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.     

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.     

d-β-HYDROXYBUTYRATE: A MAJOR PRECURSOR OF AMINO ACIDS IN DEVELOPING RAT BRAIN

Abstract— D-β-hydroxybutyrate (β-OHB) was compared to glucose as a precursor for brain amino acids during rat development. In the first study [3-¹⁴C]β-OHB or [2-¹⁴C]glucose was injected subcu-taneously (01 μCi/g body wt) into suckling rats shortly after birth and at 6. 11, 13, 15 and 21 days of age. Blood and brain tissue were obtained 20 min later after decapitation. The specific activity of the labelled precursor in the blood and in the brain tissue was essentially the same for each respective age suggesting that the labelled precursor had equilibrated between the blood and brain pools before decapitation. [3-¹⁴C]β-OHB rapidly labelled brain amino acids at all ages whereas [2-¹⁴C]glucose did not prior to 15 days of age. These observations are consistent with a maturational delay in the flux of metabolites through glycolysis and into the tricarboxylic acid cycle. Brain glutamate, glutamine, asparate and GABA were more heavily labelled by [3-¹⁴C]β-OHB from birth-15 days of age whereas brain alanine was more heavily labelled by [2-¹⁴C]glucose at all ages of development. The relative specific activity of brain glutamine/glutamate was less than one at all ages for both labelled precursors suggesting that β-OHB and glucose are entering the‘large’glutamate compartment throughout development. In a second study, 6 and 15 day old rats were decapitated at 5 min intervals after injection of the labelled precursors to evaluate the flux of the [¹⁴C]label into brain metabolites. At 6 days of age, most of the brain acid soluble radioactivity was recovered in the glucose fraction of the [2-^,4C]glucose injected rats with 72, 74, 65 and 63% after 5, 10, 15 and 20 min. In contrast, the 6 day old rats injected with [3-¹⁴C]β-OHB accumulated much of the brain acid soluble radioactivity in the amino acid fraction with 22, 47, 57 and 54% after 5, 10, 15 and 20 min. At 15 days of age the transfer of the [¹⁴C]label from [2-¹⁴C]glucose into the brain amino acid fraction was more rapid with 29, 40, 45, 61 and 73% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. There was almost quantitative transfer of [¹⁴C]label into the brain amino acids of the 15-day-old [3-¹⁴C]β-OHB injected rats with 66, 89, 89, 89 and 90% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. The calculated half life for /?-OHB at 6 days was 19 8 min and at 15 days was 12-2 min. Surprisingly, the relative specific activity of brain GABA/glutamate was lower at 15 days of age in the [3-¹⁴C]β-OHB injected rats compared to the [2-¹⁴C]glucose injected rats despite a heavier labelling of brain glutamate in the [3-¹⁴C]β-OHB injected group. We interpreted these data to mean that β-OHB is a less effective precursor for the brain glutamate ‘subcompartment’ which is involved in the synthesis of GABA.     

Metabolism of U14C leucine and U14C valine by adult female Glossina morsitans during pregnancy

After U¹⁴C leucine or U¹⁴C valine injections into haemolymph of adult female Glossina morsitans during late pregnancy, radioactivity was detected in the post-parturient female and its larval offspring in the injected material, lipids, and a range of non-essential amino acids. The level of radioactivity recorded from the third instar larva was higher than that remaining in the injected adult, and the activity was higher in amino acids than in the lipid fraction. Radiometric analysis of oöcyte and intra-uterine progeny 24 hr after haemocoelic administration to females of labelled leucine or valine revealed a pattern of radioactivity coincident with growth characteristics of these young stages. Rate of leucine uptake by the in utero third instar larva was slightly higher than that of valine, and this instar continues feeding even only a few hours before parturition. For both labelled materials, expired carbon dioxide and excreta from remales in early pregnancy showed significantly higher radioactivity than those in late pregnancy. Uric acid is the main nigrogenous waste of leucine and valine metabolism, though small amounts of these amino acids are also lost during excretion, with valine elimination being higher than leucine.     

