Concentrations Of L-serine Research Articles

High concentrations of neutral amino acids activate NMDA receptor currents in rat hippocampal neurons

High concentrations (30 μM–5 mM) of the neutral amino acids l-serine, l-cysteine, l-alanine, l-proline and glycine elicited inward current responses when applied to hippocampal neurons patch clamped at −60 mV in the presence of 1–10 μM glycine and 1 μM strychnine. The amplitude of the response to l-serine increased in a concentration-dependent fashion within the range 0.1–10 mM (EC50, 2.6 mM). l-Serine (1 mM) currents were attenuated by Mg2+ (100 μM) and completely blocked by the competitive N-methyl-d-aspartate (NMDA) antagonist 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) (30 μM). The CPP block could be overcome by raising the concentration of l-serine. We conclude that high concentrations of some neutral amino acids activate NMDA receptor-coupled ion channels by acting as agonists at the NMDA recognition site.

Purification and properties of an iron‐sulfur‐containing and pyridoxal‐phosphate‐independent l‐Serine dehydratase from Peptostreptococcus asaccharolyticus

L-Serine dehydratase with a specific activity of 15 nkat/mg protein was present in the anaerobic eubacterium Peptostreptococcus asaccharolyticus grown either on L-glutamate or L-serine. The enzyme was highly specific for L-serine with the lowest Km = 0.8 mM ever reported for an L-serine dehydratase. L-Threonine (Km = 22 mM) was the only other substrate. V/Km for L-serine was 500 times higher than that for L-threonine. L-Cysteine was the best inhibitor (Ki = 0.3 mM, competitive towards L-serine). The enzyme was purified 400-fold to homogeneity under anaerobic conditions (specific activity 6 mukat/mg). PAGE in the presence of SDS revealed two subunits with similar intensities (alpha, 30 kDa; beta, 25 kDa). The molecular mass of the native enzyme was estimated as 200 +/- 20 kDa (gel filtration) and 180 kDa (gradient PAGE). In the absence of oxygen the enzyme was moderately stable even in the presence of sodium borohydride or phenylhydrazine (5 mM each). However, by exposure to air the activity was lost, especially when the latter agent was added. The enzyme was reactivated by ferrous ion under anaerobic conditions. The inability of several nucleophilic agents to inactivate the enzyme indicated the absence of pyridoxal phosphate. This was confirmed by a microbiological determination of pyridoxal phosphate. However, the enzyme contained 3.8 +/- 0.2 mol Fe and 5.6 +/- 0.3 mol inorganic sulfur/mol heterodimer (55 kDa) indicating the presence of an [Fe-S] center. The enzyme was successfully applied to measure L-serine concentrations in bacterial media and in human sera.

Behavior of L-threonine-degrading enzymes during liver regeneration.

We examined the effects of a two-thirds hepatectomy in the adult rat on the activities of the three L-threonine-degrading enzymes, L-threonine dehydratase, L-threonine aldolase and L-threonine dehydrogenase. Noticeable variations were observed which did not occur in either sham-operated or turpentine-treated rats and were not linked to food intake. They were considered specific to the regenerating liver. When the reactions were followed in vitro, L-threonine deaminase and L-threonine aldolase were significantly lower for the first 12-24 h: L-threonine dehydrogenase decreased only after 48 h. These results are linked to a decrease in the enzyme concentration in the tissue. L-Serine and L-threonine liver concentrations increased 2-3-fold during the same periods. When the activities were evaluated in vivo, the levels of the first two enzymes remained constant for 24 h, but increased after 48 h; L-threonine dehydrogenase increased between 12 and 48 h. The in vivo activity of the enzymes was reflected by total L-threonine degradation, which had a single sharp peak at 48 h. The asynchronous variations in enzyme activity are related to the differences in protein metabolism which occur in the regenerating liver, and are the consequence of a new transient differential control. The changes observed are significant in liver regeneration; they regulate the consumption and the serum and liver levels of L-serine and L-threonine, setting them aside for protein synthesis. They minutely control the flux of amino acids toward gluconeogenesis, since, during the first 48 h after partial hepatectomy, the production of glucose is ensured principally by lactate; the contribution of L-threonine seems to be more significant only at 48 h. These findings are useful in the study of the regulation of the enzymes involved in amino acid metabolism during liver regeneration.

