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PubMed2004

D-Serine as a glial modulator of nerve cells.

Until the last decade, it was widely accepted that D-amino acids had no functional role in higher organisms, but that they were restricted to lower organisms, such as bacteria, where they are integrated into the proteoglycans of the cell wall. However, D-serine proved to be an effective coagonist at the "glycine-binding" site of the N-methyl-D-aspartate (NMDA) glutamate receptors, and this observation led to chemical analyses that have now revealed the presence of high levels of D-serine in the central nervous system, including many regions of the brain and retina. D-Serine has been localized to astrocytes and can be released by glutamate through stimulation of AMPA receptors. A new enzyme, serine racemase has been localized to glial cells and converts L-serine to D-serine. Degradation of D-serine takes place through D-amino acid oxidase, an enzyme once thought to metabolize D-amino acids from external sources. Although the "glycine-binding" site of NMDA receptors was initially regarded as a saturated site, evidence in many brain regions has established that this site is not saturated and is therefore modulated by interactions between glial cells and neurons. In some, but not all, studies, D-serine enhances NMDA-mediated currents; a light-evoked enhancement to NMDA currents has been reported in the retina. D-serine also plays a role in synaptic and cellular development, particularly in the cerebellum, where the normal developmental sequences underlying synapse formation onto Purkinje cells and the migration of granule cells are dependent on NMDA receptors during a time when high levels of D-serine are expressed in the Bergmann glia and other cerebellar astrocytes. D-serine must be added to the list of agents through which glial cells participate in controlling the excitability of neurons.

Animals↗
PubMed1987

Reactions of O-acyl-L-serines with tryptophanase, tyrosine phenol-lyase, and tryptophan synthase.

The reactions of tryptophanase, tyrosine phenol-lyase, and tryptophan synthase with a new class of substrates, the O-acyl-L-serines, have been examined. A method for preparation of O-benzoyl-L-serine in high yield from tert.-butyloxycarbonyl (tBoc)-L-serine has been developed. Reaction of the cesium salt of tBoc-L-serine with benzyl bromide in dimethylformamide gives tBoc-L-serine benzyl ester in excellent yield. Acylation with benzoyl chloride and triethylamine in acetonitrile followed by hydrogenolysis with 10% palladium on carbon in trifluoroacetic acid gives O-benzoyl-L-serine, isolated as the hydrochloride salt. O-Benzoyl-L-serine is a good substrate for beta-elimination or beta-substitution reactions catalyzed by both tryptophanase and tyrosine phenol-lyase, with Vmax values 5- to 6-fold those of the physiological substrates and comparable to that of S-(o-nitrophenyl)-L-cysteine. Unexpectedly, O-acetyl-L-serine is a very poor substrate for these enzymes, with Vmax values about 5% of those of the physiological substrates. Both O-acyl-L-serines are poor substrates for tryptophan synthase, measured either by the synthesis of 5-fluoro-L-tryptophan from 5-fluoroindole and L-serine catalyzed by the intact alpha 2 beta 2 subunit or by the beta-elimination reaction catalyzed by the isolated beta 2 subunit. With all three enzymes, the elimination of benzoate appears to be irreversible. These results suggest that the binding energy from the aromatic ring of O-benzoyl-L-serine is used to lower the transition-state barrier for the elimination reactions catalyzed by tryptophanase and tyrosine phenol-lyase. Our findings support the suggestion (M. N. Kazarinoff and E. E. Snell (1980) J. Biol. Chem. 255, 6228-6233) that tryptophanase undergoes a conformational change during catalysis and suggest that tyrosine phenol-lyase also may undergo a conformational change during catalysis.

Catalysis↗
PubMed1994

Distribution of free D-serine in vertebrate brains.

