Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Glyceric Acids”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 973 records · Page 54Linked to original sources
PubMed1977

Chemical inhibition of the glycolate pathway in soybean leaf cells.

Isolated soybean (Glycine max [L.] Merr.) leaf cells were treated with three inhibitors of the glycolate pathway in order to evaluate the potential of such inhibitors for increasing photosynthetic efficiency. Preincubation of cells under acid conditions in alpha-hydroxypyridinemethanesulfonic acid increased (14)CO(2) incorporation into glycolate, but severely inhibited photosynthesis. Isonicotinic acid hydrazide (INH) increased the incorporation of (14)CO(2) into glycine and reduced label in serine, glycerate, and starch. Butyl 2-hydroxy-3-butynoate (BHB) completely and irreversibly inhibited glycolate oxidase and increased the accumulation of (14)C into glycolate. Concomitant with glycolate accumulation was the reduction of label in serine, glycerate, and starch, and the elimination of label in glycine. The inhibitors INH and BHB did not eliminate serine synthesis, suggesting that some serine is synthesized by an alternate pathway. The per cent incorporation of (14)CO(2) into glycolate by BHB-treated cells or glycine by INH-treated cells was determined by the O(2)/CO(2) ratio present during assay. Photosynthesis rate was not affected by INH or BHB in the absence of O(2), but these compounds increased the O(2) inhibition of photosynthesis. This finding suggests that the function of the photorespiratory pathway is to recycle glycolate carbon back into the Calvin cycle, so if glycolate metabolism is inhibited, Calvin cycle intermediates become depleted and photosynthesis is decreased. Thus, chemicals which inhibit glycolate metabolism do not reduce photorespiration and increase photosynthetic efficiency, but rather exacerbate the problem of photorespiration.

Journal Article↗
PubMed1995

Posttranscriptional and posttranslational control of enolase expression in the facultative Crassulacean acid metabolism plant Mesembryanthemum Crystallinum L.

During the induction of Crassulacean acid and metabolism by environmental stresses in the common ice plant (Mesembryanthemum crystallinum L.), enzyme activities involved in glycolysis and gluconeogenesis, including enolase (2-phospho-D-glycerate hydrolase), increase significantly. In this study, we describe two nearly identical cDNA clones (Pgh1a and Pgh1b) encoding enolase from the common ice plant. This cytoplasmically localized enzyme is encoded by a gene family of at least two members. The polypeptides encoded by these cDNAs share a high degree of amino acid sequence identity (86.7-88.3%) with other higher plant enolases. Enolase activity increased more than 4-fold in leaves during salt stress. This increase was accompanied by a dramatic increase in Pgh1 transcription rate and the accumulation of enolase transcripts in leaves. Pgh1 transcript levels also increased in leaves in response to low temperature, drought, and anaerobic stress conditions and upon treatment of unstressed plants with the plant growth regulators abscisic acid and 6-benzylaminopurine. In roots, enolase transcripts increased in abundance in response to salt, low and high temperature, and anaerobic stresses. Surprisingly, we observed no increase in enolase protein levels, despite the increased levels of mRNA and enzyme activity during salt stress. The stress-induced increase in enolase activity is therefore due to posttranslational regulation of steady-state enzyme pools. Our results demonstrate that the stress-induced shift to Crassulacean acid metabolism in the ice plant involves complex regulatory control mechanisms that operate at the transcriptional, posttranscriptional, and postranslational levels.

Amino Acid Sequence↗
PubMed1982

Characterization of the Formation and Distribution of Photosynthetic Products by Sedum praealtum Chloroplasts.

