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PubMed2005

Thermal destabilization of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans upon phosphate binding in the active site.

Catalysis by the NADP-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans, a member of the aldehyde dehydrogenase (ALDH) family, relies on a local conformational reorganization of the active site. This rearrangement is promoted by the binding of NADP and is strongly kinetically favored by the formation of the ternary complex enzyme.NADP.substrate. Adiabatic differential scanning calorimetry was used to investigate the effect of ligands on the irreversible thermal denaturation of GAPN. We showed that phosphate binds to GAPN, resulting in the formation of a GAPN.phosphate binary complex characterized by a strongly decreased thermal stability, with a difference of at least 15 degrees C between the maximum temperatures of the thermal transition peaks. The kinetics of phosphate association and dissociation are slow, allowing both free and GAPN.phosphate complexes to be observed by differential scanning calorimetry and to be separated by native polyacrylamide electrophoresis run in phosphate buffer. Analysis of a set of mutants of GAPN strongly suggests that phosphate is bound to the substrate C-3 subsite. In addition, the substrate analog glycerol-3-phosphate has similar effects as does phosphate on the thermal behavior of GAPN. Based on the current knowledge on the catalytic mechanism of GAPN and other ALDHs, we propose that ligand-induced thermal destabilization is a mechanism that provides to ALDHs the required flexibility for an efficient catalysis.

Binding Sites↗
PubMed1996

Phosphate-binding sites in phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

The two anion-binding sites of the glycolytic glyceraldehyde-3-phosphate dehydrogenase (GraP-DH), the Ps and Pi sites, were originally proposed by Moras et al. [Moras, D., Olsen, K.W., Sabesan, M.N., Buehner, M., Ford, G.C. & Rossmann, M. G. (1975) J. Biol. Chem. 250, 9137-9162] to bind the C3 phosphate of the glyceraldehyde 3-phosphate and the inorganic phosphate respectively. Ps site mutants T179A, and T179M, and R231L, and the Pi site mutants T150A and T208 of the Bacillus stearothermophilus GraP-DH were constructed by site-directed mutagenesis and their kinetic properties were determined and compared with those of mutants R195L and R231G, already described [Corbier, C., Michels, S., Wonacott, A. & Branlant, G. (1994) Biochemistry 33, 3260-3265]. Taking advantage of the opportunity to study both the oxidoreduction and the phosphorylation step independently and the fact that the phosphorylation becomes rate determining for most of the mutants, the relative energetic contribution of each mutated amino acid to the phosphorylation step was evaluated. It was concluded that (a) Ps amino acids contribute more than the Pi amino acids to the stabilisation of the transition state relative to the ground state and (b) the side chain of arginine contributes more than that of the threonine residue. It was also concluded that the differences observed in the efficiency of the phosphorylation step for Ps and Pi mutants is a consequence of the orientation of the thioester bond of the thioacyl] intermediate relative to the attacking inorganic phosphate and not of a change in the intrinsic electrophilic property of the thioacyl intermediate. Furthermore, the kinetic results on the overall steps leading to the acyl-enzyme formation provided supplementary evidence that the C3 phosphate moiety of the glyceraldehyde 3-phosphate interacts with the Pi site during these steps and thus are consistent with the findings of Skarzynski et al. [Skarzynski, T., Moody, P. C. E. & Wonacott, A. J. (1987) J. Mol Biol. 193, 171-183] and Corbier et al. [Corbier, C., Michels, S., Wonacott, A. & Branlant, G. (1994) Biochemistry 33, 3260-3265] that recommended the reconsideration of the first definition of the Ps and Pi sites.

Binding Sites↗
PubMed1989

Sodium-phosphate cotransport in OK cells: inhibition by PTH and "adaptation" to low phosphate.