Free D-serine distribution in vertebrate brains was investigated. In various brain regions of the lower vertebrate species, carp, frog and chick, free D-serine levels were low. On the contrary, in the mammals, mouse, rat and bull, the contents of free D-serine were high in the forebrain (around 400 nmol/g wet weight, and the ratio of D-serine to L-serine, was D/L = 0.4), and low in the hindbrain. In developing mice, D-serine levels in the cerebrum increased with age and attained the adult level (D/L = 0.40) 8 weeks after birth. In the cerebellum and brain stem, the free D-serine levels increased with age until 2 weeks, followed by a decrease to the adult levels: the D/L ratios remained constant until 2 weeks of age, then decreased to 0.03 in the cerebellum and 0.12 in the brain stem. Free D-serine was shown not to be of microbial origin using germ-free mice. In the rat forebrain, D-serine was evenly distributed in two cerebral regions, namely frontal and occipital lobes. The D/L ratios in other regions of forebrain, hippocampus and hypothalamus, were comparable to the cerebrum (D/L = 0.4), while that in the olfactory bulb was lower (D/L = 0.12). In the rat cerebrum, the D-serine content in the grey matter was significantly higher than that in the white matter. The contents of free D-serine in bovine cerebrum and cerebellum were similar to those in other mammalian brains, but the D/L ratio for bovine cerebral grey matter was lower than that for the cerebral white matter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Expression and utilization of chymotrypsin-like but not trypsin-like serine protease enzymes by nonspecific T killer cells activated by anti-CD3 monoclonal antibody.

Coculture of purified murine T cells with anti-CD3 monoclonal antibody (145-2C11) results in the induction of nonspecific cytotoxic T lymphocytes (CTL) with MHC-unrestricted cytolytic activity against a range of tumor targets. Serine proteases associated with effector cell granules are among the molecules postulated to play a role in cell-mediated cytolysis. The present study examines the ability of exogenous serine protease substrates to inhibit anti-CD3-activated cytotoxic T (ACT) cell-mediated killing of P815 mastocytoma and YAC1.2 lymphoma target cells. The chymotrypsin substrate N-acetyl-L-tyrosine ethyl ester (ATEE) was found to significantly inhibit ACT cell-mediated cytolysis. In contrast, the trypsin substrate N-benzoyl-L-arginine ethyl ester (BAEE) had little, if any, effect on ACT cell-mediated cytolysis. These effects were observed with both target cell populations. Conjugate inhibition studies performed with ATEE indicated that a chymotrypsin-like serine protease is involved in a postbinding event during cytolysis. Pretreatment of either target or effector cells with ATEE prior to cytolytic assay revealed that the chymotrypsin-like serine protease involved in cytotoxicity is of effector cell origin. Northern blot analysis of total RNA extracted from ACT cells revealed the presence of transcripts coding for CCP1 and CCP2 serine proteases known to be involved in antigen-specific CTL function, but little or no expression of the HF serine protease which has also been implicated in antigen-specific CTL killing. CCP2 exhibits chymotrypsin-like activity while HF displays trypsin-like activity. On the other hand, the CCP1 gene product has protease activity which resembles neither chymase nor tryptase activities. Thus, the level of mRNA expression for these serine proteases is consistent with our earlier observations, using the serine protease substrates, that a chymotrypsin-like serine protease but not a trypsin-like serine protease is involved in ACT cell-mediated cytolysis. "Lymphocyte panning" of ACT cells revealed abundant CCP1 and moderate CCP2 mRNA expression in CD4- and CD8+ anti-CD3-activated T cells with strong tumoricidal activity. CD8- anti-CD3-activated T cells with moderate cytolytic activity also expressed substantial levels of CCP1 and CCP2 mRNA, suggesting that both CD4- CD8- and CD4- CD8+ ACT cells participate in killing tumor targets. In contrast, CD4+ anti-CD3-activated T cells lacked both cytolytic activity and significant CCP1 and CCP2 mRNA expression. These findings are consistent with the involvement of chymotrypsin-like, as well as other, serine proteases in CTL-mediated lysis.

Animals↗
PubMed1984

Impaired glycine-serine conversion and increased plasma taurine levels in episodic psychotic patients with psychedelic symptoms.