Photoassimilation of (14)CO(2) by intact chloroplasts from the Crassulacean acid metabolism plant Sedum praealtum was investigated. The main water-soluble, photosynthetic products were dihydroxyacetone phosphate (DHAP), glycerate 3-phosphate (PGA), and a neutral saccharide fraction. Only a minor amount of glycolate was produced. A portion of neutral saccharide synthesis was shown to result from extrachloroplastic contamination, and the nature of this contamination was investigated with light and electron microscopy. The amount of photoassimilated carbon partitioned into starch increased at both very low and high concentrations of orthophosphate. High concentrations of exogenous PGA also stimulated starch synthesis.DHAP and PGA were the preferred forms of carbon exported to the medium, although indirect evidence suported hexose monophosphate export. The export of PGA and DHAP to the medium was stimulated by high exogenous orthophosphate, but depletion of chloroplastic reductive pentose phosphate intermediates did not occur. As a result only a relatively small inhibition in the rate of CO(2) assimilation occurred.The rate of photoassimilation was stimulated by exogenous PGA, ribose 5-phosphate, fructose 1,6-bisphosphate, fructose 6-phosphate, and glucose 6-phosphate. Inhibition occurred with phosphoenolpyruvate and high concentrations of PGA and ribose 5-phosphate. PGA inhibition did not result from depletion of chloroplastic orthophosphate or from inhibition of ribulose 1,5-bisphosphate carboxylase. Exogenous PGA and phosphoenolpyruvate were shown to interact with the orthophosphate translocator.

Journal Article↗
PubMed1987

Gluconeogenesis from serine in rabbit hepatocytes.

L-Serine alone is not gluconeogenic in isolated rabbit hepatocytes, whereas in rat liver this amino acid has been reported to yield as much glucose as does L-lactate itself. The current study has been an investigation into the explanation of the difference between the two species. Hepatocytes were isolated from 48-h-starved, 750- to 1000-g male rabbits, and the viability of each preparation was judged by ATP levels (2.4 +/- 0.2 mumol/g wet wt) at the beginning and end of the incubation as well as gluconeogenesis from 10 mM L-lactate (0.83 +/- 0.08 mumol/min/g wet wt). L-Serine alone produced virtually no glucose or pyruvate accumulation above baseline. Hydroxypyruvate, however, did appear in the incubation mixture. When L-serine and pyruvate were combined to test the functional activity of L-serine:pyruvate aminotransferase (EC 2.6.1.51), however, gluconeogenesis remained at the rate produced by pyruvate alone (0.61 +/- 0.04 mumol/min/g wet wt). On the other hand, the combination of L-serine and L-lactate produced rates of glucose accumulation 35% above that of L-lactate alone. The combination of L-lactate plus hydroxypyruvate produced nearly maximal rates (1.39 +/- 0.08 mumol/min/g wet wt), approaching those achieved by a physiologic ratio (10:1) of L-lactate and pyruvate. Hydroxypyruvate itself was only moderately gluconeogenic (0.44 +/- 0.04 mumol/min/g wet wt). That a reduction of the cytoplasmic free [NAD+]/[NADH] ratio by L-lactate was not its only contribution to L-serine utilization was suggested by the fact that ethanol completely eliminated gluconeogenesis from virtually all precursors (or combinations) tested, with the exception of hydroxypyruvate. It has been concluded from the data that, probably in contrast to the rat, the major pathway for the entrance of L-serine into gluconeogenesis in rabbit hepatocytes is through the pathway initiated by L-serine: pyruvate aminotransferase and that L-lactate is an important participant (i) by generating cytoplasmic reducing equivalents (NADH), (ii) by supplying pyruvate for the transaminating reaction itself, and, perhaps, (iii) by preventing hydroxypyruvate from being reduced by L-lactate dehydrogenase (EC 1.1.1.27) to L-glycerate.

Animals↗
PubMed1989

Enzymological characterization of a feline analogue of primary hyperoxaluria type 2: a model for the human disease.