ÈVIDENCE FOR THE COMPARTMENTATION OF GLUTAMATE METABOLISM IN ISOLATED RAT RETINA

—Glucose is a major precursor of glutamate and related amino acids in the retina of adult rats. ¹⁴C from labelled glucose appears to gain access to a large glutamate pool, and the resulting specific activity of glutamate labelled from glucose is always higher than that of glutamine or the other amino acids. Radioactive acetate appeared to label a small glutamate pool. The specific activity of glutamine labelled from acetate relative to that of glutamate was always greater than 1.0. Other precursors of the small glutamate pool were found to include glutamate, aspartate, GABA, serine, leucine and sodium bicarbonate. The level of radioactivity present in retinae incubated with [U-¹⁴C]glucose or [1-¹⁴C]sodium acetate was reduced in the presence of 10^?5m -ouabain. Under these conditions, the relative specific activity of glutamine labelled from [1-¹⁴C]sodium acetate was lowered, but it was raised when [U-¹⁴C]glucose was used as substrate. Ouabain also considerably reduced the synthesis of GABA from [1-¹⁴C]sodium acetate. In all cases ouabain caused a fall in the tissue levels of the amino acids. Aminooxyacetic acid (10^?4m ) almost completely abolished the labelling of GABA from both [U-¹⁴C]glucose and [1-¹⁴C]sodium acetate, while the RSA of glutamine labelled from the latter substrate was significantly increased. Aminooxyacetic acid raised the tissue concentration of glutamate, but caused a fall in the tissue concentrations of glutamine, aspartate and GABA. The results suggest that there are separate compartments for the metabolism of glutamate in retina and that these can be modified in different ways by different drugs.     

ENHANCED CEREBRAL PROTEIN SYNTHESIS IN DEVELOPING HYPOTHYROID RATS: EVIDENCE FOR DELAYED MATURATION

(1) Neonatal hypothyroidism resulted in a 40% increase in the incorporation of [¹⁴C]leucine into protein by cerebral cortical slices from 25-day-old rats. The uptake of the [¹⁴C]-labelled amino acid into the acid-soluble free amino acid pool was similar in hypothyroid and control groups which excluded the possibility that transport differences contributed to the observed differences in incorporation. (2) The conversion of [¹⁴C]leucine in the free amino acid pool to other metabolites was substantially greater in the hypothyroid state compared to euthyroid controls. (3) The correction of the incorporation data for radioactivity associated with [¹⁴C]leucine in the precursor pool, provided an estimate of cerebral protein synthetic rate which was markedly higher in thyroid hormone-deficient-rats compared to litter mate controls. (4) The administration of L-thyroxine to hypothyroid animals for two successive days essentially returned the accelerated metabolism of the precursor pool leucine to normal but failed to ameliorate the increased incorporation into protein. (5) Incubations conducted in the presence of high exogenous leucine levels, to eliminate possible differences in intracellular free amino acid pool size, provided additional evidence for an increased rate of cerebral protein synthesis in 25-day-old hypothyroid rats compared to controls. (6) The results are compatible with a retardation in the normal developmental decline in the rate of cerebral protein synthesis associated with hypothyroidism.     

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.     

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.     

INCORPORATION OF 14C FROM [U-14C]GLUCOSE INTO FREE AMINO ACIDS IN MOUSE BRAIN LOCI IN VIVO UNDER NORMAL CONDITIONS

By macroautoradiography and by GLC separation, differences in the uptake of radioactive carbon from [U-¹⁴C]glucose into free amino acids (glutamate + glutamine, aspartate + asparagine, GABA, alanine and glycine) in mouse cerebral neocortex, hippocampus, thalamus and hypothalamus were investigated. (1) The autoradiographical densities in the thalamus, cerebral neocortex and hippocampus measured with a microdensitometer were higher than that in the hypothalamus at 5 min after subcutaneous injection. At 180 min, densities in the cerebral neocortex, hippocampus and hypothalamus were higher than that in thalamus. (2) The free amino acid levels determined by GLC varied with each brain region. (3) The specific radioactivity (d.p.m./μmol) of alanine in each brain region was higher than that of the other amino acids at 5 min after the injection. The specific radioactivity of GABA in the brain regions was clearly higher than that of (glutamate + glutamine), (aspartate + asparagine) and glycine at 5 and 15 min. (4) The autoradiographical data were in good agreement with the chemical data at 5 min but were different at 180 min. (5) Variations in specific radioactivity of each free amino acid among brain regions at 5 min were influenced greatly by existing free amino acid concentrations in each region.     

