Turnover of dhurrin in green sorghum seedlings

The turnover of dhurrin in green seedlings of Sorghum bicolor (Linn) Moench var Redland x Greenleaf, Sudan 70 has been investigated using glyphosate and pulse-labeling studies with ¹⁴C-tyrosine and [¹⁴C]shikimic acid. The rate of dhurrin breakdown was 4.8 nanomoles per hour in the shoot and 1.4 nanomoles per hour in the root. The rate of dhurrin accumulation in the shoot of 4- to 5-day-old seedlings was high but decreased with age until at the peak period of dhurrin accumulation, the rates of dhurrin synthesis and breakdown were equal. Using a first order equation (an approximation) the rate of dhurrin synthesis (which equals accumulation plus breakdown rates) was 17.4 nanomoles per hour in the shoot and 4.1 nanomoles per hour in the root. In both tissues, the breakdown rate was between 27 and 34% of their synthetic capacity within the experimental period. Dhurrin synthesis in green sorghum seedlings occurred in both the light and dark photoperiods but was faster in the dark period. The result is discussed in relation to the possible metabolic roles of the turnover.     

Compartments of protein metabolism in the developing brain.

We investigated whether the higher rate of amino acid incorporation into immature than into mature brain protein is due to (a) rapid growth, (b) a small rapidly metabolized protein pool, or (c) a higher turnover rate of most of the protein. We measured net growth and the incorporation of [14C]tyrosine or [14C]valine into brain proteins in young rats and mice. The specific activity of the free amino acid pool was kept constant in the tyrosine experiments. Incorporation of tyrosine into protein was continued for up to 30 h by which time the specific activity of protein-bound amino acid reached 1/3 of that of the free (precursor) amino acid. The growth (accretion) of brain proteins was approx. 0.635% per h in mice and rats in the 1-4 day period after birth. In previous studies we found that the turnover rate of the bulk (about 96%) of adult brain proteins is below 0.3% per h. Because of the presence of a small (about 4%) active pool the average turnover rate is 0.6% per h. The present experiments show a degradation rate of 0.7-1.1% per h in the brain proteins of the young. This high metabolic rate is not due to a small rapidly degraded fraction of protein. The very rapid protein fraction previously seen in adult rats is either very small (below 1%) or absent in the young. Thus most of the proteins in the immature brain during the rapid growth phase are formed and broken down at a rate that is approximately three times higher than that of the bulk of proteins in the adult brain. The small active protein pool in the adult on the other hand has a metabolic rate higher than that of the immature brain proteins.     

Cerebral lipid metabolism in experimental hyperphenylalaninaemia: incorporation of 14C-labelled glucose into total lipids

—1. Effects of the administration of phenylalanine to rats on incorporation in vivo or in vitro of [U-¹⁴C]glucose into cerebral lipids were studied during the first 5–10 days of postnatal development. In addition, the effects of added phenylalanine and its deaminated metabolites on incorporation of [U-¹⁴C]glucose by homogenates into lipids of developing rat brain were investigated. Hyperphenylalaninaemia reduced incorporation both in vivo and in vitro of [U-¹⁴C]glucose into cerebral lipids. 2. Phenylalanine or tyrosine added in vitro at concentrations equivalent to those in the brain of the hyperphenylalaninaemic rat (0-1 μmole/ml incubation medium) did not inhibit incorporation of [U-¹⁴C)glucose into lipids, although at much higher concentrations of phenylalanine (36 μumoles/ml incubation medium) slight inhibition (10 per cent) of incorporation of [U-¹⁴C]glucose into lipids was observed. 3. In contrast, the deaminated metabolites in general exerted greater inhibitory effects at lower concentrations. Phenyllactic acid, in comparison to phenylpyruvic and phenyl-acetic acid, was the most potent inhibitor of the incorporation in vitro of [U-¹⁴C]glucose into cerebral lipids. These results indicated that these metabolites of phenylalanine were the more potent inhibitors of cerebral lipid metabolism in immature animals.     

Defect in translation of poliovirus RNA at the restrictive temperature.

