon into amino acids reflects the enlargement, during maturation, of the metabolic compartments which are associated with neuronal processes.