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PubMed1993

Metabolism and transport of maternal serine by the ovine placenta: glycine production and absence of serine transport into the fetus.

The present study compares the transplacental transport of L-[l-13C]serine and L-[l-13C]leucine in sheep. An in vivo preparation using twin gestations was set up such that the arterial circulation to one uterine horn, including its placenta and fetus, was infused with tracer serine and leucine while the umbilical circulations of both fetuses were sampled. Uterine and umbilical blood flows were measured in each horn. Plasma serine enrichments were 14.6 +/- 2.7% and 4.3 +/- 1.6% in the uterine veins draining the experimental and control horns, respectively. Fetal plasma leucine enrichments in the umbilical veins were 50 and 55% of the uterine venous enrichments in the control and experimental fetuses, respectively. By contrast, during 280 min of infusion, there was no detectable serine enrichment in either fetal circulation. However, significant plasma glycine enrichment was present in the fetal circulation of the experimental horn and venous glycine enrichments in the experimental horn were significantly greater than arterial glycine enrichments for both the umbilical (p < 0.02) and uterine (p < 0.001) circulations. We conclude that under conditions in which leucine transport is easily demonstrable there is no significant transplacental transport of maternal serine and that maternal plasma serine is used within the uteroplacental tissues for producing glycine, some of which is delivered into the fetal circulation.

Animals↗
PubMed1991

Insulin-stimulated serine and threonine phosphorylation of the human insulin receptor. An assessment of the role of serines 1305/1306 and threonine 1348 by their replacement with neutral or negatively charged amino acids.

Insulin promotes insulin receptor beta-subunit phosphorylation on tyrosine, serine, and threonine residues in a variety of cells, including simian COS cells which transiently express human insulin receptors following transfection with a cDNA encoding the wild-type receptor protein. To examine the potential roles of serines 1305 and 1306 and threonine 1348 as sites of insulin-stimulated phosphorylation in these cells, these residues (i.e. either serines 1305 and 1306, or threonine 1348) were replaced with neutral (alanine) or negatively charged (aspartate) amino acids. Following transient expression of each of these mutant receptors in COS cells, two-dimensional phosphopeptide mapping reveals that threonine 1348 is the major, if not the only, insulin-stimulated threonine phosphorylation site. In contrast, while serines 1305 and/or 1306 are phosphorylated in an insulin-dependent manner, these sites comprise only a minor proportion of insulin receptor serine phosphorylation in these cells. Substitution of either serines 1305 and 1306 or threonine 1348 with neutral or negatively charged amino acids has no effect on insulin-stimulated tyrosine autophosphorylation of these mutant receptors in intact cells. Furthermore, insulin-stimulated exogenous protein-tyrosine kinase activity of the mutant receptors is unaffected, as assessed following either phosphorylation of receptors in intact cells or following immunopurification of receptors and their autophosphorylation in vitro.

Amino Acid Sequence↗
PubMed1994

Site-directed mutagenesis of conserved serines in rat histidase. Identification of serine 254 as an essential active site residue.

We have identified serine 254 as an essential residue in rat histidase (histidine ammonia-lyase, EC 4.3.1.3). Histidase and phenylalanine ammonia-lyase are the only two enzymes that have been postulated to require the modified amino acid, dehydroalanine, for enzyme activity. In the bacterial peptides nisin and subtilin, and in the pyruvoyl enzymes, the precursor for dehydroalanine is a serine. To determine whether serine may be the dehydroalanine precursor in rat histidase, we substituted four highly conserved serines with alanines, and expressed the mutated histidase cDNAs in COS cells, which have no endogenous histidase activity. Substitution of serines 223, 254, 508, and 533 resulted in the production of approximately equal amounts of histidase protein with histidase activities of 2.4, 0.0, 75, and 16%, respectively. The abrogation of histidase activity by the substitution of alanine for serine 254, together with the modification by L-cysteine of the corresponding residue in Pseudomonas putida histidase (Hernandez, C., Stroh, J. G., and Phillips, A. T. (1993) Arch. Biochem. Biophys. 307, 126-132), is strong evidence that this residue is the precursor of the essential electrophilic moiety of histidase.