Utilization in vivo of glucose and volatile fatty acids by sheep brain for the synthesis of acidic amino acids

1. Free glutamic acid, aspartic acid, glutamic acid from glutamine and, in some instances, the glutamic acid from glutathione and the aspartic acid from N-acetyl-aspartic acid were isolated from the brains of sheep and assayed for radioactivity after intravenous injection of [2-¹⁴C]glucose, [1-¹⁴C]acetate, [1-¹⁴C]butyrate or [2-¹⁴C]propionate. These brain components were also isolated and analysed from rats that had been given [2-¹⁴C]propionate. The results indicate that, as in rat brain, glucose is by far the best precursor of the free amino acids of sheep brain. 2. Degradation of the glutamate of brain yielded labelling patterns consistent with the proposal that the major route of pyruvate metabolism in brain is via acetyl-CoA, and that the short-chain fatty acids enter the brain without prior metabolism by other tissue and are metabolized in brain via the tricarboxylic acid cycle. 3. When labelled glucose was used as a precursor, glutamate always had a higher specific activity than glutamine; when labelled fatty acids were used, the reverse was true. These findings add support and complexity to the concept of the metabolic `compartmentation' of the free amino acids of brain. 4. The results from experiments with labelled propionate strongly suggest that brain metabolizes propionate via succinate and that this metabolic route may be a limited but important source of dicarboxylic acids in the brain.     

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.     

STUDIES ON THE MECHANISM OF DEMYELINATION: TRIETHYL TIN-INDUCED DEMYELINATION

The metabolism of myelin undergoing breakdown as a result of edema induced by chronic administration of triethyl tin (TET) dissolved in the drinking water (10 mg/l.) was examined. The spinal cord showed more edema and loss of myelin than the brain. Uptake in vitro of [1-¹⁴C]acetate into myelin lipids of slices of brain or spinal cord from TET-treated rats was depressed until 4–5 weeks after the beginning of the regime, then rose to above normal levels. The uptake of [l-¹⁴C]leucine into myelin protein rose within several weeks of TET treatment to levels averaging over 300 per cent of normal and remained high even after the TET was removed. The high levels of [l-¹⁴C]leucine incorporation were inhibited by cycloheximide and were not explained by an increase in the size of the free amino acid pool. The three classes of myelin proteins, basic, proteolipid protein, and Wolfgram protein shared in the increased incorporation. Spinal cord myelin showed the greatest metabolic response, brain stem myelin less, and myelin from the forebrain was minimally affected by the TET treatment. Myelin prelabelled by intracisternal injection of [l-¹⁴C]acetate and [l-¹⁴C]leucine before the onset of TET administration showed faster turnover in myelin proteins in relation to the myelin lipids than the control in the most severely affected animals, but not in others less affected. A ‘floating fraction’ was observed floating on 10.5% (w/v) sucrose during the myelin purification. This fraction showed metabolic characteristics typical of myelin, and myelin-labelling studies at various stages of the animal's development showed it to be derived from recently synthesized myelin. The floating fraction from the brain contained less cerebroside and more lecithin than myelin, while the spinal cord floating fraction composition was much like that of myelin. The floating fractions contained less protein typical of myelin (basic and proteolipid protein) and more highmolecular-weight protein which may have been derived from contaminating microsomes. The floating fraction was presumed to be partially deproteinated myelin. The use of TET-treatment as model for demyelination as a result of edema and proceeding in the absence of macrophages is discussed.     

THE EFFECT OF MORPHINE ADMINISTRATION ON THE INCORPORATION OF [14C]LEUCINE INTO PROTEIN IN CELL-FREE SYSTEMS FROM RAT BRAIN AND LIVER

—Ribosomes isolated from the brains of rats treated with morphine in vivo were less active in promoting the incorporation of [¹⁴C]leucine into protein than ribosomes isolated from untreated rats. This inhibitory phenomenon was studied in relation to dose of morphine, time after drug administration and the pharmacological responses of hypothermia and analgesia. The inhibition of [¹⁴C]leucine incorporation into brain proteins in vitro was transient after a single injection of morphine and dose-dependent, and related to the hypothermic response, but not prevented by keeping the rats at an ambient temperature which prevented hypothermia. The incorporation of [¹⁴C]leucine into protein by liver ribosomes was also inhibited in preparations from morphine treated rats.     

