Separate binding sites in rat brain synaptic membranes for l-cysteine sulfinate and for l-glutamate

Binding of l-[³H]cysteine sulfinic acid (CSA) and l-[³H]glutamate were compared in various subcellular fractions and in the presence of a variety of pharmacological and ionic manipulations in order to test the possibility that the two amino acids possessed separate binding sites.The specific l-[³H]cysteine sulfinate binding was found to be enriched maximally in medium and high density synaptic membranes, while the crude mitochondrial synaptosomal fraction displayed the highest l-[³H]glutamate binding. The ratio of l-[³H]cysteine sulfinate binding/l-[³H]glutamate binding was variable across brain regions. Several compounds differentially affected l-[³H]cysteine sulfinate and l-[³H]glutamate binding. l-cysteine sulfinate was the most potent displacer regardless of the binding considered. Finally, while cations produced qualitatively similar effects on the binding of the two amino acids, quantitative differences were evident.In sum, these data revealed the complexity of l-[³H]cysteine sulfinate and l-[³H]glutamate binding. They suggest the existence of several binding sites and that some of these are shared by both substances. However, the results also indicate that separate binding sites for the two amino acids exist in synaptic membrane, giving further support to the hypothesis that cysteine sulfinate serves a neurotransmitter role in the central nervous system.     

Metabolic studies of rat liver plasma membranes using d-[l-C]glucosamine

The metabolism of plama membranes of rat liver cells was studied using d-[l-¹⁴C]glucosamine. The labelling of plasma membranes occurred more slowly than that of microsomes, reaching a maximum at about 3 h after injection compared to 1.5 h for microsomes, and the radioactivity decayed with a half-life of 37 h which is close to the value obtained using [guanidino-¹⁴C]arginine to label proteins. Hexosamine and sialic acid of plasma membranes were found to metabolize at practically equal rates.     

Studies on l-ascorbic acid biosynthesis and metabolism in Parthenocissus quinquefolia L. (vitaceae)

Parthenocissus quinquefolia (L.) Planch., commonly known as Virginia Creeper, is a vitaceous tartrate-accumulating vine that exhibits C-4/C-5 cleavage of l-ascorbic acid (AA) to produce l-tartaric acid (TA) from the C₄ fragment and carbohydrate pool material from the C₂ fragment. Experiments in which detached leaves were supplied d-[5-³H,1-¹⁴C]glucose or d-[5-³H,6-¹⁴C]glucose yielded AA devoid of ³H whereas the l-threonic acid (ThA) and TA recovered from the same tissues still retained some ³H. These comparative experiments also indicated that the ThA was derived from carbons 3 through 6 of d-glucose. ThA was shown to be a natural constituent of P. quinquefolia but apparently not an intermediate between AA and TA. Results are consistent with a biosynthetic pathway from d-glucose to AA that involves a hydrogen-exchanging epimerization at C-5 as reported earlier for the geraniaceous plant Pelargonium crispum, but differing from P.crispum in biosynthesis and metabolism of ThA.When l-[6-¹⁴C]idonate or its lactone was supplied to P. quinquefolia leaves, about 80% of the ¹⁴C appeared in the carbohydrates, an observation remarkably similar to previous observations with [6-¹⁴C]AA-labeled leaves. l-Idonate and its lactone appear to have an intermediate role in AA metabolism in vitaceous plants.     

A new assay for l-asparagine synthetase

A fast, relatively inexpensive method of measuring the enzymatic formation of l-asparagine from l-aspartate is presented. This radiochemical assay requires simple manipulations making it ideal for working with large numbers of samples, while maintaining high sensitivity and reproducibility. A mechanism similar to the enzymatic β-decarboxylation of aspartate is utilized but in a nonenzymatic reaction. In the presence of pyridoxal and Al³⁺ ions, the ¹⁴C of l-[4-¹⁴C]aspartate is decarboxylatd while l-[4-¹⁴C]asparagine remains intact. This assay is shown to be suitable for measuring mammalian l-asparagine synthetase activity, while not requiring the isolation of assay enzymes.     