Translation of the RNA of LSc type 1 poliovirus was examined in vivo at the restrictive temperature (39 °C). During the first two hours of infection at 39 °C the levels of viral polyribosomes were 50% lower than at 35 °C (permissive temperature). During the third hour of infection at 39 °C, only 4 to 10% of the control levels of polyribosomes were observed. Three experiments indicate that the elongation of viral peptides was not occurring properly at 39 °C. First, cultures incubated at 39 °C during the third hour of infection with both [³⁵S]methionine and [³H]uridine exhibit a fourfold increase in the ratio of viral protein/viral RNA in the polyribosome region of sucrose gradients in comparison to controls kept at 35 °C. However, at both temperatures the relative size distribution of polyribosomes was similar. Second, the ratios of released protein/nascent protein after 90-second and 5-minute pulses with [³⁵S]methionine indicate that elongation of peptide chains was inhibited at 39 °C. Third, when initiation of synthesis of viral protein was blocked with 150 mM-NaCl, the polyribosomes disaggregated four to five times more rapidly at 35 °C than at 39 °C. The data indicate that translation of viral RNA is inhibited at the restrictive temperature because of a reduced rate of elongation of viral proteins. The reduced rate of peptide chain elongation at 39 °C was fully reversible when cultures were shifted to 35 °C in the presence of 150 mm-NaCl. The latter finding indicates a conformational change in viral protein at 39 °C.     

THE EFFECT OF LIMB ISCHAEMIA ON THE TURNOVER OF NORADRENALINE IN THE HYPOTHALAMUS AND BRAIN STEM OF THE RAT

The effect of ischaemic limb injury on the turnover of noradrenaline in the hypothalamus and brain stem has been studied in rats. There are theoretical reasons for thinking that these regions are activated in trauma and previous work showed that during limb-ischaemia the concentration of noradrenaline in the hypothalamus decreased by 27 per cent. The tourniquets were applied to both hind-limbs 1 h after the injection of [¹⁴C]-tyrosine when the labelling of the noradrenaline was maximal. During 4 h limb ischaemia the endogenous tyrosine concentration in the plasma decreased while that in the hypothalamus first rose and then fell. Changes in a similar direction in the brain stem were not statistically significant. Limb ischaemia did not affect the decline in the specific activity of the plasma or tissue tyrosine. It was concluded that the injury increased the utilization of tyrosine by the body. During the 4 h bilateral hind-limb ischaemia the rate of decline of [¹⁴C]noradrenaline was significantly increased in the brain stem but not in the hypothalamus. Conditions in the brain stem were sufficiently close to ‘steady-state’ to be able to conclude that the injury increased the metabolism of noradrenaline in the brain stem. Conditions in the hypothalamus were too complicated for definite conclusions to be drawn. The possible reasons for this and the limitations of this method for studying noradrenaline turnover are discussed.     

BIOCHEMICAL EFFECTS OF THYROID DEFICIENCY ON THE DEVELOPING BRAIN

Abstract— The effects of neonatal thyroidectomy on some constituents of the cerebrum, cerebellum and liver of the rat have been studied during the first 7 weeks of life. In the normal rat between the 6th and 14th post-natal days the RNA content per unit of DNA in the brain increased by 70 per cent. Although the brain continued to grow from the 14th to the 35th day, the amount of RNA relative to DNA decreased by about 20 per cent. The ratio of protein to DNA increased during the whole period studied and in the cerebral cortex it was more than trebled between the age of 6 and 35 days. The growth of the cerebellum extended over a longer period than that of the cerebrum, its weight increasing by 88 per cent between the ages of 14 and 35 days as compared with a cerebral increase of 34 per cent. The DNA content showed a 50 per cent increase during this period. Qualitatively these maturational changes were not affected by neonatal thyroidectomy. Quantitative changes, which applied equally to the cerebral cortex and brain as a whole, were observed. At the age of 35 days, the weights of the cerebral hemispheres and cerebellum were reduced by thyroidectomy by 20 per cent; the overall DNA content per organ did not change, but the amounts of protein and RNA relative to DNA decreased significantly. It is therefore inferred that thyroid deficiency affects the size of the cells in brain and cerebellum rather than their total number. Conversely, the cell population of the liver was only a quarter of that in the control. There was a small but significant decrease in the hepatic protein and RNA content in the hypothyroid animal. The activities of the following enzymes which served as markers for subcellular fractions in homogenates of cerebral cortex were determined: lactate dehydrogenase for the supernatant, glutamate dehydrogenase for the mitochondrial and glutamate decarboxylase for the synaptosomal fractions. When the activities were expressed on a fresh weight basis a significant decrease by comparison with the control values was observed only in the case of glutamate decarboxylase (—15 per cent at the age of 17–32 days); when the activities were based on DNA content all values were reduced, probably as a result of the general decrease in cell size. Pyrimidine metabolism of brain and liver, studied after the administration of [6-¹⁴C]-orotic acid, was not affected in either tissue by neonatal thyroidectomy. A small but significant reduction in the incorporation of labelled pyrimidine nucleotides in liver RNA was observed, but no significant decrease in the incorporation in cerebral RNA was found in the hypothyroid rats.     