Enzymology of long-chain base synthesis by liver: Characterization of serine palmitoyltransferase in rat liver microsomes

Serine palmitoyltransferase [palmitoyl-CoA: l-serine C-palmitoyltransferase (decar-boxylating) EC 2.3.1.50]catalyzes the initial and committed step in the biosynthesis of the long-chain bases of sphingolipids. A simple assay, based upon the incorporation of [ 3H]serine into the chloroform-soluble product 3-ketosphinganine, has been developed and demonstrated to be valid for analyzing this enzyme in rat liver microsomes. More than 75% of the serine palmitoyltransferase of rat liver was associated with the microsomal subfraction. The dependencies of activity on the incubation time, pH, temperature, other assay components (e.g., dithiothreitol, EDTA, and pyridoxal 5′-phosphate), and the concentrations of microsomal protein, l-serine, and palmitoyl-CoA were investigated. The requirement of pyridoxal 5′-phosphate for activity was established by formation of the apoenzyme by dialysis against cysteine, and recovery of full activity upon reconstitution with the coenzyme. Activities with fatty acyl-CoA's of varying alkyl chain length were distributed nearly symmetrically around a maximum at 16 carbons (palmitoyl-CoA) for the fully saturated substrates. Less activity was obtained with the CoA thioesters of cis-unsaturated fatty acids, but trans-9-hexadecenoyl-CoA yielded essentially the same activity as palmitoyl-CoA. Hence, this enzyme is capable of initiating the synthesis of the major long-chain bases, as well as compounds that may constitute the unidentified bases reported in analyses of mammalian sphingolipids.

Total parenteral nutrition of premature infants: metabolic effects of an exogenous supply of L-aspartic acid and L-glutamic acid

Within the scope of clinically indicated total parenteral nutrition of premature infants, a comparative randomized study was performed to examine--by means of nitrogen-balance studies and determination of the free amino acids in the serum--the metabolic effects of absent or parallel intake of 1.140 mumol L-aspartic acid plus 2.160 mumol L-glutamic acid per kg body weight per day in complete L-amino acid solutions with a comparative E/T-ratio and with identical intake of all other nutrients adapted to the requirement. 1. The nitrogen balance level was not affected by the absent or parallel intake of the dicarbonic acids. 2. Intravenous intakes of glycine plus L-serine, which are higher than 2.5 mmol per kg body weight and day, caused statistically significant increased serum concentrations of glycine and L-serine. Such intakes are obviously above the physiologic regulation range. 3. The absent intake of L-aspartic acid and L-glutamic acid resulted in parallel, statistically significant reduced serum concentrations of aspartic acid and asparagine as well as in homeostatic serum concentrations of glutamic acid and glutamine. Despite the only 15-20% higher intake of proline, alanine and arginine under the infusion regimen lacking dicarbonic acids, there was a parallel, statistically significant marked increase in the serum concentrations of proline, alanine, arginine and methionine as well as a statistically significant marked decrease in those of taurine. Under the infusion regimen containing dicarbonic acids exclusively, constant homeostatic serum concentrations of these amino acids as well as of aspartic acid and glutamic acid were measured. 4. A direct or indirect effect of the exogenous supply of L-aspartic acid and/or L-glutamic acid on the homeostasis of aspartic acid and asparagine, on the endogenous turnover of L-alanine and L-proline as well as on the physiologic course of the Krebs-Henseleit cycle and of the "transsulfuration pathway" must be discussed. 5. Since the supply rates of L-aspartic acid plus L-glutamic acid chosen in series 2 (when continuously administered during 24-hour periods) apparently do not cause any disturbance in amino-acid homeostasis, it is established that under the nutritional conditions given this intake lies within the respective physiologic regulation range and therefore is atoxic.

Enzymatic methods for the determination of l-serine concentration and l-[ 14C]serine specific radioactivity in blood plasma

Methods are desribed for the use of l-serine dehydratase purified from Clostridium acidiurici for the determination of l-serine concentration and l[ 14C]serine specific radioactivity in sheep plasma. A spectrophotometric assay using this enzyme accurately measured the concentration of l-serine in standard solutions and in a commercially available mixture of amino acids and related compounds. This assay was shown to be suitable for measurement of plasma l-serine concentrations in excess of 30 μ m. The reverse isotope dilution method was used for plasma l-[ 14C]serine specific radioactivity measurements. Carrier l-serine was added to plasma and separated from neutral and anionic compounds using ion-exchange chromatography. The l-serine was then converted to pyruvate with l-serine dehydratase and this was purified as the phenylhydrazone derivative. After recrystallization, drying and weighing, the derivative was assayed for radioactivity. The accuracy of this method was verified by adding l-[U- 14C]serine to plasma and comparing the experimentally determined l-[ 14C]serine specific radioactivity with the calculated value. The method yielded a value which was 98.6 ± 0.8% (5) of this calculated value.