Coronatine biosynthesis: Incorporation of l-[U-14C]isoleucine and l-[U-14C] threonine into the 1-amido-1-carboxy-2-ethylcyclopropyl moiety

Addition of either l-[U-¹⁴C]threonine or l-[U-¹⁴C]isoleucine to 2.7-day-old shaking liquid cultures of Pseudomonas syringae pv. atropurpurea resulted in incorporation of radioactivity into coronatine, but not into N- coronafacoylvaline, another phytotoxin excreted by P.s. atropurpurea. In contrast, addition ofl-[U-¹⁴C]valine did not lead to incorporation of radioactivity into coronatine, but instead into coronafacoylvaline. Acid hydrolysis of the purified [¹⁴C] coronatine obtained after incorporation of either [¹⁴C]isoleucine or [¹⁴C]threonine demonstrated that > 94% of the radioactivity was present in the 1-amido-1-carboxy-2-ethylcyclopropyl moiety of coronatine, and < 6 % was in the coronafacoyl moiety. These findings are used to propose a biosynthetic pathway for coronatine.     

The metabolism of l[3-3H]lactate by isolated hamster liver cells

The rate of tritium removal from l[3-³H]lactate by hamster liver cells is faster than the analytical rate of lactate utilization, or the rate of ¹⁴C disappearance from l[U-¹⁴C]lactate, with the result that the ³H/¹⁴C ratio in residual lactate from l-[U-¹⁴C,3-³H]lactate decreases. However, addition of low concentrations (0.1 to 1.0 mM) of l-cycloserine, a glutamate pyruvate transaminase inhibitor, nearly equalizes the rates of isotope utilization from l-[3-³H]lactate and l-[U-¹⁴C]lactate. The results suggest a very limited rate of recycling of phosphoenolpyruvate back to pyruvate during gluconeogenesis from lactate in fasted hamster liver cells.     

Metabolism of 14C-labelled D-glucose, D-fructose and allitol by Itea plants

The metabolism of D-[1-¹⁴C]glucose, D-[6-¹⁴C]glucose, D-[1-¹⁴C]fructose and D-[6-¹⁴C]fructose by leafy spurs of Itea plants results in rapid incorporation of label into allitol and D-allulose. The patterns of labelling found in the allitol and D-allulose are discussed, a direct interconversion from D-glucose and D-fructose being indicated. Allitol has been found to be an active metabolite in Itea plants.     

l-DOPA (l-3,4-dihydroxyphenylalanine) uptake by human red blood cells

The uptake of l-DOPA (l-3,4-dihydroxyphenylalanine) was studied in normal human red blood cells in vitro using l-[3-¹⁴C]DOPA. Uptake was slow, tending towards a distribution ratio close to unity with a half-time to equilibrium of one hour. Uptake was not Na⁺-dependent. Concentration dependence studies showed both saturable and non-saturable components of uptake, and inhibition studies using l-leucine and l-tryptophan suggest that the L and T systems of red cell amino acid uptake are involved. A powerful inhibitor of both systems, 3,4-dihydroxy-2-methylpropriophenone (U-0521), is described. It is concluded that uptake is by carrier-mediated facilitated diffusion via the L and T systems for which l-DOPA has low affinity.     

Preparation of radioactive acetyl-l-carnitine by an enzymatic exchange reaction

A rapid method for the preparation of [1-¹⁴C]acetyl-l-carnitine is described. The method involves exchange of [1-¹⁴C]acetic acid into a pool of unlabeled acetyl-l-carnitine using the enzymes acetyl-CoA synthetase and carnitine acetyltransferase. After isotopic equilibrium is attained, radioactive acetylcarnitine is separated from the other reaction components by chromatography on Dowex 1 (Cl^?) anion exchange resin. One of the procedures used to verify the product [1-¹⁴C]acetyl-l-carnitine can be used to synthesize (3S)-[5-¹⁴C]citric acid.     

Differences in the incorporation pattern of labelled cinnamic acid and l-phenylalanine into the anthocyanins of Petunia hybrida

The incorporation of phenylalanine-[¹⁴C] into anthocyanins of petals of Petunia hybrida is greater than that of cinnamic acid-[¹⁴C]. Moreover, there is a preferential incorporation of phenylalanine-[¹⁴C] into delphinidin 3-monoglucoside, as compared with the incorporation into cyanidin and peonidin 3-monoglucosides.     

