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PubMed1978

An improved quantitative assay of galactose-1-phosphate uridyltransferase activity in erythrocytes based on the determination of glucose 1-phosphate generation.

An improved quantitative method for measuring galactose-1-phosphate uridyltransferase (EC 2.7.7.12, Gal-PUT) activity in erythrocytes was developed based on the detection of glucose 1-phosphate generated under the catalytic influence of the enzyme. This is achieved by incubating the enzyme with galactose 1-phosphate and uridyldiphosphoglucose during 15 min, followed by deproteinisation. The glucose 1-phosphate generated is quantitated subsequently by measuring NADPH formation from added NADP+ in a second incubation step with added phosphoglucomutase (EC 2.7.5.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Gal-PUT activity is calculated from the increment in absorption at 340 nm. Because it is technically a simple assay that is sensitive, specific and not affected by UDPgalactose-4-epimerase (EC 5.1.3.2) activity in the erythrocytal lysates it is suggested to be the method of choice for measuring Gal-PUT activity. Activities in erythrocytes of controls varied from 264 to 556 U/kg hemoglobin; in obligate heterozygotes from 53 to 190 U/kg Hb and in homozygous deficient patients less than 5 U/kg Hb was measured at 37 degrees C.

Erythrocytes↗
PubMed1992

Interaction of mannose-6-phosphate with the hysteretic transition in glucose-6-phosphate hydrolysis in intact liver microsomes.

We showed previously that glucose-6-phosphatase activity was characterised in intact liver microsomes by a hysteretic transition between a rapid and a slower catalytic form of the enzyme. We have now further investigated the substrate specificity of these two kinetic forms. It was found that the pre-incubation of intact microsomes with mannose-6-phosphate or glucose-6-phosphate (50 microM for 30 s) suppressed the burst in glucose-6-phosphatase activity, that the hysteretic transition was reversible and that mannose-6-phosphate inhibited glucose-6-phosphate hydrolysis during the first seconds of incubation, but not anymore after the burst. Our results indicate (i) that mannose-6-phosphate is recognised by the enzyme and can promote the hysteretic transition and (ii) that the transient phase is part of the catalytic mechanism itself.

Animals↗
PubMed1995

Identification of galactitol 2-phosphate and galactitol 3-phosphate in the lens of galactose-fed rats.

Production of unusual phosphorylated metabolites in the lens is one of several changes caused by hyperglycemia. Sorbitol 3-phosphate (Sor-3P) and fructose 3-phosphate (Fru-3P) are two such compounds identified in the diabetic lens, and galactitol 2-phosphate (Gal-2P) and galactitol 3-phosphate (Gal-3P) are identified here in the galactosemic lens. These new compounds are the first example of galactitol metabolism in mammalian tissue other than liver. Sor-3P and Fru-3P are also present in the galactosemic lens, apparently synthesized directly from their precursors, sorbitol and fructose, which are elevated in the lens due to increased flux of glucose through the aldose reductase (AR) pathway. The NADPH necessary to support this increased flux is derived from activation of the hexose monophosphate shunt (HMPS), which is clearly demonstrated by a large increase in the concentration of sedoheptulose 7-phosphate (Sed-7P), a HMPS-specific metabolite. Additionally, during 3 weeks of galactose feeding, there is a dramatic increase in lenticular concentrations of galactitol, sorbitol, galactose, and fructose and a sharp decrease in inositol. Glucose remains unchanged. A precipitous loss of both phosphorylated and nonphosphorylated metabolites occurs after 3 weeks, possibly due to lens rupture.

Animals↗
PubMed1985

Determination of 18O/16O ratio in inorganic phosphate as an oncogenic marker and indicator for disturbances in phosphate metabolism.