Amino Acid Sequence↗
PubMed1993

Yeast MCK1 protein kinase autophosphorylates at tyrosine and serine but phosphorylates exogenous substrates at serine and threonine.

The product of the Saccharomyces cerevisiae MCK1 gene is a protein kinase that phosphorylates poly (Glu,Tyr) in vitro and is itself phosphorylated at both tyrosine and serine in vivo. To characterize the substrate specificity of Mck1, the enzyme was purified to apparent homogeneity from the soluble fraction of yeast cell extracts by ammonium sulfate precipitation, followed by ion exchange chromatography (Q- and S-Sepharose), dye-ligand affinity chromatography (Orange A-agarose), adsorption chromatography (hydroxylapatite), and ion exchange fast protein liquid chromatography (Mono-S). In the absence of an exogenous substrate, purified Mck1 was able to autophosphorylate on tyrosine and serine. A catalytically inactive mutant (K68R in conserved kinase domain II) expressed in an mck1 delta strain did not contain detectable phosphotyrosine, confirming that the tyrosine phosphorylation observed in vivo is due to autophosphorylation, but did contain phosphoserine, suggesting that Mck1 is a target for other cellular protein kinases. Purified Mck1 phosphorylated a variety of proteins in heat-inactivated yeast extracts, primarily on serine (and threonine). The purified enzyme also used a number of mammalian proteins as phosphoacceptors, including myelin basic protein (MBP), microtubule-associated protein 2 (MAP-2), and tau protein. All of these substrates were phosphorylated on either serine or threonine (or both). Mck1 isolated from yeast extracts by immunoprecipitation with an anti-Mck1 antibody directed against its C terminus also phosphorylated MBP at serine. In the same immune complex kinase assay, the K68R mutant did not detectably phosphorylate MBP, indicating that the serine-specific phosphotransferase activity of Mck1 is intrinsic and not due to contamination by an associated kinase. These findings demonstrate that Mck1 is a member of a novel class of protein kinases that displays the ability to phosphorylate all three hydroxyamino acids in proteins.

Chromatography, Liquid↗
PubMed1996

PRK1 phosphorylates MARCKS at the PKC sites: serine 152, serine 156 and serine 163.

The 80kDa Myristolated Alanine-Rich C-Kinase Substrate (MARCKS) is a major in vivo substrate of protein kinase C (PKC). Here we report that MARCKS is a major substrate for the lipid-activated PKC-related kinase (PRK1) in cell extracts. Furthermore, PRK1 is shown to phosphorylate MARCKS on the same sites as PKC in vitro. Thus, control of MARCKS phosphorylation on these previously identified 'PKC' sites may be regulated under certain circumstances by PRK as well as PKC mediated signalling pathways. The implications for MARCKS as a marker of PKC activation and as a point of signal convergence are discussed.

Amino Acid Sequence↗
PubMed2006

Capillary electrophoresis method for determination of D-serine and its application for monitoring of serine racemase activity.

Serine racemase (SR) is an enzyme responsible for the biosynthesis of D-serine, the coagonist of the N-methyl-D-aspartate receptor, in the brain. Therefore, it has been suggested as a possible therapeutic target for the treatment of various neurodegenerative diseases. To develop a potent inhibitor of SR, a simple, sensitive, fast, and robust assay is needed. In this paper, a new CE method for the determination of D-serine is described. Serine enantiomers are resolved in the form of o-phthaldialdehyde (OPA)/2-mercaptoethanol (2-ME) derivatives in an alkaline BGE composed of 50 mM sodium tetraborate, pH 9.7, and containing 40 mM 2-hydroxypropyl-gamma-CD as a chiral selector. The problem of time-limited stability of OPA/2-ME derivatives has been overcome by employing in-capillary derivatization of the sample, i.e., the derivatization reaction was carried out directly in the separation capillary in the first phase of the CE run. UV-absorption detection at 230 nm allowed concentration detection limit of 3 microM. Baseline resolution of D- and L-serine derivatives was achieved in less than 10 min. This fact, together with the simple sample pretreatment, allowed application of the method to medium-throughput monitoring of SR activity, such as the screening of potential SR inhibitors. A good agreement was achieved between the developed CE method and the previously established HPLC method for determination of the inhibition constant, K(i), of a new SR inhibitor, L-erythro-3-hydroxyaspartate.