Effects of temperature on L-leucine transport in toadfish liver in vivo

The effect of body temperature in the 4–30°C range on L-leucine uptake by toadfish liver in vivo was examined by means of a single-injection pulse technique. The ratio of [¹⁴C]leucine to [³H]mannitol or [³H]inulin in blood leaving the liver was measured as a function of time after hepatic portal vein injection. Recoveries of the two isotopes in liver and [¹⁴C]leucine incorporation into protein were determined.The Q₁₀ value for influx was 3.8, that for efflux 2.8. At all temperatures, the leucine influx was 8–10-times higher than its incorporation into protein. The directly energy-linked reactions appear to be the main site of increased temperature sensitivity at low temperatures.     

Stereochemistry of the decarboxylation of l-ornithine with ornithine decarboxylase from mouse kidney

Using highly purified ornithine decarboxylase isolated from androgen-treated mice, [1R-²H]putrescine was generated by the decarboxylation of l-ornithine in D₂O, and [1S-²H]putrescine was generated from [2-²H]ornithine by carrying out the decarboxylation in H₂O. Chirality of the putrescines was then determined from the 200-MHz ¹H NMR spectra of their bis-camphanamides in the presence of Eu(fod)₃. These results demonstrated that decarboxylation had taken place with retention of configuration.     

Distribution of radiolabeled l-glutamate and d-aspartate from blood into peripheral tissues in naive rats: Significance for brain neuroprotection

Excess l-glutamate (glutamate) levels in brain interstitial and cerebrospinal fluids (ISF and CSF, respectively) are the hallmark of several neurodegenerative conditions such as stroke, traumatic brain injury or amyotrophic lateral sclerosis. Its removal could prevent the glutamate excitotoxicity that causes long-lasting neurological deficits. As in previous studies, we have established the role of blood glutamate levels in brain neuroprotection, we have now investigated the contribution of the peripheral organs to the homeostasis of glutamate in blood. We have administered naive rats with intravenous injections of either l-[1-¹⁴C] Glutamic acid (l-[1-¹⁴C] Glu), l-[G-³H] Glutamic acid (l-[G-³H] Glu) or d-[2,3-³H] Aspartic acid (d-[2,3-³H] Asp), a non-metabolized analog of glutamate, and have followed their distribution into peripheral organs. We have observed that the decay of the radioactivity associated with l-[1-¹⁴C] Glu and l-[G-³H] Glu was faster than that associated with glutamate non-metabolized analog, d-[2,3-³H] Asp. l-[1-¹⁴C] Glu was subjected in blood to a rapid decarboxylation with the loss of ¹⁴CO₂. The three major sequestrating organs, serving as depots for the eliminated glutamate and/or its metabolites were skeletal muscle, liver and gut, contributing together 92% or 87% of total l-[U-¹⁴C] Glu or d-[2,3-³H] Asp radioactivity capture. l-[U-¹⁴C] Glu and d-[2,3-³H] Asp showed a different organ sequestration pattern. We conclude that glutamate is rapidly eliminated from the blood into peripheral tissues, mainly in non-metabolized form. The liver plays a central role in glutamate metabolism and serves as an origin for glutamate metabolites that redistribute into skeletal muscle and gut. The findings of this study suggest now that pharmacological manipulations that reduce the liver glutamate release rate or cause a boosting of the skeletal muscle glutamate pumping rate are likely to cause brain neuroprotection.     

Phenylalanine ammonia-lyase: Mirror-image packing of d- and l-phenylalanine and d- and l-transition state analogs into the active site

The binding of substrate and product analogs to phenylalanine ammonia-lyase (EC 4.3.1.5) from maize has been studied by a protection method. The ligand dissociation constants, K_L, were estimated from the variation with [L] of the pseudo-first-order rate constants for enzyme inactivation by nitromethane. The phenylalanine analogs d- and l-2-aminooxy-3-phenylpropionic acid showed K_L, values over 20,000-fold lower than the K_m for l-phenylalanine. From these and other K_L values it is deduced that when the enzyme binds l-phenylalanine the structural free energy stored in the protein is higher than when it binds the superinhibitors. Models for binding d- and l-phenylalanine and the superinhibitors are described. The enantiomeric pairs are considered to have similar K_L values because they pack into the active site in a mirror-image relationship. If the elimination reaction approximates to the least-motion course deduced on stereoelectronic grounds, the mirror-image packing of the superinhibitors into the active site mimics the conformation inferred for a transition state in the elimination. It appears, therefore, that structural changes take place in the enzyme as the transition state conformation is approached causing stored free energy to be released. This lowers the activation free energy for the elimination reaction and accounts for the strong binding by the above analogs.     

l-Alanine as a precursor of ethylamine in Camellia sinensis

After absorption of ammonium nitrogen, nitrogen-deficient Camellia sinensis synthesized theanine following synthesis of glutamic acid and alanine. The rate of incorporation of ¹⁴C from l-alanine U-¹⁴C into theanine was faster than from acetaldehyde 1–2¹⁴4C. Incorporation of ¹⁴C from l-alanine U-¹⁴C into the ethylamide of theanine was prevented by adding an excess of ethylamine to the culture solution. Green seedlings converted alanine to ethylamine more rapidly than did etiolated seedlings.     