Sodium-phosphate (Na-Pi) cotransport is principally regulated by parathyroid hormone (PTH) and by the intrinsic ability to "adapt" to ambient phosphate concentration. In the present study, these two control mechanisms were examined in a cloned opossum kidney (OK) cell line. PTH inhibited Na-Pi cotransport, half-maximal inhibition at 5 x 10(-12) M, by fractionally similar amounts irrespective of the initial transport rates predetermined by "adaptation" to media phosphate concentration. At maximal concentrations of PTH (10(-8) M), the residual Na-Pi cotransport activity was higher in cells exposed to low-phosphate media. Cells preexposed to PTH (10(-8) M) or dibutyryl adenosine 3'-5'-cyclic monophosphate (DBcAMP) (10(-5) M) and forskolin (10(-5) M) increase transport (adaptation) by fractionally similar amounts as control cells for any given external phosphate concentration. The protein kinase C inhibitor, staurosporine, prevented PTH action but did not alter the ability to adapt Na-Pi cotransport in response to low media phosphate concentration. These data support the notion that regulation of Na-Pi cotransport by PTH and the adaptive response to available media phosphate concentration are distinct regulatory control mechanisms.

Alkaloids↗
PubMed1984

Retinyl palmitate, retinyl phosphate, and dolichyl phosphate of postnuclear membrane fraction from hepatoma, host liver, and regenerating liver: marginal vitamin A status of hepatoma tissue.

The retinyl palmitate content of the postnuclear membrane fraction from 10 Morris hepatomas, their host rat livers, one acetylaminofluorene-induced rat liver hepatoma, and the host liver and of regenerating rat liver was measured by reverse-phase high-pressure liquid chromatography of the chloroform:methanol extracts. Membranes from the hepatoma tissue contained less than detectable levels of retinyl acyl esters, whereas membranes from host liver tissue and regenerating liver contained levels of retinyl palmitate within normal ranges. The amount of cellular retinol-binding protein was also decreased considerably in cytosols from 9618 and 7777 hepatomas. The ratio of endogenous retinyl phosphate to the polyisoprenoid dolichyl phosphate available for mannosylation in an assay containing postnuclear membranes and guanosine dephospho[14C] mannose was decreased by a factor of 3 to 10 in hepatoma tissue. Such change in ratio was not attributable to specific changes in retinyl phosphate mannose-synthesizing activity, but it appeared to be related to the vitamin A deficiency condition of the membrane from tumors. As for membranes from vitamin A-deficient liver tissue, postnuclear membranes from rat cystic hepatocarcinoma, Morris 7777, 3924A1-1, and 5123D-1-2 transplantable rat hepatomas and guinea pig line 10 hepatoma all synthesized a mannolipid with intermediate hydrophobic properties between retinyl phosphate mannose and dolichyl phosphate mannose and not normally found in liver tissue. These alterations in patterns of lipid intermediates may be responsible for altered glycosylation of glycoproteins in neoplastic cells. In conclusion, the present investigation establishes that hepatoma cell membrane is in a status of vitamin A and of retinyl phosphate depletion, while dolichyl phosphate contents appear similar to host liver membrane.

Animals↗
PubMed1997

Phosphate excretion and phosphate transporter messenger RNA in uremic rats treated with phosphonoformic acid.

The prevention of phosphate retention in chronic renal disease may reduce both renal osteodystrophy and disease progression. We evaluated the expression of the sodium-dependent phosphate transporter, NaPi-2, and the response to phosphonoformic acid (PFA) in rats with 5/6 nephrectomy-induced renal failure. Partial nephrectomy resulted in a significant proteinuria and reduced renal function. In addition, there was an approximately 50% reduction in the expression of NaPi-2 mRNA. Treatment of rats for 48 hr with PFA (0.6% in glucose drinking fluid) had no effect on NaPi-2 mRNA; however, PFA resulted in a significant increase in fractional phosphate excretion in both normal (7 +/- 0.5% vs. 3 +/- 0.2%) and uremic (60 +/- 4% vs. 36 +/- 4%) rats. Plasma phosphate concentration was higher in uremic rats (2.5 +/- 0.1 mM) compared with normal rats (1.9 +/- 0.04 mM) but not in uremic rats treated with PFA (2.1 +/- 0.04 mM). These data suggest that PFA can increase renal phosphate excretion independent of changes in phosphate transporter expression and prevent phosphate retention.