Animals↗
PubMed1989

Characterization of sites of serine phosphorylation in human placental insulin receptor copurified with insulin-stimulated serine kinase activity by two-dimensional thin-layer peptide mapping.

Insulin receptor was copurified from human placenta together with insulin-stimulated kinase activity that phosphorylates the insulin receptor on serine residues. Analysis of phosphorylated insulin receptor by two-dimensional tryptic peptide mapping showed that sites of insulin stimulated serine phosphorylation in the insulin receptor were recovered in the same peptides as those known to be phosphorylated on serine in vivo in response to insulin. This indicates that the serine kinase copurified with the insulin receptor represents a physiologically important enzyme involved in the insulin triggered serine phosphorylation of the insulin receptor in vivo.

Humans↗
PubMed1998

Interaction of insulin receptor substrate-1 (IRS-1) with phosphatidylinositol 3-kinase: effect of substitution of serine for alanine in potential IRS-1 serine phosphorylation sites.

Serine and threonine phosphorylation has been shown to down-regulate insulin signaling at multiple steps, including the receptor and downstream molecules such as insulin receptor substrate-1 (IRS-1). To further address the mechanism of this regulation at the level of IRS-1, we constructed a double serine mutant of IRS-1: S662A/S731A-IRS-1. The serines 662 and 731 mutated to alanine are surrounding tyrosines Y658 and Y727, respectively. These tyrosines are comprised in YXXM motifs, which are potential binding sites for the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase. In a first series of experiments using the yeast two-hybrid system, we show that IRS-1 interacts with p85alpha, and this interaction depends on tyrosine phosphorylation, as shown with the IRS-1 mutant F18 and 3Y-IRS-1. F18-IRS-1 contains 18 potential tyrosine phosphorylation sites mutated to phenylalanine; three of them, i.e. Y608, 628, and 658, which are potential binding sites for p85alpha, have been added back in the 3Y-IRS-1 mutant. The tyrosine phosphorylation of IRS-1, which is required for the interaction with p85alpha, is thought to occur via endogenous yeast kinases that phosphorylate IRS-1 at least on these PI 3-kinase-binding sites. Next, we show that not only p85alpha but also p55PIK, another regulatory subunit of PI 3-kinase, interacts with IRS-1 in yeast. Interestingly, for both regulatory subunits their interaction with IRS-1 is up-regulated by mutating serines 662 and 731 on IRS-1. In a previous study we found that insulin-stimulated PI 3-kinase activity was increased not only in the presence of S662A/S731A-IRS-1 but also under resting conditions compared with the activity seen with WT-IRS-1. Here we demonstrate in 293-EBNA cells overexpressing S662A/S731A-IRS-1 that insulin-stimulated protein kinase B activity is not augmented, whereas without insulin treatment, basal activity is increased compared with that in cells overexpressing wild-type IRS-1. In conclusion, we have shown that 1) potential serine phosphorylation sites on IRS-1, which are adjacent to YXXM binding motifs for PI 3-kinase, negatively regulate binding of IRS-1 to PI 3-kinase regulatory subunits; and 2) these modulations affect protein kinase B activity.

Alanine↗
PubMed1990

Insulin-sensitive phosphorylation of serine 1293/1294 on the human insulin receptor by a tightly associated serine kinase.