A disturbance of serine-glycine metabolism has been demonstrated in patients suffering from episodic acute psychosis, characterized by sensory perceptual distortions in whom oral loading with either serine or glycine induced psychedelic symptoms. Fasting plasma amino acid levels in oral serine-responsive patients were compared with those in glycine-responsive and manic-depressive patients. Only serine-responsive patients had a decreased plasma serine and increased plasma taurine concentration, while glycine responsive patients showed decreased alanine and manic-depressive patients an increased plasma glycine level. Amino acid analyses of plasma samples obtained after serine loading in patients and controls suggest that serine-responsive patients had an impaired conversion of glycine into serine. The results suggest that the higher plasma taurine level in serine-responsive patients is caused by an increased synthesis of taurine from homocysteine and serine. The limiting capacity of methyltetrahydrofolate-homocysteine methyltransferase coupled to increased formation of taurine may be responsible for the serine-evoked psychotic syndrome.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed2005

D-serine efficacy as add-on pharmacotherapy to risperidone and olanzapine for treatment-refractory schizophrenia.

BACKGROUND: D-serine, a selective full agonist at the glycine site of N-methyl-D-aspartate glutamate receptor, might presently be the compound of choice for counteracting the hypothesized dysfunction of this receptor class in schizophrenia. Studies performed with Taiwanese patients indicate that D-serine significantly improves schizophrenia symptoms when used as adjuvant to conventional neuroleptics but not to clozapine. We assessed the efficacy and safety of D-serine adjuvant treatment for Occidental schizophrenia patients treated with newer atypical antipsychotics. METHODS: Thirty-nine risperidone- or olanzapine-treated schizophrenia patients participated in a double-blind, placebo-controlled, 6-week crossover trial with 30 mg/kg/day D-serine added to their antipsychotic medication. Measures of clinical efficacy and side effects were determined biweekly throughout the study. Clinical laboratory parameters and amino acid serum levels were monitored. RESULTS: D-serine administration induced increased serine serum levels (p < .001) and resulted in significant (p < .001) improvements in negative, positive, cognitive, and depression symptoms, as measured by the Positive and Negative Syndrome Scale. For approximately one third of the sample, D-serine treatment resulted in significant (>20%) reductions in Brief Psychiatric Rating Scale total scores. D-serine was well tolerated, and no detrimental changes in clinical laboratory parameters were noted. CONCLUSIONS: These findings 1) indicate that risperidone and olanzapine efficacy might be augmented with D-serine adjuvant treatment; 2) confirm D-serine efficacy against main schizophrenia symptom domains; and 3) warrant the assessment of D-serine antipsychotic monotherapy for this illness.

Adult↗
PubMed2005

Preferred stereoselective brain uptake of d-serine--a modulator of glutamatergic neurotransmission.

Although it has long been presumed that d-amino acids are uncommon in mammalians, substantial amounts of free d-serine have been detected in the mammalian brain. d-Serine has been demonstrated to be an important modulator of glutamatergic neurotransmission and acts as an agonist at the strychnine-insensitive glycine site of N-methyl-d-aspartate receptors. The blood-to-brain transfer of d-serine is thought to be extremely low, and it is assumed that d-serine is generated by isomerization of l-serine in the brain. Stimulated by the observation of a preferred transport of the d-isomer of proline at the blood-brain barrier, we investigated the differential uptake of [3H]-d-serine and [3H]-l-serine in the rat brain 1 h after intravenous injection using quantitative autoradiography. Surprisingly, brain uptake of [3H]-d-serine was significantly higher than that of [3H]-l-serine, indicating a preferred transport of the d-enantiomer of serine at the blood-brain barrier. This finding indicates that exogenous d-serine may have a direct influence on glutamatergic neurotransmission and associated diseases.

Animals↗
PubMed2003

Serine metabolism in Lactobacillus plantarum.