Characterization of phosphatidylserine synthase from saccharomyces cerevisiae and a mutant defective in the enzyme

The membrane fraction of exponentially growing cells of Saccharomyces cerevisiae was found to exhibit phosphatidylserine synthase activity. The enzyme was solubilized by Triton X-100 and chromatographed on a Sepharose 6B column. The enzyme had a pH optimum between 8.0 and 8.5. The apparent K m values for CDP-diacylglycerol and L-serine were 0.12 and 13 mM, respectively. Triton X-100 stimulated the enzyme. Mg 2+ or Mn 2+ was required for the activity. Ca 2+ was inhibitory at relatively low concentrations. The enzyme was highly specific to L-serine. Labeling experiments showed that the enzyme synthesized phosphatidylserine by transferring the phosphatidyl moiety to L-serine. A mutant of S. cerevisiae defective in phosphatidylserine synthase was isolated. The strain required ethanolamine for its growth. Ethanolamine could be substituted by choline or high concentrations of L-serine. The mutant showed normal levels of CDPdiacylglycerol-inositol 3-phosphatidyltrans-ferase and phosphatidylethanolamine methyltransferase activities.

Genetic and biochemical properties of Escherichia coli mutants with defects in serine chemotaxis.

In Escherichia coli, taxis to certain chemoeffectors is mediated through an intrinsic membrane protein called methyl-accepting chemotaxis protein I (MCP I), which is the product of the tsr gene. Mutants were selected that are defective in taxis toward all MCP I-mediated attractants (alpha-aminoisobutyrate, L-alanine, glycine, and L-serine) but are normal to MCP I-mediated repellents and to chemoeffectors mediated by other MCPs. The mutants could be divided into two classes based on their ability to respond to various concentrations of L-serine. Two MCP I-mediated L-serine systems appear to function in the wild type: one of high and one of lower affinity. The mutations responsible for the serine taxis defects map at about 99 min on the E. coli chromosome and are not complemented by episomes carrying mutations in the tsr gene; this suggests that they are defective in tsr function. Low concentrations of L-[14C]serine specifically bound to wild-type membranes with a Km of 5 microM; in contrast, there was greatly decreased binding to vesicles prepared from the new mutants or from the tsr mutant AW518. Binding of labeled serine to wild-type vesicles was inhibited by MCP I-mediated attractants, but not by MCP II-mediated attractants. The data suggest that MCP I may function as the L-serine chemoreceptor in E. coli.

L-Azaserine toxicity in established cell lines : Correlation with γ-glutamyl transpeptidase activity

Four established cell lines derived from pig kidney (LLC-PK 1), human Wilms' tumor (TuWi), rat liver (ARL 6), and hamster lung (V79) were found to contain widely divergent levels of γ-glutamyl transpeptidase (GGT) which decreased in the order given from 96 nmole p-nitroaniline released/min/mg protein for LLC-PK 1 to 0.5 nmole for V79, independent of cell density. When lines were treated with l-azaserine for short periods, a positive correlation was observed between cellular GGT levels and sensitivity to azaserine toxicity. Equimolar concentrations of l-serine and l-glutamine were non-toxic. Azaserine-resistant strains were isolated from TuWi cultures exposed to l-azaserine for 24 h. Most exhibited reduced levels of GGT. Those strains expressing the lowest GGT activities also showed the greatest resistance to azaserine toxicity following short-term re-exposure to l-azaserine. These results indicate that GGT is an important determinant in cell sensitivity to l-azaserine toxicity and suggest that heightened susceptibility is a consequence of active transport of the diazo-amino acid.

Initial kinetic characterization of the multienzyme complex, cysteine synthetase

Cysteine synthetase from Salmonella typhimurium LT-2 displays a saturation curve for sulfide identical to that obtained with uncomplexed O-acetylserine sulfhydrylase, indicating substrate inhibition with a K m of 0.1 ± 0.017 m m and a K 1 of 0.303 ± 0.194 m m. With both l-serine and acetyl CoA, however, cysteine synthetase exhibits two intermediary plateaus in the respective saturation curves. The time course of cysteine synthetase activity when the reaction is started by adding enzyme displays a pronounced lag phase. This lag is explained as being due to the buildup of a sufficient concentration of O-acetyl- l-serine to permit binding to O-acetylserine sulfhydrylase. This conclusion is substantiated by the fact that plots of 1 τ against concentrations of both l-serine and acetyl CoA reflect the saturation curves for these substrates. In addition, the incubation of the complex with l-serine and acetyl CoA results in the accumulation of the intermediate products of the reaction sequence, CoA and O-acetyl- l-serine. Dissociation of the multienzyme complex under these conditions was ruled out by Sephadex G-200 chromatography of the complex after incubation with assay levels of the substrates of the reaction. Aggregation of cysteine synthetase was detected using disc gel electrophoresis and confirms earlier reports [Kredich, N. M., and Tomkins, G. M. (1966) J. Biol. Chem. 241, 4955–4965]. Aggregation of O-acetylserine sulfhydrylase was also detected using the same technique.