In these studies we demonstrate that insulin stimulates both tyrosine and serine phosphorylation of the insulin receptor after its partial purification on wheat germ-agarose, and after affinity purification on insulin-agarose. Analysis of the serine phosphate incorporated into partially purified or highly purified insulin receptor suggests that an insulin-sensitive serine kinase (IRSK) copurifies with the insulin receptor. Following trypsin digestion, reversed-phase high pressure liquid chromatography (HPLC) analysis of the phosphorylated, affinity-purified insulin receptor preparation reveals phosphopeptide profiles similar to those of trypsin-digested receptors immunoprecipitated from 32P-labeled fibroblasts overexpressing the human insulin receptor. The major insulin-stimulated HPLC phosphopeptide peak from insulin receptors labeled in intact cells contains a hydrophilic phosphoserine-containing peptide which rapidly elutes from a C18 column. HPLC and two-dimensional separation indicate that the same phosphopeptide is obtained when affinity-purified insulin receptors are phosphorylated by IRSK. The serine containing tryptic peptide within the cytoplasmic domain of the human insulin receptor predicted to elute most rapidly upon HPLC had the sequence SSHCQR corresponding to residues 1293-1298. A synthetic peptide containing this sequence is phosphorylated by the insulin receptor/IRSK preparation. After alkylation and trypsin digestion, the synthetic phosphopeptide comigrates with the alkylated, tryptic phosphopeptide derived from insulin receptor phosphorylated in vitro by IRSK. We propose that serine 1293 or 1294 of the human insulin receptor is a major site(s) phosphorylated on the insulin receptor in intact cells and is phosphorylated by IRSK. Furthermore, insulin added directly to affinity-purified insulin receptor/IRSK preparations stimulates the phosphorylation of synthetic peptides corresponding to this receptor phosphorylation site and another containing threonine 1336. Kemptide phosphorylation is not stimulated by insulin under these conditions. No phosphorylation of peptide substrates for Ca2+/calmodulin-dependent protein kinase, protein kinase C, casein kinase II, or cGMP-dependent protein kinase by IRSK is detected. These data indicate that IRSK exhibits specificity for the insulin receptor and may be activated by the insulin receptor tyrosine kinase in an insulin-dependent manner.

Amino Acids↗
PubMed1997

The conserved serine-threonine-serine motif of the carnitine acyltransferases is involved in carnitine binding and transition-state stabilization: a site-directed mutagenesis study.

There has been speculation that the carnitine acyltransferase reaction mechanism may involve the formation of an acyl-serine intermediate. A serine-threonine-serine (STS) motif that is conserved throughout the carnitine acyltransferase family, and is present also in the choline acetyltransferases, contains the only two conserved serines. The functional role of this motif in carnitine octanoyltransferase was probed by using a site-directed mutagenesis strategy to generate all seven possible alanine substitutions: single, double and triple mutants. Kinetic analyses of these mutant enzymes demonstrated that the STS motif is not essential for catalysis, thereby excluding an acyl-serine intermediate from the reaction mechanism. The kinetic analyses support, however, substantial roles for the STS motif in carnitine binding and transition-state stabilization.

Amino Acid Sequence↗
PubMed1993

A new investigation of copper(II)-serine, copper(II)-histidine-serine, copper(II)-asparagine, and copper(II)-histidine-asparagine equilibria under physiological conditions, and implications for simulation models relative to blood plasma.

Some years ago, the application of computer modeling to metal speciation in biofluids was questioned based on the discrepancy between the simulated distribution of copper(II) in blood plasma and related experimental results obtained by Neumann and Sass-Kortsak in reconstituted serum. A recent investigation of the relevant copper(II)-amino acid equilibria reconciled these conflicting data, confirming that the reliability of computer models crucially depends on the data on which they are based. Since then, however, some of the constants of the copper-serine system used in that study have been suspected to be overestimated. This work thus reports the redetermination of copper-serine and copper-histidine-serine formation constants under physiological conditions. In addition, serine being close to asparagine in Neumann and Sass-Kortsak's classification, copper-asparagine and copper-histidine-asparagine equilibria have also been reinvestigated. For asparagine complexes, former constants have been basically confirmed. In contrast, all constants relative to serine have effectively been found lower than the previous ones. The effects of these new data on the stimulated distribution of plasma copper are only minor, but a better agreement is observed relative to Neumann and Sass-Kortsak's models in reconstituted serum.