This study investigated the metabolism of (L-) serine by Lactobacillus plantarum B3089 isolated from cheese. Serine was deaminated by growing cells to ammonia with the corresponding formation of acetate and formate. Serine was also deaminated by non-growing cells to ammonia but with the formation of acetate only (no production of formate). Phosphoserine and threonine were not catabolised. It is proposed that serine was deaminated by serine dehydratase (deaminase) to ammonia and pyruvate. Pyruvate was further catabolised predominantly to acetate, carbon dioxide and formate in growing cells, catalysed by pyruvate-formate lyase and pyruvate oxidase; some of the pyruvate was converted to acetoin. In non-growing cells, however, pyruvate-formate lyase was inactive and pyruvate oxidase degraded the pyruvate to acetate and carbon dioxide. Serine dehydratase activity could not be detected in cell-free extracts, presumably because of enzyme instability. The growth of L. plantarum was neither enhanced nor stimulated by serine under the current conditions. Whereas there was little difference in serine utilisation between pH 7.0 and pH 5.8, serine utilisation was decreased by 30% at pH 5.0. NaCl of up to 4% (w/v) concentration had little effect on serine utilisation. Serine had no impact on lactose metabolism. Lactose was fermented mainly to lactate (73%) with the remainder converted to an unidentified polysaccharide (27%).

Cheese↗
PubMed2026

Serine: From Metabolic Intermediate to Signaling Entity.

Serine, a nonessential amino acid classically defined as a precursor for protein synthesis and one-carbon metabolism, is increasingly recognized as a signaling metabolite that links the cellular metabolic status to regulatory decision-making. Intracellular serine availability is shaped by nutrient conditions, glycolytic flux, and activity of the serine synthesis pathway, and these fluctuations are sensed to elicit coordinated metabolic and signaling responses. This review discusses mechanisms by which serine modulates cell growth and stress responses, with particular emphasis on its interaction with central nutrient-sensing pathways, including mTORC1 and the integrated stress response. In parallel, serine-driven one-carbon metabolism is examined for its role in supporting nucleotide biosynthesis, methylation reactions, and redox homeostasis through folate-dependent pathways and NADPH generation, thereby coupling anabolic processes to the maintenance of redox balance and genome integrity. In addition to intracellular functions, serine contributes to intercellular signaling. Conversion of l-serine to d-serine mediates neuromodulatory activity via N-methyl-d-aspartate receptors, while serine availability also influences immune cell function, inflammatory signaling, and host-microbe interactions. Dysregulation of serine metabolism and signaling is further considered in the context of disease states, including cancer, neurodegeneration, and metabolic disorders. Together, these observations support a framework in which serine functions as an information-bearing metabolic signal that coordinates the biosynthetic capacity with cellular adaptation and intercellular communication.

Serine↗
PubMed2006

Thermal formation of homochiral serine clusters and implications for the origin of homochirality.

Spontaneous assembly of amino acids into vapor-phase clusters occurs on heating the solid compounds in air. In comparison to the other amino acids, serine forms clusters to an unusual extent, showing a magic number octamer on sublimation; this octamer can be ionized and characterized by mass spectrometry. Two isomers of the vapor-phase serine octamer are generated, the minor one at 130 degrees C and the major at 220 degrees C. The higher temperature cluster shows a strong homochiral preference, as confirmed by isotopic labeling experiments. This serine cluster, like that generated earlier from solution in electrospray ionization experiments, undergoes gas-phase enantioselective substitution reactions with other amino acids. These reactions transfer the chirality of serine to the other amino acid through enantioselective incorporation into the octamer. Other serine pyrolysis products include alanine, glycine, ethanolamine, and small dipeptides, and many of these, too, are observed to be incorporated into the thermally formed serine octamers. Chiral chromatographic analysis confirmed that L-serine sublimation produced DL-alanine, glycine, and ethanolamine, while in the presence of hydrogen sulfide, L-serine yielded L-cysteine. The data demonstrate that sublimation of serine under relatively mild conditions yields chirally enriched serine octamers and that the chiral preference of the starting serine can be transferred to other compounds through cluster-forming chemical reactions.

Gas Chromatography-Mass Spectrometry↗
PubMed2000

Production and characterization of monoclonal antibodies to serine proteinase allergens in Penicillium and Aspergillus species.