Effect of thyroid hormone on metabolic compartmentation in the developing rat brain

1. The effects of treatment with thyroid hormone (tri-iodothyronine) and of neonatal thyroidectomy on the cerebral metabolism of [U-¹⁴C]leucine were investigated during the period of functional maturation of the rat brain extending from 9 to 25 days after birth. 2. Age-dependent changes in the labelling of brain constituents under normal conditions appear to depend on changes in the availability of blood-borne [¹⁴C]leucine resulting from differential rates of growth of body and brain; but developmental changes in the pool size of free leucine and in the rates of protein synthesis and oxidation of leucine are also involved. 3. Treatment with thyroid hormone had no significant effect on the conversion of leucine carbon into proteins and lipids; and the age-dependent changes in the concentration and specific radioactivity of leucine were similar to controls. On the other hand there was an acceleration in the conversion of leucine carbon into amino acids associated with the tricarboxylic acid cycle. These observations indicate that leucine oxidation was the process mainly affected. 4. The specific radioactivity of glutamine relative to that of glutamate was used as an index of metabolic compartmentation in brain tissue. Treatment with thyroid hormone advanced the development of metabolic compartmentation. 5. Neonatal thyroidectomy led to a marked decrease in the conversion of leucine carbon into proteins and lipids and to a significant increase in the amount of ¹⁴C combined in the amino acids associated with the tricarboxylic acid cycle. The age-dependent increase in the glutamate/glutamine specific-radioactivity ratio was strongly retarded. 6. The increased conversion of leucine carbon into cerebral amino acids applied to glutamate and aspartate, but not to glutamine and γ-aminobutyrate. This observation facilitated the understanding of the effects of thyroid deprivation on brain metabolism and provided new evidence for the allocation of morphological structures to the metabolic compartments in brain tissue. 7. In contrast with the marked effects of the thyroid state on metabolic compartmentation, it had relatively little effect on the developmental changes in the concentration of amino acids in the brain. 8. The rate of conversion of leucine carbon into the `cycle amino acids' both under normal conditions and in thyroid deficiency indicated a special metabolic relationship between glutamate and aspartate on the one hand, and glutamine and γ-aminobutyrate on the other.     

Studies on the incorporation of ( 14 C)amino acids into protein by isolated rat brain nuclei

—Various parameters of the in vitro incorporation of [¹⁴C]amino acids into protein by cell nuclei isolated and purified from rat brain and liver were investigated. Nuclei purified through 2.2 m sucrose solution were capable of amino acid incorporation in vitro; and washing procedure to eliminate hypertonic sucrose before incubation was essential since sucrose in high concentration was inhibitory. Microbial contamination was found to be a serious source of error and the use of sterile conditions for incubation were necessary to obtain reproducible and valid results. Using completely sterile conditions, Na ⁺, K⁺, RNase, DNase, puromycin, cycloheximide and chloramphenicol were without any effect on the ability of brain and liver nuclei to incorporate labelled amino acids into protein. Results of time-course and preincubation experiments revealed that some factors essential for amino acid incorporation pass out of the nucleus into the medium. In addition, approximately 15 per cent of the labelled nuclear proteins with higher specific radioactivity was recovered in the incubation medium. Incorporation of [¹⁴C]leucine was proportional to the concentration of labelled amino acid and to the number of nuclei, and it is suggested that carefully controlled conditions of incubation are essential to obtain valid comparisons between different types of nuclei in terms of their relative abilities to incorporate amino acids in vitro. No evidence was obtained indicating isotope dilution phenomenon in these experiments. Whether or not in vitro incorporation of amino acid by nuclei represents protein synthesis is discussed.     

Protein metabolism in goldfish brain

Abstract— Protein metabolism of goldfish brain was studied in vivo by means of intraperitoneal or intracranial injections of [³H]leucine and compared with concomitant studies in the mouse. Heterogeneity of turnover values was observed. Long turnover times were seen relative to other organs examined. The free amino acid pools of goldfish brain were determined, and the fate of tritium from labelled leucine was followed at various times after injection. Following ‘chasing’ with large amounts of unlabelled leucine or protein inhibitors shortly after isotope injection, further incorporation was arrested, but examination of the labelled protein over a period of 2 weeks indicated a slow decay, similar to that seen without ‘chasing’. Possible use of ‘pulse-chase’ experiments in vivo in animals is discussed in relation to behavioural studies.     