This paper concerns an enzymological investigation into a putative feline analogue of the human autosomal recessive disease primary hyperoxaluria type 2. The hepatic activities of D-glycerate dehydrogenase, using both D-glycerate and hydroxypyruvate as substrates, and glyoxylate reductase, which are the deficient enzyme activities in human primary hyperoxaluria type 2, were markedly depleted in four affected cats (0-6% of controls). The activities of a number of other enzymes, lactate dehydrogenase, glutamate dehydrogenase, D-amino acid oxidase, aspartate:2-oxoglutarate amino-transferase, glutamate:glyoxylate aminotransferase and alanine:glyoxylate aminotransferase (the deficient enzyme in primary hyperoxaluria type 1) were unaltered. The intracellular distribution of D-glycerate dehydrogenase and glyoxylate reductase in cat liver was shown to be cytosolic, as they are in human liver. The activities of D-glycerate dehydrogenase and glyoxylate reductase were determined in unaffected related cats and putative heterozygotes were identified. The correlation between D-glycerate dehydrogenase and glyoxylate reductase activities in the related cats and their combined deficiency in the affected cats confirmed previous suggestions that they are identical gene products.

Alcohol Oxidoreductases↗
PubMed1994

A recombinant bisphosphoglycerate mutase variant with acid phosphatase homology degrades 2,3-diphosphoglycerate.

To date no definite and undisputed treatment has been found for sickle cell anemia, which is characterized by polymerization of a deoxygenated hemoglobin mutant (HbS) giving rise to deformed erythrocytes and vasoocclusive complications. Since the erythrocyte glycerate 2,3-bisphosphate (2,3-DPG) has been shown to facilitate this polymerization, one therapeutic approach would be to decrease the intraerythrocytic level of 2,3-DPG by increasing the phosphatase activity of the bisphosphoglycerate mutase (BPGM; 3-phospho-D-glycerate 1,2-phosphomutase, EC 5.4.2.4). For this purpose, we have investigated the role of Gly-13, which is located in the active site sequence Arg9-His10-Gly11-Glu12-Gly13 in human BPGM. This sequence is similar to the Arg-His-Gly-Xaa-Arg* sequence of the distantly related acid phosphatases, which catalyze as BPGM similar phosphoryl transfers but to a greater extent. We hypothesized that the conserved Arg* residue in acid phosphatase sequences facilitates the phosphoryl transfer. Consequently, in human BPGM, we replaced by site-directed mutagenesis the corresponding amino acid residue Gly13 with an Arg or a Lys. In another experiment, we replaced Gly13 with Ser, the amino acid present at the corresponding position of the homologous yeast phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1). Mutation of Gly13 to Ser did not modify the synthase activity, whereas the mutase and the phosphatase were 2-fold increased or decreased, respectively. However, replacing Gly13 with Arg enhanced phosphatase activity 28.6-fold, whereas synthase and mutase activities were 10-fold decreased. The presence of a Lys in position 13 gave rise to a smaller increase in phosphatase activity (6.5-fold) but an identical decrease in synthase and mutase activities. Taken together these results support the hypothesis that a positively charged amino acid residue in position 13, especially Arg, greatly activates the phosphoryl transfer to water. These results also provide elements for locating the conserved Arg* residue in the active site of acid phosphatases and facilitating the phosphoryl transfer. The implications for genetic therapy of sickle cell disease are discussed.

2,3-Diphosphoglycerate↗
PubMed1990

Analogs of oxalacetate as potential substrates for phosphoenolpyruvate carboxykinase.

Structural analogs of the substrate oxalacetate were examined as potential substrates and inhibitors for chicken liver mitochondrial phosphoenolpyruvate (P-enolpyruvate) carboxykinase. Steady-state kinetics were employed to characterize the inhibitory effects of these substrate analogs with the enzyme. Assays were carried out in both carboxylation and decarboxylation reaction directions. Pyruvate, beta-hydroxypyruvate, beta-mercaptopyruvate, beta-fluoropyruvate, DL-lactate, glycolate, glycoaldehyde, glyoxylate, glyphosate, and DL-aspartate showed no inhibitory effects by steady-state kinetics. Oxalate, acetopyruvate, and DL-, D-, and L-glycerate exhibited weak noncompetitive inhibition of the P-enolpyruvate carboxykinase-catalyzed reaction. DL-3-Nitro-2-hydroxypropionic acid, 3-nitro-2-oxopropionic acid, DL-malate, malonate, tartronate, and alpha-ketobutyrate all show weak inhibition with estimated inhibition constants greater than 20 nM. Several of these compounds were investigated by 31P NMR to determine if they function as phosphoryl acceptors for GTP. None of the compounds tested act as phosphoryl acceptors in the enzyme-catalyzed reaction. Chicken liver mitochondrial phosphoenolpyruvate carboxykinase shows a remarkably high degree of specificity at the binding site of oxalacetate.