Animals↗
PubMed1965

THE CONVERSION OF PYRIDOXINE PHOSPHATE INTO PYRIDOXAL PHOSPHATE IN ESCHERICHIA COLI.

1. Evidence is presented for the presence of pyridoxine phosphate oxidase in aqueous extracts of Escherichia coli. Some comparison is made with pyridoxamine phosphate oxidase. 2. Isoniazid and iproniazid were found to combine with pyridoxal phosphate, but isoniazid did not combine with either pyridoxamine phosphate or pyridoxine phosphate. Both oxidase activities were somewhat inhibited by benzylamine and putrescine, but not by phenethylamine or cadaverine. 3. The significance of pyridoxine phosphate oxidase in cell metabolism is discussed.

Amines↗
PubMed2000

N-Bromoacetylethanolamine phosphate as a probe for the identification of a liver microsomal glucose-6-phosphate transporter peptide in rats and Ehrlich ascites tumor-bearing mice.

Hepatic microsomal glucose-6-phosphatase is a multicomponent system composed of substrate/product translocases and a catalytic subunit. Previously we (Foster et al. (1996) Biochim. Biophys. Acta 12, 244-254) demonstrated that N-bromoacetylethanolamine phosphate (BAEP) is a time-dependent, irreversible inhibitor of glucose-6-phosphate hydrolysis in intact but not disrupted microsomes. We proposed that BAEP manifests its inhibitory effect by binding with a glucose-6-phosphate translocase protein of the glucose-6-phosphatase system. Here we provide additional evidence that BAEP inhibits glucose-6-phosphate transport in microsomal vesicles and utilize [(32)P]BAEP as an affinity label in the identification of a glucose-6-phosphate transport protein. In this study, we identify 51-kDa rat and mouse liver microsomal proteins involved in glucose-6-phosphate transport into and out of microsomal vesicles by utilizing (1) an Ehrlich ascites tumor-bearing mouse model, which displays a decreased sensitivity to the time-dependent inhibitory effect of BAEP, and (2) another glucose-6-phosphate translocase inhibitor, tosyl-lysine chloromethyl ketone, in conjunction with [(32)P]BAEP as an affinity label.

Affinity Labels↗
PubMed1981

Plasma membrane phosphate transport and extracellular phosphate concentration in the regulation of cellular respiration in isolated perfused rat heart.

The role of extracellular Pi and transmembrane fluxes across the sarcolemma in the regulation of cellular respiration was studied in isolated Langendorff-perfused rat hearts. Extracellular phosphate did not significantly affect the oxygen consumption or cellular phosphorylation potential of the myocardium. K+-induced arrest was used to change the mechanical work load of the heart. Arresting the heart caused a rapid decrease in the unidirectional efflux of phosphate determined by in vitro prelabelling of the intracellular phosphate compounds with 32P and determining the specific radioactivity of the gamma-P of ATP, and the label appearance into the perfusion medium. At normal or elevated perfusate phosphate concentration there was a fairly slow net uptake of phosphate. The decrease in phosphate fluxes upon the K+-induced arrest was probably not due to a decrease in the transmembrane Na+ or K+ gradients because a further increase in the perfusate K+ concentration caused an increase in the K+ efflux to the levels observed in contracting hearts. The use of higher than normal concentrations of phosphate necessitated a lowering of the extracellular Ca2+ concentration, which caused a diminution of the oxygen consumption, accompanied by mitochondrial flavoprotein in the heart. This finding suggested that the extracellular Ca2+ concentration may be involved in the substrate level regulation of mitochondrial metabolism.