BACKGROUND: Alkaline and/or vacuolar serine proteinases are major allergens in prevalent airborne Penicillium and Aspergillus species. OBJECTIVE: The object of this study is to generate and characterize monoclonal antibodies against these serine proteinase allergens. METHODS: BALB/c mice were immunized individually with the Penicillium citrinum culture medium or the crude extract and culture medium preparations of Aspergillus fumigatus. Hybridoma cells that secrete monoclonal antibodies against serine proteinase allergens were selected by immunoblotting. Antigens in three different Penicillium (P. citrinum, P. notatum and P. oxalicum) and two different Aspergillus species (A. fumigatus, and A. flavus) recognized by these monoclonal antibodies were analysed by sodium dodecyl sulphate and two-dimensional polyacrylamide gel electrophoresis immunoblotting and N-terminal amino acid sequence analysis. RESULTS: Four (PCM8, PCM10, PCM16 and PCM39) and one (FUM20) monoclonal antibodies against serine proteinase allergens were generated after fusion of NS-1 cells with spleen cells obtained from BALB/c mice immunized with antigens from P. citrinum and A. fumigatus, respectively. Immunoblotting results showed that PCM8 reacted with an alkaline serine proteinase allergen in P. citrinum and P. notatum. PCM10 and PCM39 reacted with the alkaline serine proteinase in two Penicillium (P. citrinum, P. notatum) and two Aspergillus species (A. fumigatus, and A. flavus) tested. PCM16 reacted with the alkaline serine proteinase allergen in P. citrinum, A. fumigatus and A. flavus but not with that in P. notatum. MoAb FUM20 reacted with the alkaline serine proteinase allergen in two Aspergillus species (A. fumigatus and A. flavus) but not with that in two different Penicillium species (P. citrinum, P. notatum) tested. Among these five monoclonal antibodies generated, only PCM39 and FUM20 can react with the vacuolar serine proteinase allergen in P. notatum, P. oxalicum and in A. fumigatus. The 35 kDa P. citrinum component that reacted with FUM20 has an N-terminal amino acid sequence of DSPSVEKNAP. CONCLUSION: Five monoclonal antibodies against different epitopes of the serine proteinase major allergens in prevalent Penicillium and Aspergillus species were generated in the present study. Antibodies obtained may be useful in the characterization and standardization of serine proteinase allergens in crude fungal extracts.

Allergens↗
PubMed1996

Formation of D-serine from L-phosphoserine in brain synaptosomes.

Although glycine has been assumed to be the sole endogenous coagonist at NMDA-associated glycine receptors, recent descriptions of endogenous D-serine in the brain indicate that this assumption is probably not valid. D-Serine is a stereospecific agonist of the NMDA-associated glycine receptor, with an affinity equal to or greater than that of glycine but with no affinity for the strychnine-sensitive glycine receptor. In the current studies, we assessed the levels and metabolic sources of D-serine in rat neocortical synaptosomal preparations. Previous studies have demonstrated that CNS serine and glycine are synthesized de novo primarily via a phosphorylated pathway, originating with the glycolytic intermediate phosphoglycerate. The rate-limiting step in the synthesis of serine is the hydrolysis of phosphoserine by phosphoserine phosphatase (EC 3.1.3.3). In synaptosomal preparations we have demonstrated high endogenous levels of D-serine and the uptake of L-phosphoserine along with its hydrolysis to both L-serine and D-serine, which are preferentially released into the medium. Experiments with both intact and lysed synaptosomal preparations demonstrated hydrolysis of D-phosphoserine to only D-serine and inhibition of hydrolysis by the phosphoserine phosphatase inhibitor 2-amino-3-phosphonopropionic acid (AP3). The lack of stereospecificity for synaptosomal hydrolysis of phosphoserine and the inhibitory actions of AP3 are consistent with the presence of phosphoserine phosphatase in synaptosomes and further indicate that epimerization of serine can occur during or subsequent to the hydrolysis of L-phosphoserine but not D-phosphoserine. In conclusion, these studies demonstrate that phosphoserine phosphatase may be an important enzyme in regulating the steady-state levels of D-serine in neocortical synaptosomes.

Alanine↗
PubMed1997

Characterization of 5,7-dichlorokynurenate-insensitive D-[3H]serine binding to synaptosomal fraction isolated from rat brain tissues.