Animals↗
PubMed1990

Biochemical effects of fluoride on oral bacteria.

Fluoride inhibition of carbohydrate metabolism by the acidogenic plaque microflora is well-established, although it has not always been appreciated that oral bacteria vary considerably in their susceptibility to fluoride. Early studies demonstrated that the F-induced reduction in acid production was due, in part, to the inhibition of the glycolytic enzyme, enolase, which converts 2-P-glycerate to P-enolpyruvate. The decreased output of PEP in the presence of F, in turn, results in the inhibition of sugar transport via the PEP phosphotransferase system (PTS). Bacterial accumulation of fluoride involves the transport of HF, a process requiring a transmembrane pH difference or pH gradient, which is generated only by metabolically active cells. The uptake of HF into the more alkaline cytoplasm results in the dissociation of HF to H+ and F- and, if allowed to continue, the accumulation of protons acidifies the cytoplasm, causing a reduction in both the proton gradient and enzyme activity. Current information indicates that in addition to enolase, F- also inhibits the membrane-bound, proton-pumping H+/ATPase, which is involved in the generation of proton gradients through the efflux of protons from the cell at the expense of ATP. Thus, fluoride has the dual action of dissipating proton gradients and preventing their generation through its action on H+/ATPase. The collapse of transmembrane proton gradient, in turn, reduces the ability of cells to transport solutes via mechanisms involving proton motive force. In spite of these known effects on the bacterial cell, there is no general agreement that the anti-microbial effects of F contribute to the anti-caries effect of fluoride.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteria↗
PubMed1999

Metabolic flux analysis for serine alkaline protease fermentation by Bacillus licheniformis in a defined medium: effects of the oxygen transfer rate.

The metabolic fluxes through the central carbon pathways in the bioprocess for serine alkaline protease (SAP) production by Bacillus licheniformis were calculated by the metabolic flux-based stoichiometric model based on the proposed metabolic network that contains 102 metabolites and 133 reaction fluxes using the time profiles of citrate, dry cell, organic acids, amino acids, and SAP as the constraints. The model was solved by minimizing the SAP accumulation rate in the cell. The effects of the oxygen-transfer rate (OTR) on the metabolic fluxes were investigated in a defined medium where citrate was used as the sole carbon source. The central pathways were active for the growth and the SAP synthesis in all the periods of the bioprocess at low (LOT), medium (MOT), and high (HOT) oxygen-transfer conditions. The flux partitioning in the TCA cycle at alpha-ketoglutarate towards glutamate group and at oxalacetate (OA) toward aspartic acid group amino acids were dependent on the OTR. The flux of the anaplerotic reaction that connects the TCA cycle either from malate or OA to the gluconeogenesis pathway via the main branch point pyruvate (Pyr) was also influenced by the OTR. With the decrease in the OTR, the intracellular flux values after glycerate 3-phosphate (PG3) in the gluconeogenesis pathway and the specific growth rate decreased. The total ATP-generation rate increased with the increase in OTR. The pathway towards the aspartic acid family amino acids which is important for sporulation that precedes the SAP synthesis were all active throughout the bioprocess. Metabolic flux analysis results at LOT, MOT, and HOT conditions encourage the design of an oxygen-transfer strategy in the bioreactor; moreover, asparagine synthetase or aspartate kinase could be the potential metabolic engineering sites due to the low value of the flux from the branch point aspartate toward asparagine.

Adenosine Triphosphate↗
PubMed1982

Organic acidurias: approach, results and clinical relevance.