Asparagine↗
PubMed1979

L-serine dehydratase and L-serine-pyruvate aminotransferase activities in different animal species.

1. A rough inverse correlation between liver serine dehydratase activity and species body size was observed with mammals. This was not found for glutamate, malate or lactate dehydrogenase, fumarase or aspartate-2-oxoglutarate aminotransferase. Serine dehydratase (and certain liver aminotransferases showing a similar species-size dependence) may have a role in the stimulation of heat production. 2. Cold-exposed rats showed a 3-4 fold increase in serine dehydratase activity. 3. No liver serine dehydratase was measurable in amphibia. 4. In mammals high liver serine-pyruvate aminotransferase is associated with a flesh-eating dietary habit. High activity was found in amphibia and goldfish. A gluconeogenic role is suggested.

Animals↗
PubMed1999

Insulin and insulin antagonists evoke phosphorylation of P20 at serine 157 and serine 16 respectively in rat skeletal muscle.

We show here that insulin and insulin antagonists differentially modify phosphorylation of three phospho-isoforms of P20 (termed S1, S2 and S3) in rat skeletal muscle. Precise phosphorylation sites of S1 and S2 were mapped to serine 157 and serine 16 respectively. Insulin evoked phosphorylation of P20 at serine 157 through the phosphatidylinositol (PI) 3-kinase pathway. Epinephrine and calcitonin gene-related peptide decreased phosphorylation at serine 157 and increased phosphorylation at serine 16 and other unidentified sites. These results demonstrate that the PI-3-kinase pathway of anabolic insulin and the cAMP pathway of catabolic hormones converge on P20 and suggest a potential role of this protein in regulating energy metabolism of skeletal muscle.

Amino Acid Sequence↗
PubMed1968

An application of diagonal electrophoresis to the selective purification of serine phosphate peptides. Serine phosphate peptides from ovalbumin.

A diagonal-electrophoresis method for the selective purification of serine phosphate peptides was applied to tryptic, chymotryptic and peptic digests of oxidized ovalbumin. This method is based on the release of the phosphate group bound to serine by treatment with alkaline phosphatase on paper. The identified serine phosphate peptides were purified by paper electrophoresis at pH6.5 and 2.0, dephosphorylation with bacterial alkaline phosphatase, and paper electrophoresis at pH2.0 again, in that order. The presence of two groups of serine phosphate peptides was apparent from the amino acid composition. One group contained no lysine, cysteic acid, proline, leucine or isoleucine (sequence 1) and the other had all those amino acids (sequence 2). Further degradation with subtilisin of those peptides and ;dansyl'-Edman sequence analysis established their partial sequences. The proposed sequences are as follows (with ;SerP' representing serine phosphate): sequence 1, -Ala-Gly-Arg-Glu-Val-Val-Gly-SerP-Ala-Glu-Ala-Gly-Asp-Val-Ala-Ala-Ser-(Val,Glx(2),Ser,Phe)-Arg-; sequence 2, -Asp-Lys-Leu-Pro-Gly-Phe-Gly-Asp-SerP-Ile-Glx-Ala-Glx-CySO(3)H-Gly-(Thr,Ser,Val)-(Asp,His,Val)-. The partial sequence of one of the phosphopeptides, Asp-(Glu,Ile,SerP), reported by Flavin (1954) was used to establish the proposed sequence 2.

Alkaline Phosphatase↗
PubMed1982

L-serine degradation in Escherichia coli K-12: a combination of L-serine, glycine, and leucine used as a source of carbon.