Biopterin

The active synthesis of [¹⁴C]7,8-dihydrobiopterin (BH₂) from intraventricularly administered U-[¹⁴C]GTP was demonstrated in rat brain. The identity of [¹⁴C]BH₂ isolated from brain was confirmed by mass fragmentography. Evidence is presented that [¹⁴C]BH₂ in brain was not synthesized in the peripheral organs. The rate of cerebral synthesis of [¹⁴C]BH₂ from [¹⁴C]GTP was maximal at 2 h; it was 0.53 nmol/g per h, which is consistent with the estimated turnover rate of cerebral BH₂ (0.43 nmol/g per h). Intraventricularly injected 2,4-diamino-6-hydroxypyrimidine (DAOPyr) and 6-thioguanosine were effective inhibitors of the synthesis. U-[¹⁴C]dGTP and 8-[¹⁴C]GTP, when given intraventricularly, did not yield [¹⁴C]BH₂. Simultaneous intraventricular injection of U-[¹⁴C]GTP and DAOPyr resulted in the accumulation of a compound with properties identical to a formamidopyrimidine derivative isolated from the nonenzymatic hydrolysis of GTP. The data from preliminary experiments demonstrated the synthesis of [¹⁴C]BH₂ from U-[¹⁴C]GTP incubated with 12,000g supernatants of rat brain homogenates.     

In vitro studies of cerebral cortical RNA and nucleotide metabolism in hypothyroidism.

—Thyroid hormone deficiency induced during the neonatal period in the rat, resulted in an enhanced incorporation of [2-¹⁴C]uridine and [8-¹⁴C]adenosine in vitro into cerebral cortical RNA at 25 days of age. An examination of the acid-soluble pool constituents separated by polyethyleneiminecellulose TLC, revealed that all phosphorylated derivatives were more highly labelled compared to controls. These differences were not apparent at a lower incubation temperature (4°C). When the average specific activity of precursor pool ATP labelled from adenosine was utilized for the calculation of the rate of RNA synthesis, no change was observed in hypothyroidism. The results are compatible with a maturational-dependent increase in nucleoside transport and rate of phosphorylation in hypothyroidism which is reflected in the stimulated incorporation into cerebral RNA. The apparent normal rate of RNA synthesis coupled with a diminished cellular RNA concentration in thyroid hormone deficiency, suggests an increased RNA turnover. Experiments with actinomycin D revealed no apparent difference in the rate of decay of rapidly-labelled (nuclear) RNA. The possibility is discussed that the processing of nuclear RNA, the formation of stable ribosomal complexes and events at the translational level are subject to modification in developing hypothyroid rats.     

Rapid transport of protein in the optic system of the goldfish

Abstract— Several amino acids, particularly [³H]proline and [³H]asparagine specifically and efficiently labelled rapidly transported proteins in the goldfish optic nerve and tectum after intraocular injection. Studies with these amino acids showed that the rapidly transported proteins moved as a discrete band at a rate which was temperature-dependent, and was equal to 70-100 mm per day at 20°C. Transported protein in the optic tectum was 80 per cent particulate and was found in synaptosomal, mitochondrial, and myelin fractions, but not in purified nuclei or ribosomes.     

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.     

METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

• 1 The metabolism of two ¹⁴C-labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro.
• 2 The metabolism of [U-¹⁴C]mannose was essentially identical to that of glucose; oxygen consumption and CO₃ production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration.
• 3 With [U-¹⁴C]fructose, maximal oxygen consumption and CO₃ production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3-¹⁴C]pyruvate into CO₂ or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO₂ from [U-¹⁴C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent.
• 4 By comparison with [1-¹⁴C]glucose, incorporation of radioactivity from [1-¹⁴C]-glucosamine into lactate, CO₂, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine-1-phosphate, glucosamine-6-phosphate, and an unidentified metabolite, all accumulated.