More than twenty-five inherited organic acidurias have been identified during the last fifteen years. This remarkable development is due mainly to the introduction of gas chromatography, and gas chromatography combined with mass spectrometry, in paediatric laboratories for metabolic disease. The chemical approach is determined mainly by physical properties of the acid, such as their extractability and volatility. Most progress has been made with extractable acids. The techniques used for derivatization are mentioned, such as trimethylsilylation, methylation and the preparation of asymmetric derivatives for the separation of optical enantiomers. Metabolite patterns may be so characteristic that the underlying enzyme defect can be deduced. Examples are the leucine degradation defects, all encountered in the authors' laboratory: branched-chain ketoaciduria; isovaleric acidaemia; 3-methylcrotonylglycinuria; 3-methylglutaconic aciduria; and 3-hydroxy-3-methylglutaric aciduria. These abnormalities are discussed. D-glyceric aciduria is shown as an example of a not yet fully understood organic aciduria. The clinical approach varies. Metabolic acidosis is an indication for organic acid analysis in urine and plasma, but in many defects there is no acidosis, or only a transient one caused by secondary metabolites, such as lactic and 3-hydroxybutyric acids. Gas chromatography is an obligatory routine investigation in screening programmes for inborn errors of metabolism, especially for the examination of acutely ill neonates and premature babies.

Child↗
PubMed1980

Gas chromatographic determination of extracellular metabolites produced by baker's yeast during glucose-induced acidification.

Gas chromatographic separation of metabolites extruded during the medium acidification by resting baker's yeast supplied with glucose is described. Silylation with bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane and a direct chromatography on Chromosorb G-AW-DMCS with 2.5% SE-52 (lactic, pyruvic, succinic, glyceric, fumaric, malic), polyols (glycerol, arabinitol) and sugars (glucose). The yeast was found to extrude glycerol, lactic, malic, and succinic acid.

Chromatography, Gas↗
PubMed1986

Kinetic properties and essential amino acids of the 2,3-bisphosphoglycerate synthase-phosphatase from pig skeletal muscle.

Histidine, arginine and lysine residues are essential for the multifunctional 2,3-bisphosphoglycerate synthase-phosphatase purified from pig skeletal muscle. The synthase, phosphatase and phosphoglycerate mutase activities of the enzyme are concurrently lost upon treatment with diethylpyrocarbonate, phenylglyoxal and trinitrobenzenesulfonate. The phosphatase activity shows hyperbolic kinetics. In contrast, the synthase activity shows a nonhyperbolic pattern which fits to a second-degree polynomial. The Km values for glycerate 1,3-P2, glycerate 3-P and glycerate 2,3-P2 are similar to those of the enzyme from mammalian erythrocytes.

Amino Acids↗
PubMed1982

[In vivo and in vitro actions of biscarbamates on the photosynthetic activity of chloroplasts].

The "photosynthetic inhibition" component in the whole context of plant toxicity, when different concentrations of the bis-carbamate phenmedipham are supplied through the roots or foliar application to spinach plants grown in hydroponic media have been determined. Chloroplasts were isolated after eight days of the herbicide addition, and then determined: electron transport H2O leads to NADP+, H2O leads to ferrycyanide and ascorbate/DPIP leads to NADP+, cyclic and non cyclic photophosphorilation, CO2 assimilation rate and intermediate patterns of CO2 fixation. We have also determined in foliar disks the O2 evolving and the CO2 assimilation capabilities. Type A and type B chloroplasts showed increased inhibition, respectively, of the Phot. II dependent electron transport chains H2O leads to NADP+ and H2O leads to ferricyanide, to the extent that the phenmedipham concentration increased in the hydroponic media and the spraying solution, so that a 50% inhibition of both processes was obtained at 100 microM and 10 microM, respectively, against 0.2 microM in the in vitro experiments. Non cyclic photophosphorylation showed a stronger inhibition than the cyclic one. Concerning the Phot. I dependent electron transport ascorbate/DPIP leads to NADP+, the chloroplast preparations showed a negligible inhibition. We have found a synergistic effect of the above two factors on the CO2 assimilation. The intermediate patterns of CO2 assimilation showed a decrease of the 3C-compounds P-glycerate and trioses-P, with a parallel increase of the sugar mono and diphosphates as well as disaccharides and amino acids.