EFFECTS OF SODIUM FLUOROACETATE ON THE METABOLISM OF N-ACETYLASPARTATE AND ASPARTATE IN MOUSE BRAIN

A subconvulsant dose of sodium fluoroacetate inhibited the metabolic utilization of intracerebrally-administered N-acetyl-l -[U-¹⁴C]asparticacid and the labelling of glutamine from this precursor in mouse brain, but not the labelling of glutamate or aspartate. A convulsant dose also inhibited the utilization of l -[U-¹⁴C]aspartic acid. When intraperitoneal injection of a convulsant dose of sodium fluoroacetate was followed by intracerebral injection of N-acetyl-l -[U-¹⁴C]asparticacid, the levels of N-acetylaspartate, aspartate and glutamate in brain were lowered, while the glutamine content was increased. The specific radioactivity of glutamine relative to that of glutamate was much lower when these compounds were labelled from l -[U-¹⁴C]aspartic acid than when N-acetyl-l -[U-¹⁴C]aspartic acid was used as the precursor. Intracerebral injection of tracer amounts of l -[U-¹⁴C]aspartic acid reduced the content of N-acetylaspartate in brain and raised the glutamine content. Sodium fluoroacetate had no additional effect on the relative specific radioactivity of glutamine or the content of N-acetylaspartate, aspartate, glutamate or glutamine when l -[U-¹⁴C]aspartic acid was the precursor. We consider the results to be consistent with a selective inhibition both by sodium fluoroacetate and by exogenous aspartic acid of the tricarboxylic acid cycle in brain associated with the biosynthesis of glutamine. We suggest that the activity of this pathway may regulate the metabolism of N-acetylaspartate and aspartate.     

EFFECT OF UNDERNUTRITION ON AMINO ACID COMPARTMENTATION IN THE DEVELOPING RAT BRAIN

—Rat pups undernourished through 21 days of age show abnormal patterns of cerebral amino acid metabolism. The pattern of incorporation of radioactivity from l -[U-¹⁴C]leucine into amino acids derived from tricarboxylic acid cycle intermediates was altered, with significantly more ¹⁴C being incorporated into glutamate and aspartate in the underfed rats than in controls. Glutamate compartmentation, manifested in the ratio of specific radioactivities of glutamine to glutamate, developed more slowly in the. diet-restricted group. These results are similar to those seen in neonatally-thyroidectomized rats and suggest decreased growth of neuronal processes. This impairment of amino acid metabolism returns to normal after a 7-week period of adequate nutrition.     

THE EFFECT OF METHIONINE SULPHOXIMINE ON THE INCORPORATION OF LABELLED GLUCOSE, ACETATE, PHENYLALANINE AND PROLINE INTO GLUTAMATE AND RELATED AMINO ACIDS IN THE BRAINS OF MICE

—The incorporation of radioactivity from labelled glucose, acetate, phenylalanine and proline into glutamate, aspartate and glutamine was measured in mice treated with methionine sulphoximine and in the control animals. The labelled precursors were injected and their incorporation determined before the onset of convulsions. The incorporation of radioactivity from labelled glucose into the dicarboxylic amino acids was reduced, in particular the incorporation into glutamine. The incorporation of radioactivity from labelled acetate and phenylalanine into glutamate and aspartate was increased by methionine sulphoximine, while the incorporation into glutamine was not changed very much. The labelling of glutamine, relative to glutamate, was reduced with all precursors, indicating that glutamine synthetase was inhibited in vivo by methionine sulphoximine. It is very likely that methionine sulphoximine affects many aspects of energy metabolism in brain; in particular the metabolism of glucose seems to be inhibited, while the rate of conversion of substrates other than glucose seems to be increased.     

Measurement of protein turnover in rat brain

Abstract— Degredation rates of rat brain proteins were measured by following the decay in specific radioactivity of carboxyl labelled aspartate and glutamate over a 17-day period. Initial labelling of these amino acids was achieved by a single intraperitoneal injection 0f NaH¹⁴CO₃. The non-linear decay curve for total brain proteins could be approximated by assuming that the mixture contained two classes of proteins with half-lives of 3.3 and 8.7 days, respectively. Half-lives of 2.5 and 7.7 days were estimated for such protein classes in the microsomal fraction. The half-lives of soluble proteins, synaptic membranes, cell body and synaptic mitochondria were 3.1, 5.8, 5.6 and 8.4 days, respectively. Identical results were obtained if the change in specific activity of intact protein labeled by NaH¹⁴CO₃ was followed. Two-fold slower decay rates were obtained when brain proteins were labeled with a pulse of [4,5-³H]leucine or [l-¹⁴C]leucine. Half-lives calculated for the two classes of proteins in whole brain were 8.4 and 16.5 days, respectively with [4,5-³H]leucine and 8.9 and 14.2 days, respectively with [1-¹⁴C]leucine. These results indicate the very significant reutilization of this amino acid in brain. Sodium [¹⁴C]bicarbonate is a more satisfactory isotopic precursor for accurate assessment of rates of protein turnover in brain.