Bibliothek

Notizen: —The Na+ requirement of amino acid transport was measured in brain slices. The tissue was first washed free of Na+ and then Na+ was replaced by one of the following: choline, Li+, Rb+, or mannose. Amino acid uptake was measured at different times (5–120 min) and at low (10-7–10-5m) and high (10-3m) concentrations. Most of the Na+ could be washed out of the tissue; this also decreased K+ levels despite increased K+ in the medium. K+ tissue levels were partially restored when Na+ was added.The absence of Na+ abolished the uptake of Glu, Asp, GABA, Gly, Tau and Pro. Most of the neutral amino acids (Ala, Val, Trp, His) were very strongly inhibited by the absence of Na+ under most experimental conditions. Basic amino acids (Arg, Lys) were not completely inhibited, in that 30 per cent of the equilibrium uptake remained and some of the basic amino acid influx was independent of the Na+ tissue level. The uptake of amines (tyramine, cadaverine, putrescine) did not require Na+, and often was greater in the absence of Na+.We conclude that amino acid uptake in brain slices is Na+ dependent, although the absence of Na+ may affect transport indirectly.

Notizen: Abstract— Adult mice were fed standard diets that were enriched with selected amino acids, i.e. 3% methionine, 6% valine, or 8% lysine. These diets caused the following changes in the amino acid pool of the brain measured at 7 and 21 days. The high methionine diet resulted in 50-fold higher levels of methionine and cysteine and somewhat lower levels of serine and glutamine. The valine and lysine-enriched diets also caused 2- to 4-fold increases in valine and lysine contents of brain, respectively. In spite of the large changes in amino acid levels, however, there were essentially no changes in aspartate: α-ketoglutarate, alanine: α-ketoglutarate, ornithine: α-ketoglutarate, methionine: α-ketoglutarate, and the branched chain aminotransferase activities of brain 3, 10, and 21 days after the onset of the dietary regimen. In contrast, these diets produced significant changes in some of these enzyme activities in liver. Changes in liver included a 2-fold increase in ornithine and alanine aminotransferase activities with the methionine-enriched diet. Liver ornithine aminotransferase activity also increased slightly in animals fed the valine-enriched or lysine-enriched diet.

Notizen: Peptide and peptidyl-peptide hydrolase activities were measured in the cerebral cortex, adenoand neurohypophysis, anterior and posterior regions of the hypothalamus to assess regional differences in peptide hormone turnover. In general all enzyme activities were higher in the pituitary except for the monoacyl arylamidase which was lower, and neutral proteinase which was distributed more evenly in all regions. Of the enzymes measured the highest activities occurred in the presence of the di- and tripeptide substrates (aminopeptidases); this activity was some 20-60-fold higher than the acid and neutral proteinases. Comparison of the peptide-amide substrates revealed the highest activity with the monoacyl derivative which was five-fold higher than the dipeptidyl, and 50-fold higher than the tripeptidyl (hormonal) factor Pro-Leu-Gly. NH2. Analysis of the breakdown products of the hormonal factor indicated inactivation by N-terminal release of Pro, Leu with Gly. NH2 as the final product. Regional differences in enzyme content in neurosecretory areas suggest that this plays a role in the turnover of specific hormones.

Notizen: 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.

Notizen: Abstract— The aminotransferase activity of homogenates of brains from adult and neonatal rats has been investigated. Aminotransferase activity was demonstrated wtih 15 of 22 amino acids incubated with seven keto acids. The basic amino acids exhibited little or no activity.〈list xml:id="l1" style="custom"〉1The greatest activity was obtained when glutamate or aspartate was incubated with α-ketoglutarate or oxaloacetate. Significant activity was also observed when the neutral aliphatic and aromatic amino acids were incubated with these two keto acids.2Activity with pyruvate was obtained principally upon incubation with glutamate and alanine. Most of the other amino acids that underwent transamination with α-ketoglutarate also did so with pyruvate, although at a lower rate.3When phenylpyruvate was added to the medium, glutamate, phenylalanine and tyrosine transaminated most actively.4Incubations with 11 amino acids and glyoxylic acid demonstrated aminotransferase activity, with glutamate and ornithine being the most active substrates.5α-Ketoisocaproate and α-ketoisovalerate accepted amino groups primarily from the branched-chain amino acids. Except for glutamate, activity with other amino acids was low or not detectable.6A comparison of aminotransferase activity in the newborn brain with that in the adult brain showed that the greatest change in activity occurred for glutamate with pyruvate or for alanine with α-ketoglutarate, these activities increasing about 10-fold from birth to adulthood; during this time activities with most other amino acids increased two- to threefold. Amino transfers from the branched-chain amino acids showed no increase with maturation, and some reactions, such as that with methionine and a number of keto acids, decreased from birth to adulthood.7Our results correspond in general to previous studies of aminotransferase activity in brain and in liver. However, our study also indicates a possible second aminotransferase acting on the branched-chain amino acids, the presence of aminotransferase activity for methionine and asparagine, and relatively high aminotransferase activity for glutamine or ornithine when incubated with glyoxylic acid rather than other keto acids. Moreover, phenylpyruvate and glyoxylate are active in amino transfers and may serve as substrates for a number of aminotransferases.