Carbamates↗
PubMed1981

The effects of anions, substrates, metal ions and sulfhydryl reagents on the proteolytic susceptibility of yeast phosphoglycerate kinase.

With the aim of confirming our previous spectrophotometric binding studies ((1978) Eur. J. Biochem. 85, 345-350 and (1980) Eur. J. Biochem. 104, 249-254) and of ascertaining the full physiological significance of ion binding, we investigated the effects of ions and thiol reagents on the proteolysis of yeast phosphoglycerate kinase (ATP: 3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3). The single non-essential thiol of the enzyme was modified with 5,5'-dithiobis(2-nitrobenzoic acid) or 2-chloromercuri-4-nitrophenol. Both modifications greatly increased the susceptibility of the kinase to inactivation by trypsin or yeast proteinase A, when compared with that of the native kinase. Electrophoresis in sodium dodecyl sulfate (SDS) revealed that limited proteolysis had occurred. The time courses for the proteolysis and loss of catalytic activity were followed and the active and inactive fragments identified. The molecular masses of the major proteolytic fragments differed with the two endopeptidases. Substrate and non-substrate anions in a concentration-dependent fashion, protected the native and mercurial-labelled kinase from inactivation by trypsin or yeast proteinase A. However, Zn2+, in a concentration-dependent fashion, increased the susceptibility of the native kinase to inactivation by each endopeptidase. The time courses for the inactivation and for the proteolysis allowed the active and inactive fragments to be identified. Zn2+ decreased the rate of inactivation of the mercurial-labelled kinase by proteinase A. The effects of these ions were detected at concentrations compatible with occupancy of an anion binding site and a low affinity Zn2+ binding site, both of which have been indicated from our previous binding studies.

Anions↗
PubMed1997

Induction of heat shock proteins DnaK, GroEL, and GroES by salt stress in Lactococcus lactis.

The bacterium Lactococcus lactis has become a model organism in studies of growth physiology and membrane transport, as a result of its simple fermentative metabolism. It is also used as a model for studying the importance of specific genes and functions during life in excess nutrients, by comparison of prototrophic wild-type strains and auxotrophic domesticated (dairy) strains. In a study of the capacity of domesticated strains to perform directed responses toward various stress conditions, we have analyzed the heat and salt stress response in the established L. lactis subsp. cremoris laboratory strain MG1363, which was originally derived from a dairy strain. After two-dimensional separation of proteins, the DnaK, GroEL, and GroES heat shock proteins, the HrcA (Orf1) heat shock repressor, and the glycolytic enzymes pyruvate kinase, glyceral-dehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase were identified by a combination of Western blotting and direct N-terminal amino acid sequencing of proteins from the gels. Of 400 to 500 visible proteins, 17 were induced more than twofold during heat stress. Two classes of heat stress proteins were identified from their temporal induction pattern. The fast-induced proteins (including DnaK) showed an abruptly increased rate of synthesis during the first 10 min, declining to intermediate levels after 15 min. GroEL and GroES, which also belong to this group, maintained a high rate of synthesis after 15 min. The class of slowly induced proteins exhibited a gradual increase in the rate of synthesis after the onset of stress. Unlike other organisms, all salt stress-induced proteins in L. lactis were also subjected to heat stress induction. DnaK, GroEL, and GroES showed similar temporal patterns of induction during salt stress, resembling the timing during heat stress although at a lower induction level. These data indicate an overlap between the heat shock and salt stress responses in L. lactis.

Amino Acid Sequence↗
PubMed1999

The gene encoding hydroxypyruvate reductase (GRHPR) is mutated in patients with primary hyperoxaluria type II.