To explore target sites for endogenous D-serine that are different from the glycine site of the N-methyl-D-aspartate (NMDA) type glutamate receptor, we have studied the binding of D-[3H]serine to the synaptosomal P2 fraction prepared from the rat brain and peripheral tissues in the presence of an excess concentration (100 microM) of the glycine site antagonist 5,7-dichlorokynurenate (DCK). Nonspecific binding was defined in the presence of 1 mM unlabeled D-serine. Association, dissociation, and saturation experiments indicated that D-[3H]serine bound rapidly and reversibly to a single population of recognition sites in the cerebellar P2 fraction in the presence of DCK, with a K(D) of 614 nM and a Bmax of 2.07 pmol/mg of protein. D-Serine, L-serine, and glycine produced a total inhibition of the specific DCK-insensitive D-[3H]serine binding to the cerebellum with similar Ki values. Strychnine and 7-chlorokynurenate failed to inhibit the binding at 10 microM. The profiles of displacement of the DCK-insensitive D-[3H]serine binding by various amino acids and glutamate and glycine receptor-related compounds differ from those of any other defined recognition sites. DCK-insensitive D-[3H]serine binding was at high levels in the cerebral cortex and cerebellum but very low in the kidney and liver. The present findings indicate that the DCK-insensitive D-[3H]serine binding site could be a novel candidate for a target for endogenous D-serine in mammalian brains.

Animals↗
PubMed1998

Effect of serine and ethanolamine administration on phospholipid-related compounds and neurotransmitter amino acids in the rabbit hippocampus.

The report concerns mechanisms for the increase of extracellular levels of ethanolamine and phosphoethanolamine in CNS regions, such as the hippocampus, in transient brain ischemia, hypoglycemia, seizures, etc. L-Serine (2.5-10 mM), D-serine (10 mM), or ethanolamine (10 mM) was administered for 20 min via a microdialysis tubing to the hippocampus of unanesthetized rabbits. The concentrations of primary amines were determined in the dialysates. When levels were elevated 10-100 times in the extracellular fluid, L-serine caused a dose-dependent increase of the concentration of extracellular ethanolamine. Ethanolamine caused a corresponding, although somewhat smaller, increase in serine levels. Furthermore, L-serine also induced an increased concentration of phosphoethanolamine that was delayed in time relative to the peak of ethanolamine. D-Serine was as effective as L-serine in raising ethanolamine levels but had no effect on phosphoethanolamine. Ethanolamine, but not L-serine, also increased extracellular glutamate/aspartate levels in an MK-801-dependent fashion. A similar effect, but delayed in time, was observed with D-serine. These effects were inhibited by MK-801. The concentrations of other amino acids were not significantly affected. The characteristics of the effects are suggestive of base exchange reactions between serine and ethanolamine and between ethanolamine and serine glycerophospholipids, respectively, in neuronal plasma membranes.

Amino Acids↗
PubMed1982

Effect of starvation and diet composition on two pathways of L-serine metabolism in isolated rat hepatocytes.

This study examined the effects of starvation and of feeding to rats diets that contain varying protein, carbohydrate and fat levels on serine metabolism in isolated hepatocytes. The conversion of [14C]serine and [14C]lactate to 14CO2 and [14C]glucose was measured in the presence and absence of 5 mM quinolinic acid (QA) or 1 mM 3-mercaptopicolinic acid (MPA), inhibitors of phosphoenolpyruvate carboxykinase. Inclusion of MPA eliminated the contribution of the serine dehydratase-mediated pathway of serine metabolism to glucose production, allowing estimation of serine aminotransferase-mediated metabolism. Addition of MPA reduced [14C]glucose formation from [14C]serine to between 3 and 47% of control values in all dietary treatments. Addition of 10 mM threonine or 10 mM pyruvate depressed [14C]glucose production in hepatocytes from the groups fed 80% protein. Differences in serine metabolism were observed within each protein group, depending on the carbohydrate and fat ratio of the diet. These results suggest the following: 1) MPA is a more potent gluconeogenic inhibitor than QA, permitting estimation of relative flux through two pathways of serine metabolism; 2) serine metabolism occurs primarily via serine dehydratase, although the contribution of serine aminotransferase varies depending upon the nutritional state of the rat, and 3) changing a single dietary component at the expense of another may mask the intricacies of metabolic homeostasis.

Animals↗
PubMed1984

Serine uptake by luminal and basolateral membrane vesicles from rabbit kidney.