Animals↗
PubMed1990

Effect of parathyroid hormone and dietary phosphate on phosphate transport in renal outer cortical and outer medullary brush-border membrane vesicles.

Two distinctive sodium-dependent phosphate transport systems have been identified in early and late proximal tubules; a high-capacity process located only in outer cortical tissue, and a high affinity present in both outer cortical and outer medullary brush-border membranes (Km 0.1-0.25 mM). A third, sodium-independent, pH gradient-stimulated system (Vmax 4.7 +/- 0.3 nmol.mg-1.min-1, Km 0.15 +/- 0.002 mM) is present in the outer medulla, but absent in outer cortex. Brush-border vesicles were prepared from outer cortical and outer medullary tissue of pigs maintained on low (less than 0.05%), normal (0.4%), or high (4%) phosphate diets. Sodium-dependent phosphate uptake of the high-capacity system decreased (Vmax, 9.4 to 2.2 nmol.mg-1.min-1) from low to high phosphate diet, whereas uptake rates decreased about 50% in the high-affinity system. There were no changes in the respective Km values. The pH gradient-stimulated uptake also decreased (Vmax, 6.9 to 3.0 nmol.mg-1.min-1) with no change in mean Km value (0.15 +/- 0.001 mM) with dietary manipulation. Administration of 1 U parathyroid hormone prior to study resulted in a decrease in sodium-dependent uptake by 40-50% and in pH-dependent uptake (36%) with no change in the respective Km values. In conclusion, the antecedent dietary phosphate intake and parathyroid hormone administration appropriately alters phosphate uptake across the brush-border membrane of all three systems, sodium-dependent and pH gradient-stimulated phosphate transport.

Animals↗
PubMed1990

Prenatal deficiency of phosphate, phosphate supplementation, and rickets in very-low-birthweight infants.

A preliminary study showed that placental histology was abnormal for babies who subsequently had rickets. The findings--low plasma phosphate concentration; maximum percentage tubular reabsorption of phosphate; and high urinary calcium loss--are those of renal conservation of phosphate in the presence of phosphate deficiency. In a controlled trial no baby receiving phosphate supplements (50 mg per day) had radiological evidence of rickets whereas bone changes were apparent in 42% of the control group. Prenatal deficiency of phosphate, due to placental insufficiency, can be corrected by phosphate supplementation thereby preventing rickets of prematurity.

Calcium↗
PubMed1989

Cloning and characterization of a phosphate-regulated promoter involved in phosphate control of candicidin biosynthesis.

Phosphate strongly repressed the formation of p-aminobenzoic acid (PABA) synthase, an enzyme involved in candicidin biosynthesis. Expression in Streptomyces lividans of the pabS gene (encoding PABA synthase) of Streptomyces griseus is repressed by phosphate at concentrations above 0.1 mM. However, expression of the pabS gene in Escherichia coli is not regulated by phosphate. Phosphate control of the expression of the pabS gene was observed in all plasmids containing the original 4.5-kb BamHI fragment, whereas no phosphate regulation was found when an upstream 1-kb fragment that carries the pabS promoter was deleted. Using the promoter-probe plasmid pIJ424, a '114-bp' promoter was cloned. Expression of the promoterless kanamycin phosphotransferase gene when fused to the '114-bp' promoter was strongly reduced by phosphate (90% at 5 mM concentration). The '114-bp' promoter has been sequenced and the first transcribed nucleotide identified by S1 mapping. The '114-bp' fragment is A + T-rich (54%), as compared to the Streptomyces genome (70-73% GC). The presence of a phosphate control sequence (pcs) in the upstream region of the pabS gene is proposed.

Antifungal Agents↗
PubMed1985

31P NMR relaxation studies of the activation of the coenzyme phosphate of glycogen phosphorylase. The role of motion of the bound phosphate.