     

Oxidation of Fatty Acids by Leishmania braziliensis panamensis1

Heating cultures of Leishmania braziliensis panamensis (grown at 26°C) to 34°C for 1.5–12 h transformed the cells to an ellipsoidally shaped form. The heat treatment caused an increase in the rate of oxidation of both medium and long chain fatty acids but decreased the rate of oxidation of [1-¹⁴C]glucose. The rate of fatty acid oxidation continued to increase for times as long as 20 h after returning the cultures to 26°C. In both the promastigote and heat-induced ellipsoidal forms, the ratio of ¹⁴CO₂ release from [1-¹⁴C]laurate to that from [12-¹⁴C]laurate was generally larger than four, whereas this ratio from [1-¹⁴C]oleate relative to [10-¹⁴C]oleate was approximately two. These data show that metabolic and morphological differentiation begin after a short heat treatment and that some metabolic changes may continue even after the reverse transformation is initiated. The data also suggest that either the ω-terminal portion of the fatty acids is not completely oxidized to acetyl CoA and/or that there are two functional fatty acid oxidation pathways in Leishmania.     

Arginine Metabolism in Developing Soybean Cotyledons: III. Utilization

Tracerkinetic experiments were performed using l-[guanidino-¹⁴C]arginine, l-[U-¹⁴C]arginine, l-[ureido-¹⁴C]citrulline, and l-[1-¹⁴C]ornithine to investigate arginine utilization in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with carrier-free ¹⁴C compounds and incubated in sealed vials containing a CO₂ trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. After 4 hours, 75% and 90% of the ¹⁴C metabolized from [guanidino-¹⁴C]arginine and [U-¹⁴C]arginine, respectively, was in protein arginine. The net protein arginine accumulation rate, calculated from the depletion of nitrogenous solutes in the cotyledon during incubation, was 17 nanomoles per cotyledon per hour. The data indicated that arginine was also catabolized by the arginase-urease reactions at a rate of 5.5 nanomoles per cotyledon per hour. Between 2 and 4 hours ¹⁴CO₂ was also evolved from carbons other than C-6 of arginine at a rate of 11.0 nanomoles per cotyledon per hour. It is suggested that this extra ¹⁴CO₂ was evolved during the catabolism of ornithine-derived glutamate; ¹⁴C-ornithine was a product of the arginase reaction. A model for the estimated fluxes associated with arginine utilization in developing soybean cotyledons is presented.     

HE TURNOVER RATE OF TUBULIN IN RAT BRAIN

The turnover rate of tubulin in rat brain was determined from the decay in specific radioactivity of the protein after pulse-labeling. When precursors were administered by a parenteral route, the shortest half-life, 9.8 days, was obtained with [¹⁴C]NaHCO₃; the longer half-lives obtained with [U-¹⁴C]glucose or [4,5-³H]leucine suggest significant reutilization of label. Furthermore, with leucine as precursor maximal specific radioactivity of tubulin was not obtained until eight days after administration of label. Labeling and decay kinetics obtained with [4,5-³H]leucine were markedly different when the isotope was administered directly into the lateral ventricle. The difference between the turnover rates of the -α and β subunits of tubulin purified by means of high resolution polyacrylamide gel electrophoresis was not statistically significant. A half-life for tubulin of 6.2 days was measured by continuous intravenous infusion of [U-¹⁴C]tyrosine.     

RATES OF CEREBRAL GLUCOSE UTILIZATION IN RATS ANAESTHETIZED WITH PHENOBARBITONE

Abstract—

• 1 Intraperitoneal injection of phenobarbitone (250 mg/kg body wt.) into rats caused increased brain concentrations of glucose (100 per cent), glucose 6-phosphate (16 per cent) and ATP (12 per cent) and decreased concentrations of lactate (33 per cent) and ADP (15 per cent). A 31 per cent decrease in glutamate content was not statistically significant. No significant change occurred in the cerebral contents of glycogen or creatine phosphate.
• 1 The rates of increase in the brain of specific activities, in the first few minutes after systemic injection of [U-¹⁴C]glucose, of glucose, lactate, glutamate and glycogen were all halved by phenobarbitone. Calculated flux rates of ¹⁴C from glucose into metabolic intermediates and from lactate to glutamate were also decreased by 27–47 per cent; the effects on rate constants showed inconsistencies. The rate constants for conversion of glucose to lactate and to glutamate were decreased by 60–70 per cent, but that from lactate to glutamate was virtually unchanged. The rate constant for the flux from glucose to glycogen was reduced by 39 per cent, but the accumulation of glucose meant that the actual flux into glycogen increased by 20 per cent.
• 1 The results are interpreted in terms of an effect of the barbiturate not only on glucose transport, but also at an enzymic stage in glycolysis, possibly hexokinase or phosphofructokinase.