Natural stable isotope fractionation is a potential tool in investigation of metabolic process, since rigid mass balance considerations rule the changes in the isotopic ratio. As the natural changes in 13C/12C ratio of total CO2 between blood and urine served for studying renal bicarbonate reabsorption, studying changes in 18O/16O ratio of phosphate are suggested to investigate deranged phosphate metabolism. The 18O/16O ratio in serum phosphate is constant, determined by the ratio in the environmental drinking water. Therefore, measurements of this ratio in normal individuals, after modifications in phosphate metabolism and in diseases with high alkaline phosphatase activity are proposed. The main purpose of the proposed study is to assess whether measurements of 18O/16O ratio can detect malignant metastases in bones due to deranged phosphate metabolism. An assumption that these determinations might precede other tests for detecting bone metastases and can serve as an oncogenic marker is made.

Bone Neoplasms↗
PubMed2004

Kinetic measurements of phosphoglucomutase by direct analysis of glucose-1-phosphate and glucose-6-phosphate using ion/molecule reactions and Fourier transform ion cyclotron resonance mass spectrometry.

A method for the direct determination of kinetic constants for phosphoglucomutase and its phosphorylated products is described. Fourier transform ion cyclotron resonance gas-phase ion/molecule reactions between trimethyl borate and glucose phosphate, phosphorylated at either the 1 or the 6 position, generate mass spectra distinguishable with regard to product ion distribution. A multicomponent quantification method is utilized to determine the composition of a binary mixture of the two positional isomers. Using this method, the conversion between glucose-1-phosphate and glucose-6-phosphate can be directly monitored without the use of coupling enzymes. The values of K(m) for glucose-1-phosphate and glucose-6-phosphate were determined using the substrate-velocity plot and the Haldane relationship, respectively. Values of V(max) for both the forward and the reverse directions were measured, and the equilibrium constant for the reversible reaction was determined using this methodology. Kinetic parameters measured correlate well with those obtained using traditional methods. The assay was demonstrated to be accurate and particularly convenient to determine kinetic constants for enzymatic systems that involve the interconversion of phosphorylated positional isomers.

Chemistry Techniques, Analytical↗
PubMed2006

Structure of dihydroxyacetone phosphate dimethyl acetal, a stable dihydroxyacetone phosphate precursor, in the crystalline state.

Crystal and molecular structures of four different salts of a dihydroxyacetone phosphate (DHAP) precursor, its dimethyl acetal [2,2-dimethoxy-1,3-propanediol phosphate, C(5)H(13)O(7)P, (MeO)(2)DHAP]: (cha)(2)[(MeO)(2)DHAP].H(2)O (6a), (cha)[(MeO)(2)DHAP] (6b), Na(2)[(MeO)(2)DHAP].5.75H(2)O (6c) and K(2)[(MeO)(2)DHAP].H(2)O (6d), along with the cyclohexylammonium (cha) salt of its phenyl ester (cha)[(MeO)(2)DHAP(Ph)] (6e) are described. In the (MeO)(2)DHAP mono- and dianions, slightly different orientation of the phosphate group in relation to the acetal carbon atom is observed, with a delicate tendency of phosphate group to be located antiperiplanar in the monoanions and anticlinal in the dianions. The 2,2-dimethoxy-1,3-propandiol moiety, (MeO)(2)DHA, seems to be very rigid and its conformation is independent of phosphorylation, the ionization state of the inserted phosphate group and its additional substitution. The overall structures of the cyclohexylammonium (6a,b) and potassium salts (6d) have a double-layered architecture, while the sodium cation network in 6c forms the system of channels, which are filled up with the [(MeO)(2)DHAP](2-) ions. The different architectures of 6c and 6d crystals result from the different ways in which the relevant dianions coordinate to sodium and potassium ions and affect also the hydrogen bonding system observed in 6c and 6d crystals.

Acetals↗
PubMed2004

Inorganic polyphosphate in mitochondria of Saccharomyces cerevisiae at phosphate limitation and phosphate excess.