Spin-lattice and spin-spin relaxation rates (1/T1 and 1/T2) have been determined for the catalytically essential coenzyme phosphate at the active site of glycogen phosphorylase in both activated (R state) and inactive (T state) conformations of the enzyme. Dipolar contributions to 31P relaxation due to exchangeable protons on the phosphate group have been determined by measurement of relaxation rates at different concentrations of H2O and D2O, and field dependence studies have been performed to estimate the contribution of chemical shift anisotropy to the remaining 31P relaxation in D2O. At 109 MHz, dipolar relaxation from exchangeable protons was found to account for 50% of the spin-lattice relaxation for activated phosphorylase in 75% H2O, the remainder being due to chemical shift anisotropy. The spin-lattice relaxation rates in D2O for R-state glycogen phosphorylase are very similar to those measured for other proteins of very different size such as actin (Brauer, M., and B. D. Sykes, 1981, Biochemistry. 20:6767-6775), alkaline phosphatase (Coleman, J. E., I. D. Armitage, J. F. Chlebowski, J. D. Otvos, and A. J. M. S. Uiterkamp, 1979), and phosphoglucomutase (Rhyu, G. I., W. J. Ray, Jr., and J. L. Markley, 1984, Biochemistry. 23:252-260). In inactive (T state) phosphorylase the spin-lattice relaxation rates were almost an order of magnitude slower, while the spin-spin relaxation rates were essentially identical. These results have been analyzed by calculating the theoretically expected 31P relaxation rates in the presence of internal motions that are included in the relaxation calculation using the model-free approach of Lipari and Szabo (1982, J. Am. Chem. Soc. 104:4564-4559). The analysis suggests the coenzyme phosphate is relatively immobilized in the activated enzymic conformation, but in the inactive (Tstate) conformation it is considerably more mobile with a rotational correlation time one to two orders of magnitude smaller. Since the spin-lattice relaxation rate for the active R-state (immobilized) phosphate is similar to that observed in other phosphoenzymes of different size it is suggested that a librational motion on the nanosecond time scale may constitute a common spin-lattice relaxation pathway for phosphates in macromolecules. The consequences of phosphate motion in terms of recent suggestions concerning the environment and the catalytic role of the coenzyme phosphate are discussed.

Allosteric Regulation↗
PubMedDate not supplied

High phosphate requirement for oxidative phosphorylation and low affinity for phosphate transport in newborn rat liver mitochondria.

Rat liver mitochondria are not fully functional at birth. The relationship between this deficiency and the affinity for phosphate, in oxidative phosphorylation or in phosphate transport, have been studied. The phosphate concentration necessary to observe maximal rate of succinate oxidation in the presence of ADP was higher for newborn than for adult rat liver mitochondria. After preincubation of newborn rat liver mitochondria with ATP, the rate of succinate oxidation in the presence of ADP increased with phosphate concentration similarly for newborn and adult rat liver mitochondria. The maximal rate of phosphate-acetate exchange, which is an indirect measure of the rate of phosphate transport across the mitochondrial membrane, was not significantly different for adult and newborn rat liver mitochondria. On the contrary the apparent affinity for phosphate was about ten-fold lower for newborn than for adult mitochondria.

Adenosine Diphosphate↗
PubMed1983

Modification of pyruvate, phosphate dikinase with pyridoxal 5'-phosphate: evidence for a catalytically critical lysine residue.