     

Biosynthesis of myelin proteins in vitro

Abstract— The rates of uptake of DL-[1-¹⁴C]leucine into the three classes of protein in myelin isolated from slices of rat brain and spinal cord were determined. Basic protein exhibited the slowest rate of uptake; chloroform-methanol-soluble proteolipid protein exhibited intermediate rates and the insoluble protein had the most active uptake. All myelin proteins were less active than the mixture of proteins derived from the non-myelin fraction. Cyclohexi-mide (10^?3 M) and choramphenicol (5 × 10^?3 M) inhibited the incorporation of [1-¹⁴C]leucine into brain proteins by as much as 95 per cent. γ-Aminobutyric acid had no effect on the system. Chloramphenicol also inhibited the uptake of [1-¹⁴C]acetate into myelin lipids, but cycloheximide did not affect lipid synthesis. These effects were observed on both 35-day-oldand 18-month-old rats, but the biosynthetic activity was far less in myelin from the older rats. The results are discussed in relation to the structure of myelin. It is suggested that the data best fit models in which lipid and protein are in separate phases in the membrane.     

ACTIONS OF NEUROHUMORAL AGENTS AND CEREBRAL METABOLITES ON OUTPUT OF ADENINE DERIVATIVES FROM SUPERFUSED TISSUES OF THE BRAIN

—Adenine nucleotides of guinea-pig neocortical tissues were labelled by prior incubation with [¹⁴C]adenine and excess of adenine was then removed by superfusion with precursor-free media. During continued superfusion labelled adenine derivatives were released at a stable rate of about 0·05 per cent of the tissue ¹⁴C/min and this rate was increased about five-fold by electrical stimulation. Various compounds, including some known to increase the cyclic AMP content of cerebral tissues, were examined for action on the release of [¹⁴C]adenine derivatives from the tissue and also on the rates of lactate production by the tissue, both before and during electrical excitation. The tissue content of adenine nucleotides following exposure of the tissue to these compounds was also determined. Noradrenaline, γ-aminobutyrate and acetylcholine together with carbamoylcholine at the concentrations examined were without effect on the release of ¹⁴C compounds from the tissue. Also, noradrenaline and γ-aminobutyrate caused no alteration in lactate production but brought about some decrease in the adenylate energy charge of the tissue. Histamine, 100 μm , brought about a small but consistent increase (35 per cent) both in release of ¹⁴C-compounds and lactate output, while reducing the adenylate energy charge of the tissues. l -Glutamate at 5 mm decreased the tissue adenylate energy charge to a greater extent than did histamine; it increased the release of ¹⁴C-compounds seven to eight-fold and similarly increased the tissues' rates of lactate production. Lower concentrations of glutamate had smaller effects. In those cerebral tissues whose cyclic AMP content is increased by l -glutamate, the increase is probably brought about by intermediation of released adenosine.     

Ethanol inhibits phosphatidylcholine and phosphatidylethanolamine biosynthesis in human leukemic monocyte-like U937 cells

The effect of ethanol (ETOH) on the incorporation of [¹⁴C]oleic acid (18:1) into lipid in human monocyte-like U937 cells was investigated. With increasing time of exposure to ETOH, the percentage of the label distributed into neutral lipid (NL) declined from 35 per cent (3 h) to 10 per cent (24 h) accompanied by increased incorporation into phospholipid (PL). [¹⁴C] 18 : 1 was preferentially incorporated into triglyceride (TG) and phosphatidylcholine (PC), comprising over 65 per cent and 50 per cent of the label associated with NL and PL, respectively. Low concentrations of ETOH (⩽ 1·0 per cent; v/v) had no effect. At concentrations greater than 1·5 per cent, there was enhanced incorporation into TG and diacylglycerol (DAG) in a 24-h incubation period, while at 16 h the label in phosphatidylethanolamine (PE) was decreased. The effect of ETOH on the CDP-choline or ethanolamine pathway was examined by monitoring the incorporation of [³H]choline or [¹⁴C]ethanolamine into PC or PE, respectively. At low concentrations ETOH had no effect on either choline uptake or the incorporation into PC. Higher concentrations (≥ 1·5 per cent) for 3 and 6 h resulted in a slightly decreased choline uptake, and the reduction (40–50 per cent) of incorporation into PC suggests that the CDP-choline pathway was inhibited. There was a similar inhibition of the incorporation of [¹⁴C]ethanolamine into PE. When the cells were incubated for 3 h in the presence of 2 per cent ETOH and with labelled 18 : 1 and PL-base, the ratios of incorporation (base/18 : 1) into PC and PE fractions decreased, indicating that the major inhibition lay in blockage of the availability of the base moiety for PL formation. Analysis of the distribution of the label into metabolites revealed that ETOH inhibited the conversion of [¹⁴C] ethanolamine into [¹⁴C]phosphorylethanolamine. The reduction in incorporation was not due to the enhanced breakdown of base-labelled PL. Our results indicate that ETOH has an inhibitory effect on the CDP-choline or ethanolamine pathway.     