Primary hyperoxaluria type II (PH2) is a rare monogenic disorder that is characterized by a lack of the enzyme that catalyzes the reduction of hydroxypyruvate to D-glycerate, the reduction of glyoxylate to glycolate and the oxidation of D-glycerate to hydroxypyruvate. The disease is characterized by an elevated urinary excretion of oxalate and L-glycerate. The increased oxalate excretion can cause nephrolithiasis and nephrocalci-nosis and can, in some cases, result in renal failure and systemic oxalate deposition. We identified a glyoxylate reductase/hydroxypyruvate reductase (GRHPR) cDNA clone from a human liver expressed sequence tag (EST) library. Nucleotide sequence analysis identified a 1198 nucleotide clone that encoded a 984 nucleotide open reading frame. The open reading frame encodes a predicted 328 amino acid protein with a mass of 35 563 Da. Transient transfection of the cDNA clone into COS cells verified that it encoded an enzyme with hydroxy-pyruvate reductase, glyoxylate reductase and D-glycerate dehydrogenase enzymatic activities. Database analysis of human ESTs reveals widespread tissue expression, indicating that the enzyme may have a previously unrecognized role in metabolism. The genomic structure of the human GRHPR gene was determined and contains nine exons and eight introns and spans approximately 9 kb pericentromeric on chromosome 9. Four PH2 patients representing two pairs of siblings from two unrelated families were analyzed for mutations in GRHPR by single strand conformation polymorphism analysis. All four patients were homozygous for a single nucleotide deletion at codon 35 in exon 2, resulting in a premature stop codon at codon 45. The cDNA that we have identified represents the first characterization of an animal GRHPR sequence. The data we present will facilitate future genetic testing to confirm the clinical diagnosis of PH2. These data will also facilitate heterozygote testing and prenatal testing in families affected with PH2 to aid in genetic counseling.

Alcohol Oxidoreductases↗
PubMed1993

Interaction of ATP analogs with yeast 3-phosphoglycerate kinase. Affinity labeling of the hinge region.

Yeast 3-phosphoglycerate kinase is inactivated by incubation with pyridoxal 5'-diphospho-5'-adenosine (AdoP2Pxy) [Tamura, J. K., Rakov, R. D. & Gross, R. L. (1986) J. Biol. Chem. 261, 4126-4133). Incorporation of 1 mol affinity label/mol enzyme was sufficient for complete inactivation of 3-phosphoglycerate kinase. The substrate ATP affords substantial protection against inactivation. Partial protection is afforded by the substrate glycerate 3-phosphate. When AdoP2Pxy-modified phosphoglycerate kinase was reduced with [3H]NaBH4 and subjected to trypsin hydrolysis, only one radioactive peptide was isolated by reverse-phase high-performance liquid chromatography. The amino acid composition and sequence analysis of the purified radioactive peptide revealed that it spans residues 379-403 of the enzyme and Lys385 specifically reacted with the affinity label. This peptide represents the hinge region between the two domains of the protein, where the active site is also located. The fluorescence intensity of enzyme-bound AdoP2Pxy is enhanced when glycerate 3-phosphate is added, suggesting exposure of the fluorescent probe to a more hydrophobic environment. Another fluorescent analog, anthraniloyl-dATP (ant-dATP), which carries the fluorescent reporter group on the ribose ring, binds to the enzyme at two distinct sites with Kd values of 6 +/- 2 microM and 25 +/- 3 microM, as determined by steady-state anisotropy measurements. Bound ant-dATP was displaced from the enzyme by glycerate 3-phosphate and ATP, as monitored by the fluorescence anisotropy. These results suggest that both fluorescent ATP analogs bind to the active site, which is at the hinge region of the enzyme. Model-building studies showed that when AdoP2Pxy is built into the open form of the enzyme, as described in X-ray studies, the pyridoxyl group of AdoP2Pxy cannot reach Lys385 for Schiff-base formation. Labeled Lys385 is on a beta-turn immediately following helix XII, which was suggested to interact with the nucleotide and become ordered at the active site of 3-phosphoglycerate kinase [Watson, H. C., Walker, N. P. C., Shaw, P. J., Bryant, T. N., Wendell, P. L., Fothergill, L. A., Perkins, R. E., Conroy, S. C., Dobson, M. J., Tuite, M. F., Kinesman, A. J. & Kinesman, S. M. (1982) EMBO J. 1, 1635-1640]. The results presented here suggest that binding of substrates cause significant structural changes in the enzyme.

Adenosine Diphosphate↗