Pyruvate, phosphate dikinase from Bacteroides symbiosus is strongly inhibited by low concentrations of pyridoxal 5'-phosphate. The inactivation follows pseudo-first-order kinetics over an inhibitor concentration range of 0.1-2 mM. The inactivation is highly specific since pyridoxine and pyridoxamine 5'-phosphate, analogues of pyridoxal 5'-phosphate, which lack an aldehyde group, caused little or no inhibition even at high concentrations. The unreduced dikinase-pyridoxal 5'-phosphate complex displays an absorption maxima near 420 nm, typical for Schiff base formation. Following reduction of the Schiff base with sodium borohydride, N6-pyridoxyllysine was identified in the acid hydrolysate. When the enzyme was incubated in the presence of pyridoxal 5'-phosphate and reducing agent, the ATP/AMP, Pi/PPi, and pyruvate/phosphoenolpyruvate isotopic exchange reactions were inhibited to approximately the same extent, suggesting that the modification of the lysyl moiety causes changes in the enzyme that affect the reactivity of the pivotal histidyl residue. Phosphorylation of the histidyl group appears to prevent the inhibitor from attacking the lysine residue. On the other hand, addition of pyridoxal 5'-phosphate to the pyrophosphorylated enzyme promotes release of the pyrophosphate and yields the free enzyme which is subject to inhibition.

Bacteroides↗
PubMed2001

Snapshots of catalysis: the structure of fructose-1,6-(bis)phosphate aldolase covalently bound to the substrate dihydroxyacetone phosphate.

Fructose-1,6-bis(phosphate) aldolase is an essential glycolytic enzyme found in all vertebrates and higher plants that catalyzes the cleavage of fructose 1,6-bis(phosphate) (Fru-1,6-P(2)) to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). Mutations in the aldolase genes in humans cause hemolytic anemia and hereditary fructose intolerance. The structure of the aldolase-DHAP Schiff base has been determined by X-ray crystallography to 2.6 A resolution (R(cryst) = 0.213, R(free) = 0.249) by trapping the catalytic intermediate with NaBH(4) in the presence of Fru-1,6-P(2). This is the first structure of a trapped covalent intermediate for this essential glycolytic enzyme. The structure allows the elucidation of a comprehensive catalytic mechanism and identification of a conserved chemical motif in Schiff-base aldolases. The position of the bound DHAP relative to Asp33 is consistent with a role for Asp33 in deprotonation of the C4-hydroxyl leading to C-C bond cleavage. The methyl side chain of Ala31 is positioned directly opposite the C3-hydroxyl, sterically favoring the S-configuration of the substrate at this carbon. The "trigger" residue Arg303, which binds the substrate C6-phosphate group, is a ligand to the phosphate group of DHAP. The observed movement of the ligand between substrate and product phosphates may provide a structural link between the substrate cleavage and the conformational change in the C-terminus associated with product release. The position of Glu187 in relation to the DHAP Schiff base is consistent with a role for the residue in protonation of the hydroxyl group of the carbinolamine in the dehydration step, catalyzing Schiff-base formation. The overlay of the aldolase-DHAP structure with that of the covalent enzyme-dihydroxyacetone structure of the mechanistically similar transaldolase and KDPG aldolase allows the identification of a conserved Lys-Glu dyad involved in Schiff-base formation and breakdown. The overlay highlights the fact that Lys146 in aldolase is replaced in transaldolase with Asn35. The substitution in transaldolase stabilizes the enamine intermediate required for the attack of the second aldose substrate, changing the chemistry from aldolase to transaldolase.

Animals↗
PubMed1996

Role of conserved residues within the carboxy phosphate domain of carbamoyl phosphate synthetase.