GLUTAMYL, ASPARTYL AND AMIDE MOIETIES OF CEREBRAL PROTEINS: METABOLIC ASPECTS IN VITRO

Abstract— The total mixed proteins (excluding proteolipids) were isolated from cat cerebral cortex and subjected to acid and enzymic hydrolyses. Analyses on the hydrolysates were carried out by specific enzymic procedures to determine the glutamyl, glutaminyl, aspartyl and asparaginyl composition. The content of total glutamyl and total aspartyl residues was the same in all types of protein samples, with average values of 78 and 58 /miol/100 mg of protein, respectively. In biopsy samples approximately 45 per cent of each total was in the amide form. Preparation of slices of cerebral cortex for incubation was associated with deamidation in situ of 16 per cent of the protein-bound glutaminyl residues. The extent of deamidation was not increased by incubation or by prolonged hypoxia and was unaffected by prior anaesthesia or by incubation of slices with 10 mM-NH₄Cl or 40 mM-malonate. Slices prepared from animals intoxicated with methionine sulphoximine exhibited no deamidation. No deamidation was observed for slices of subcortical white matter, liver, kidney, testis or diaphragm of the cat. Cortical proteins from other species appeared to behave similarly to those of the cat. The 5-4 μmol of NH₃ released/g of fresh cortex could account for about 85 per cent of the endogenous free ammonia regularly encountered in such slices. Hence the labile fraction of protein-bound glutaminyl amide groups represents, as previously suspected, a major source of endogenous cerebral NH₃. Proteins isolated from cerebral cortical slices incubated with L-[U-¹⁴C]glutamic acid or L-[U-¹⁴C]glutamine contained 105 (±0.095) per cent of the total ¹⁴C metabolized. The ratios (x 100) of protein to free pool specific radioactivities (c.p.m.μmol) of glutamic acid and of glutamine were in the range 0-22 to 0-42, or of the same order as previously reported for other amino acids. Comparable results were obtained with proteins isolated from cerebral cortical slices incubated with 10 mM-¹⁵NH₄Cl or L-[amide-¹⁵N]glutamine or both. In the amide N of protein-bound glutaminyl residues the atoms per cent excess ¹⁵N ranged from 007 to 0-42. This degree of labelling could be accounted for completely by the turnover of the entire glutaminyl moiety, as indicated by the ¹⁴C studies. Simultaneous analyses of free pool NH₃ and glutamine suggested that transfer of glutamine from medium to slice involves deamidation as it is taken up and reamidation after entry.     

MEASUREMENT OF BRAIN PROTEIN TURNOVER WITH [14C]SODIUM BICARBONATE1

Abstract— Protein turnover in rat brain was measured over a period of 30 days by following the decay in specific radioactivity of acidic amino acids in proteins labelled by a single intraperitoneal injection of [¹⁴C]NaHCO₃. Two major populations of brain proteins can be identified from the resultant non-linear decay curve—one with an average half-life of 4 days and another with an average half-life of 12 days. The half-lives of total brain, mitochondrial, microsomal and soluble proteins determined over a period of 5 days were 3.4, 5.8, 2.8, and 2.6 days, respectively. Turnover of these same brain subcellular fractions was also measured by continuous infusion of [¹⁴C]tyrosine. The estimated half-lives were in close agreement with those obtained from the 5 day measurement of radioactive decay following a pulse label of [¹⁴C]NaHCO₃.