Isolated mitochondria of Saccharomyces cerevisiae cells grown on glucose possess acid-soluble inorganic polyphosphate (polyP). Its level strongly depends on phosphate (P(i)) concentration in the culture medium. The polyP level in mitochondria showed 11-fold decrease under 0.8 mM P(i) as compared with 19.3 mM P(i). When spheroplasts isolated from P(i)-starved cells were incubated in the P(i)-complete medium, they accumulated polyP and exhibited a phosphate overplus effect. Under phosphate overplus the polyP level in mitochondria was two times higher than in the complete medium without preliminary P(i) starvation. The average chain length of polyP in mitochondria was of <15 phosphate residues at 19.3 mM P(i) in the culture medium and increased at phosphate overplus. Deoxyglucose inhibited polyP accumulation in spheroplasts, but had no effect on polyP accumulation in mitochondria. Uncouplers (FCCP, dinitrophenol) and ionophores (monensin, nigericin) inhibited polyP accumulation in mitochondria more efficiently than in spheroplasts. Fast hydrolysis of polyP was observed after sonication of isolated mitochondria. Probably, the accumulation of polyP in mitochondria depended on the proton-motive force of their membranes.

Culture Media↗
PubMed2001

Di-tert-butyl diethylphosphoramidite as the phosphitylating reagent in the preparation of 3-deoxy-3-C-methylene-D-ribo-hexose-6-phosphate and 3-deoxy-3-C-methylene-D-erythro-pentose-5-phosphate.

3-Deoxy-3-C-methylene-D-ribo-hexose-6-phosphate and 3-deoxy-3-C-methylene-D-erythro-pentose-5-phosphate were prepared from a common intermediate 3-deoxy-3-C-methylene-1,2-O-isopropylidene-alpha-D-ribo-hexofuranose. The preparation of the phosphorylated unsaturated sugars employed di-tert-butyl diethylphosphoramidite as the phosphitylating reagent. The removal of all the protecting groups was done under acidic conditions in the ultimate step. The unsaturated sugar phosphates were competitive inhibitors but neither substrates nor inactivators of glucose-6-phosphate and ribose-5-phosphate isomerases.

Aldose-Ketose Isomerases↗
PubMed1999

Vitamin B6 biosynthesis: formation of pyridoxine 5'-phosphate from 4-(phosphohydroxy)-L-threonine and 1-deoxy-D-xylulose-5-phosphate by PdxA and PdxJ protein.

In Escherichia coli the coenzyme pyridoxal 5'-phosphate (PLP) is synthesised de novo by a pathway that is thought to involve the condensation of 4-(phosphohydroxy)-L-threonine and 1-deoxy-D-xylulose, catalysed by the enzymes PdxA and PdxJ, to form either pyridoxine (vitamin B6) or pyridoxine 5'-phosphate (PNP). Here we show that incubation of PdxJ with PdxA, 4-(phosphohydroxy)-L-threonine, NAD and 1-deoxy-D-xylulose-5-phosphate, but not 1-deoxy-D-xylulose, results in the formation of PNP. The PNP formed was characterised by (i) cochromatography with an authentic standard, (ii) conversion to pyridoxine by alkaline phosphatase treatment, and (iii) UV and fluorescence spectroscopy. Furthermore, when [2-(14)C]1-deoxy-D-xylulose-5-phosphate was used as a substrate, the radioactivity was incorporated into PNP. These results clarify the previously unknown role of PdxJ in the de novo PLP biosynthetic pathway. The sugar used as substrate by PdxJ is 1-deoxy-D-xylulose-5-phosphate rather than the previously assumed 1-deoxy-D-xylulose. The first vitamin B6 vitamer synthesised is PNP, and not pyridoxine.

Alkaline Phosphatase↗
PubMed2001

Determination of high-energy phosphate compounds and inorganic phosphate by reversed-phase high-performance liquid chromatography: evaluation of myocardial metabolic status in aerobically perfused and hypoxic mouse heart.

The present paper describes a simple HPLC method designed for measuring high-energy phosphate (HEP) compounds in a single run and inorganic phosphate (Pi) in an other short run under the same HPLC conditions. Inorganic phosphate was estimated by using thymidine phosphorylase (EC 2.4.2.4) which catalyzes a reaction involving inorganic phosphate to produce 2-deoxyribose 1-phosphate and thymine. The thymine/Pi stoichiometry was 1. The method provides a reproducible instrument for evaluating myocardial high-energy metabolism under physiological and pathological conditions.