Selenium-containing tRNAs from Clostridium sticklandii. Cochromatography of seleno-tRNA I with l-valyl-tRNA

It has been previously shown that Clostridium sticklandii specifically synthesized three readily separable ⁷⁵Se-labeled tRNAs, designated seleno-tRNAs I, II and III, and the partially purified seleno-tRNA II cochromatographed with l-prolyl-tRNA on DEAE-Sephadex A-50 (Chen, C.S. and Stadtman, T.C. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1403–1407). In the present study a highly purified ⁷⁵Se-labeled tRNA I was obtained by chromatography on benzoylated DEAE-cellulose, DEAE-Sephadex A-50 and Sepharose 4B. The ⁷⁵Se-labeled tRNA I cochromatographed with an l-valine-accepting species on DEAE-Sephadex A-50 and Sepharose 4B. Addition of a 285-fold molar excess of unlabeled l-valine to the l-valine acceptor activity assay mixture markedly decreased the amount of l-[¹⁴C]valine bound to seleno-tRNA I.     

The efflux of l-carnitine from cells in culture (CCL 27)

The efflux of l-[³H]carnitine was studied in cells from an established cell line from human heart (Girardi human heart cells, CCL 27). The cells were loaded with 4 μmol/l l-[³H]carnitine for 1 or 24 h, and the efflux of radioactivity into the medium was measured. The amount of intracellular l-[³H]carnitine retained was expressed as a function of time. The results were fitted to an exponential equation, from which efflux rate constants were computed.Increasing the extracellular concentration of butyrobetaine, l-carnitine, d-carnitine, betaine, dl-norcarnitine or 3-dimethylamino-2-hydroxypropionic acid each increased the observed efflux. This is most likely due to accelerated exchange diffusion. The substrate specificity of this accelerated exchange diffusion is different from what previously has been found in competitive uptake studies of l-carnitine. l-Carnitine was preferentially released to l-acetylcarnitine, and blocking the sulfhydryl groups with 5,5-dithiobis(2-nitrobenzoic acid) increased the efflux.     

l-2-Oxalylamino-3-aminopropionic acid,an isomer of Lathyrus sativus neurotoxin

The first chemical synthesis of l-2-oxalylamino-3-aminopropionic acid, an isomer of the Lathyrus sativus neurotoxin, is described. Studies on its biological properties are reported. Experiments with l-3-[¹⁴C-oxalyl]amino-2- aminopropionic acid show that the amount of 2-oxalylamino isomer detectable in seed extracts can be accounted for by rearrangement which occurs during isolation.     

Solvolysis of charged active esters of carboxylic acids with cyclo (l- or d-Glu-l-His)

Cyclic dipeptide cyclo(l- or d-Glu-l-His) carrying an anionic site and a nucleophilic site has been synthesized and used as a catalyst for the solvolysis of cationic esters in aqueous alcohols. In the solvolysis of 3-acyloxy-N-trimethylanilinium iodide (S⁺_n, n = 2 and 10) and Cl^?H₃N⁺(CH₂)₁₁COOPh(NO₂), no efficient nucleophilic catalysis was observed. On the other hand, in the solvolysis of Gly-OPh(NO₂)·HCl, Val-OPh(NO₂)·HCl and Leu-OPh(NO₂)·HCl a very efficient general base-type catalysis by cyclo(l-Glu-l-His) was observed. In particular, with the latter two substrates the catalysis by cyclo(l-Glul-His) was more efficient than that by imidazole, although the catalysis was not enantiomer-selective. The diastereomeric cyclic dipeptide cyclo(d-Glu-l-His) was almost inactive under the same conditions. Confomation of cyclo(l- or d-Glu-l-His) in aqueous solution was investigated and the structure/catalysis relationship is discussed.