Notizen: 〈list xml:id="l1" style="custom"〉1Slices of mouse brain were incubated with [U-14C]alanine, valine, leucine, phenylalanine, proline, histidine, lysine, arginine or aspartic acid, and the extent of metabolism was estimated by analyses utilizing paper chromatography of the tissue extracts and with an amino acid analyser.〈list xml:id="l2" style="custom"〉2The metabolism of Ala and Asp was high; of Leu and Pro, moderate; and of Lys, Arg and Phe, low; the metabolism of Val and His was not significant. The time-course of metabolism in most cases showed varying rates, indicating heterogeneous metabolic compartments for the amino acids.〈list xml:id="l3" style="custom"〉3Production of CO2 was high from Asp, moderate from Ala, and low from Leu; the other amino acids were not oxidized to CO2 to any significant extent. A large portion of the metabolized label was trapped in the form of Glu or Asp.〈list xml:id="l4" style="custom"〉4Metabolism increased with increasing concentration of amino acid to some extent and was largely inhibited by omission of glucose, by anaerobic conditions, or by cyanide. Although these conditions also inhibit uptake, the time-course and extent of inhibition uptake and metabolism were different.〈list xml:id="l5" style="custom"〉5With Asp, Ala and Phe, metabolism was lowest in slices from pons-medulla; the brain area exhibiting the highest metabolism differed for each amino acid. The metabolism of Asp was lower in brain samples from newborn than in those from adults; the metabolism of Leu was higher in slices from newborn brain.〈list xml:id="l6" style="custom"〉6The results indicate that the majority of the amino acids can be metabolized in brain tissue and that the metabolic rates are influenced by a number of factors, among them the level of amino acids and the level of available energy.

Notizen: Abstract— (1) Mouse cerebrum slices swell in tris-buffered Krebs-Ringer medium. Swelling is rapid at first, then slows to a more or less constant rate. Even after 3 hr incubation, water content/g of tissue dry wt. shows no sign of an asymptotic limit. Swelling is the same at 37° and at 0°.(2) Tissue water measured by incubation with tritiated water is equal to total tissue water measured by drying slices. Equilibration between tritiated water and tissue water is complete within 2 min.(3) Tissue liquid can be divided into three phenomenologically distinguishable compartments: first inulin space, which is the compartment permeable to inulin at both 0° and 37°; second inulin space, which is the compartment permeable to inulin at 37° but not at 0°; and 37°non-inulin space, which is the compartment impermeable to inulin at both 0° and 37°. The evidence for this is:(a) Penetration of inulin into tissue is greater at 37° than at 0°. After the first 20 min the rate of penetration at 0° is approximately equal to the rate of penetration at 37°, and only slightly less than the rate of increase of total tissue water. Therefore the smaller inulin space observed at 0° cannot be due to slower entry of inulin.(b) The inulin content of slices incubated in inulin-containing medium at 37° and cooled to 0° in the same medium is the same as the inulin content of tissue incubated at 37° without subsequent cooling. In contrast, the inulin content of tissues preincubated in inulin-free medium at 37° and then incubated in inulin-containing medium at 0° is the same as the inulin content of tissues incubated in inulin-containing medium at 0° without preincubation at 37°. Therefore the smaller inulin space at 0° than at 37°can be due neither to a reversible temperature-dependent change in the size of one single inulin space nor to an irreversible, greater swelling of a single inulin space at the higher temperature, but is due to some portion of the 37° inulin space becoming impermeable to inulin at 0°.(c) Some inulin is retained by tissue incubated with inulin at 37°, then transferred to inulin-free medium at 0°; the amount of retained inulin is equal to the difference between inulin content of tissue incubated with inulin at 37° and tissue incubated with inulin at 0°. This confirms 3b above and in addition shows that inulin which has entered the second inulin space at 37° is trapped there when this space becomes impermeable to inulin at 0°.(4) The penetration of the amino acids, L-lysine and D-glutamate at 0° is equal to the penetration of inulin at 37°. This confirms the real existence of the 37° inulin space at 0°, and shows that the barrier at 0° between the first and second inulin spaces does not exist for these substances.(5) The amino acids L-leucine and glycine penetrate total tissue water at 0°. L-leucine is actively transported at this temperature.(6) The amino acids α-aminoisobutyric acid, L-leucine, and L-lysine at 2 mm have no effect at 37° on either the inulin space or the non-inulin space.(7) The inulin space is insensitive at 37° to physiologically significant changes in the medium. In contrast, the non-inulin space is quite sensitive to these changes. Addition of D-glutamate greatly increases the non-inulin space; addition of ouabain or cyanide, or omission of glucose, increases the non-inulin space slightly; and replacement of Na+ ion by choline+ ion greatly decreases this space. These changes are independent and roughly additive.

Notizen: : The properties of the uptake of nucleosides and nucleotides by brain cells were examined in slices of mouse brain. Of the compounds tested, adenine and adenosine had the most rapid uptake and reached the highest levels. Uptake was mediated, as shown by saturability and strong inhibition, by low temperature, or by cyanide, and was only partially sodium- or calcium-dependent. The inhibition pattern by analogues indicated the presence of several uptake systems (possibly four), as shown by differences between adenine and guanine uptake, between adenine and adenosine uptake, and between adenosine and cytidine uptake. The properties of uptake systems for nucleotides and nucleosides were somewhat different from those for amino acids.