The mechanism of renal transport of L- and D-serine by membrane vesicles prepared from either whole cortex, pars convoluta or pars recta of rabbit proximal tubule was studied by a rapid filtration technique and by a spectrophotometric method using a potential-sensitive carbocyanine dye. Transport studies carried out with different salt gradients and by employing various ionophores showed that uptake of both L- and D-serine by luminal membrane vesicles from whole cortex was mediated by an Na+-dependent and electrogenic transport process. Eadie-Hofstee analysis of experimental data, obtained under extravesicular greater than intravesicular NaCl gradients, revealed the existence of multiple transport systems for L-serine but only one system for the D-isomer. The value of KA (the concentration producing a half-maximal optical response) for the D-serine transport system was calculated to be approximately 30 mM. Luminal membrane vesicles from pars convoluta take up both L- and D-serine by a single and common transport system. KA values for L- and D-serine transport were calculated to be 3.7 and 30 mM, respectively. Luminal membrane vesicles from pars recta take up L-serine by means of two transport systems, one of high affinity (KA = 0.37 mM) and the other of low affinity (KA = 10 mM). By contrast, no D-serine transport by these membrane vesicles could be detected. Uptake of L-serine by basolateral membrane vesicles is Na+ independent and electroneutral. Filtration studies showed that the transport is saturable (Km = 25-30 mM) and is inhibited by the presence of L-phenylalanine (but not by D-serine), indicating carrier-mediated uptake of L-serine.

Animals↗
PubMed1988

Characterization of a novel L-serine transport system in Escherichia coli.

A novel transport system for L-serine was found in Escherichia coli cells grown on medium containing amino acid mixture. This novel system is distinguishable from the known three transport systems for L-serine, namely, the serine-threonine system, one of the leucine-isoleucine-valine systems, and the glycine-alanine system. Uptake of L-serine via this novel system was inhibited by none of the amino acids tested, indicating that it is highly specific for L-serine. This system was induced by L-leucine, but not by L-serine. The Km for L-serine was 50 microM, and the Vmax was 23 nmol/min per mg of cell protein. Transport of L-serine via this system was strongly inhibited by KCN, an inhibitor of the respiratory chain, or by carbonyl cyanide m-chlorophenylhydrazone, an H+ conductor. Uptake of H+ was induced by L-serine influx. These results indicate that an H+-serine cotransport mechanism is operative in this novel L-serine transport system.

Alanine↗
PubMed2005

Characterization of a novel PKA phosphorylation site, serine-2030, reveals no PKA hyperphosphorylation of the cardiac ryanodine receptor in canine heart failure.

Hyperphosphorylation of the cardiac Ca2+ release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) at serine-2808 has been proposed to be a key mechanism responsible for cardiac dysfunction in heart failure (HF). However, the sites of PKA phosphorylation in RyR2 and their phosphorylation status in HF are not well defined. Here we used various approaches to investigate the phosphorylation of RyR2 by PKA. Mutating serine-2808, which was thought to be the only PKA phosphorylation site in RyR2, did not abolish the phosphorylation of RyR2 by PKA. Two-dimensional phosphopeptide mapping revealed two major PKA phosphopeptides, one of which corresponded to the known serine-2808 site. Another, novel, PKA phosphorylation site, serine 2030, was identified by Edman sequencing. Using phospho-specific antibodies, we showed that the novel serine-2030 site was phosphorylated in rat cardiac myocytes stimulated with isoproterenol, but not in unstimulated cells, whereas serine-2808 was considerably phosphorylated before and after isoproterenol treatment. We further showed that serine-2030 was stoichiometrically phosphorylated by PKA, but not by CaMKII, and that mutations of serine-2030 altered neither the FKBP12.6-RyR2 interaction nor the Ca2+ dependence of [3H]ryanodine binding. Moreover, the levels of phosphorylation of RyR2 at serine-2030 and serine-2808 in both failing and non-failing canine hearts were similar. Together, our data indicate that serine-2030 is a major PKA phosphorylation site in RyR2 responding to acute beta-adrenergic stimulation, and that RyR2 is not hyperphosphorylated by PKA in canine HF.

Animals↗