Escherichia coli K-12 strain CU1008 cannot use L-serine as the sole carbon source, but it could use L-serine as an auxiliary carbon source with glucose, L-alanine, or pyruvate and could derive energy from L-serine to support oxygen uptake. CU1008 grew with L-serine if it was also provided with glycine and leucine. These may act by increasing the available activity of L-serine deaminase; other explanations are also explored.

Energy Metabolism↗
PubMed1997

Cloning and expression of the two genes coding for L-serine dehydratase from Peptostreptococcus asaccharolyticus: relationship of the iron-sulfur protein to both L-serine dehydratases from Escherichia coli.

The structural genes sdhA and sdhB, coding for the alpha- and beta-subunits of the [4Fe-4S] cluster containing L-serine dehydratase from Peptostreptococcus asaccharolyticus, have been cloned and sequenced. Expression of modified sdhB together with sdhA in Escherichia coli led to overproduction of active His6-tagged L-serine dehydratase. E. coli MEW22, deficient in the L-serine dehydratase L-SD1, was complemented by this sdhBA construct. The derived amino acid sequence of SdhBA shares similarities with both monomeric L-serine dehydratases, L-SD1 and L-SD2, from E. coli and with a putative L-serine dehydratase from Haemophilus influenzae, which suggests that these three enzymes are also iron-sulfur proteins.

Amino Acid Sequence↗
PubMed1990

Rat serine dehydratase gene codes for two species of mRNA of which only one is translated into serine dehydratase.

With the previously obtained rat liver serine dehydratase cDNA (SDH2; Ogawa, H., Miller, D.A., Dunn, T., Su, Y., Burcham, J. M., Peraino, C., Fujioka M., Babcock, K., and Pitot, H. C. (1988) Proc. Natl. Acad. Sci. U.S. A. 85, 5809-5813) as a probe, we isolated a different species of cDNA (SDH3) from the same cDNA library from which SDH2 was obtained. Nucleotide sequence analysis has indicated that SDH3 has an open reading frame which encodes 327 amino acid residues and which is identical to that of the cDNA obtained by Noda et al. (Noda, C., Ito, K., Nakamura, T., and Ichihara, A., (1988) FEBS Lett. 234, 331-335). Primer extension analysis and RNase protection mapping clarified that the SDH3 mRNA was the major mRNA for serine dehydratase in the liver, and its transcription begins with a T residue located 23 nucleotides down-stream of a TATA-like box. In vitro transcription/translation experiment demonstrated that SDH3 encoded a polypeptide of 35 kDa, a size in agreement with that of the subunit of the purified protein, whereas SDH2, despite having a size larger than SDH3, produced a peptide of much smaller size that reacted with anti-serine dehydratase IgG. SDH2 was found to have a stop codon early in the sequence and is predicted to encode a polypeptide of 8.9 kDa. Also, SDH2 has a 5'-noncoding sequence different from that of SDH3. These results indicate that alternative transcription initiation and different modes of splicing of the primary transcripts of rat serine dehydratase gene result in the formation of two species of mRNA, of which only one is translated into the mature serine dehydratase protein.

Amino Acid Sequence↗
PubMed1978

On the appearance of Bacillus subtilis intracellular serine protease in the cell membrane and culture medium. Comparison of the enzyme and other Bacillus subtilis serine proteases.

While about 80% of the cell-bound intracellular serine protease of Bacillus subtilis A-50 have been recovered in the soluble fraction upon disruption of cells, the rest of the enzyme was found to be associated with the membrane fraction. Soluble cytoplasmic intracellular serine protease, as well as membrane-bound serine protease liberated by non-ionic detergent treatment, have been isolated in a pure state and shown to be identical. The same protease might also be found extracellularly, due presumably to cell lysis or altered membrane permeability. Intracellular serine protease of Bacillus subtilis A-50 was clearly related to Bacillus subtilis serine proteases W1 and bacillopeptidase F described as extracellular enzymes.

Amino Acids↗