Carbamoyl phosphate synthetase (CPS) catalyzes the formation of carbamoyl phosphate from glutamine, bicarbonate, and 2 mol of MgATP. The heterodimeric protein is composed of a small amidotransferase subunit and a larger synthetase subunit. The synthetase subunit contains a large tandem repeat for each of the nucleotides used in the overall synthesis of carbamoyl phosphate. A working model for the three-dimensional fold of the carboxy phosphate domain of CPS was constructed on the basis of amino acid sequence alignments and the X-ray crystal structure coordinates for biotin carboxylase and D-alanine:D-alanine ligase. This model was used to select ten residues within the carboxy phosphate domain of CPS for modification and subsequent characterization of the kinetic constants for the mutant proteins. Residues R82, R129, R169, D207, E215, N283, and Q285 were changed to alanine residues; residues E299 and R303 to glutamine; and residue N301 to aspartate. No significant changes in the catalytic constants were observed upon mutation of either R82 or D207, and thus these residues appear to be nonessential for binding and/or catalytic activity. The Michaelis constant for ATP was most affected by modification of residues R129, R169, Q285, and N301. The binding of bicarbonate was most affected by the mutagenesis of residues E215, E299, N301, and R303. The mutation of residues E215, N283, E299, N301, and R303 resulted in proteins which were unable to synthesize carbamoyl phosphate at a significant rate. All of the mutations, with the exception of the N301D mutant, primarily affected the enzyme by altering the step for the phosphorylation of bicarbonate. However, mutation of N301 to aspartic acid also disrupted the catalytic step involved in the phosphorylation of carbamate. These results are consistent with a role for the N-terminal half of the synthetase subunit of CPS that is primarily directed at the initial phosphorylation of bicarbonate by the first ATP utilized in the overall synthesis of carbamoyl phosphate. The active site structure appears to be very similar to the ones previously determined for D-alanine:D-alanine ligase and biotin carboxylase.

Acetyl-CoA Carboxylase↗
PubMed2001

Phosphate uptake and release by Acinetobacter johnsonii in continuous culture and coupling of phosphate release to heavy metal accumulation.

A strain of polyphosphate-synthesizing, phosphate-releasing Acinetobacter johnsonii was isolated from a wastewater treatment plant operating enhanced biological phosphate removal (EBPR) and was used to remove La(3+) from solution via precipitation of cell-bound LaPO(4). The effect of repeated aerobic-anaerobic cycles on the carbon and phosphate metabolism of the organism was studied in attempts to promote increased phosphate flux using a three-stage, continuous bioreactor comprising aerobic, anaerobic and settling vessels. The bioreactor was operated in two modes: In flow-through mode, cells were grown aerobically with acetate as the sole carbon source, promoting excess phosphate uptake (up to 5.0 mmol/l=3.0 mmol/g protein). Cells were diluted into the anaerobic vessel where phosphate was released (up to 1.0 mmol/l=0.3 mmol/g protein), and thence to waste. The system was initially operated to steady state in flow-through mode, then switched to recycle mode. Here the anaerobic vessel output passed to a settling vessel from which settled cells were returned to the aerobic vessel. Carbon source (acetate) was supplied only to the anaerobic vessel; increased anaerobic acetate uptake was observed during recycle, which was sustained when the system was returned to flow-through mode and was related to increased cellular lipid inclusions by flow cytometry and electron microscopy. These phenomena may represent adaptation of cells to aerobic-anaerobic cycling with aerobic carbon/energy limitation. Addition of La(3+) to the anaerobic vessel during recycle mode promoted removal of 95% of the La(3+) from a 0.1 to 0.3 mM (14-42 ppm) solution at the expense of biogenic phosphate.

Acetates↗
PubMed1973

The deoxyfluoro-D-glucopyranose 6-phosphates and their effect on yeast glucose phosphate isomerase.

1. The deoxyfluoro-d-glucopyranose 6-phosphates were prepared from the corresponding deoxyfluoro-d-glucoses and ATP by using hexokinase. 2. 3-Deoxy-3-fluoro- and 4-deoxy-4-fluoro-d-glucose 6-phosphate were substrates for glucose phosphate isomerase, and in addition the products of this reaction, 3-deoxy-3-fluoro- and 4-deoxy-4-fluoro-d-fructose 6-phosphate respectively, were good substrates for phosphofructokinase. 3. Some C-2-substituted derivatives of d-glucose 6-phosphate were found to be competitive inhibitors of glucose phosphate isomerase. 4. The possible role of the hydroxyl groups in the binding of d-glucose 6-phopshate to glucose phosphate isomerase is discussed.

Computers↗