Animals↗
PubMed1996

Comparison of the functional differences for the homologous residues within the carboxy phosphate and carbamate domains of carbamoyl phosphate synthetase.

Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from two molecules of MgATP, bicarbonate, and glutamine. It has been previously shown that the amino- and carboxy-terminal halves of the large subunit of this protein are homologous. A working model for the active site structure of the carboxy-terminal domain of the large subunit of CPS was constructed based upon amino acid sequence alignments and the previously determined three-dimensional structures of two mechanistically related proteins, biotin carboxylase and D-alanine:D-alanine ligase. The model was tested by mutation of ten amino acid residues predicted to be important for binding and/or catalysis. The mutated residues were as follows: R571, R675, R715, D753, E761, N827, Q829, E841, N843, and R845. The mutant proteins were expressed, purified to homogeneity and the catalytic properties determined for a variety of assay formats. The mutants E761A, E841Q, N843D, and R845Q were diminished in their ability to synthesize carbamoyl phosphate. The R715A, Q829A, and R675A mutants displayed elevated Michaelis constants for MgADP in the partial back reaction. The mutants E761A, N827A, E841Q, N843D, and R845Q showed significant increases in the Michaelis constants for either bicarbonate or carbamoyl phosphate. No significant alterations were noted upon mutation of either R571 or D753 to an alanine residue and thus these amino acids do not appear essential for structure or catalytic activity. These results have been utilized to further support the proposal that the C-terminal half of the large subunit of CPS is primarily responsible for the phosphorylation of the carbamate intermediate during the final formation of carbamoyl phosphate. The measured effects on the catalyic activities displayed by these mutations were found to be comparable to the previously determined effects after mutation of the homologous residues located on the N-terminal half of CPS and also for those residues mutated within D-alanine:D-alanine ligase [Shi, Y., & Walsh, C.T. (1995) Biochemistry 34, 2768-2776].

Adenosine Triphosphate↗
PubMedDate not supplied

Genotoxicity of neurotoxic triaryl phosphates: identification of DNA adducts of the ultimate metabolites, saligenin phosphates.

2-Phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is an electrophilic and neurotoxic metabolite of o-tolyl phosphates. We have investigated the genotoxicity of this saligenin phosphate and the structure of adducts formed by incubation of 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide with nucleosides and DNA. o-Tolyl phosphate was mutagenic in the Ames test (695 revertants/mumol, Salmonella typhimurium TA 100) only with metabolic activation. 2-Phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide, which is a cyclization product similar to those expected from o-tolyl phosphate, was a potent mutagen in bacteria (1452 revertants/mumol, S. typhimurium TA 100) which did not require metabolic activation. Incubation of 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide with guanosine, deoxycytidine, and deoxyadenosine resulted in formation of guanosine, deoxyuridine, and adenine adducts. These were identified as N-2-(o-hydroxybenzyl)guanosine, N-3-(o-hydroxybenzyl)deoxyuridine, N-1-(o-hydroxybenzyl)adenine, and N-3-(o-hydroxybenzyl)adenine by 1H-NMR spectroscopy, thermospray mass spectrometry, and pH-dependent electronic spectrometry. The deoxyuridine adduct is formed by an alkylation at N-3 of deoxycytidine followed by conversion of the adjacent exocyclic imino group to carbonyl (hydrolytic deamination). The formation of N-2-(o-hydroxybenzyl)-deoxyguanosine, N-3-(o-hydroxybenzyl)deoxyuridine, and N-1-(o-hydroxybenzyl)deoxyadenosine was also demonstrated when 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide was incubated with calf thymus DNA. Adducts formed with nucleosides in calf thymus DNA reacted with 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide in vitro were detected by the 32P-postlabeling technique and identified by comparison with synthetic references. DNA adducts are formed by an o-hydroxybenzylation from cyclic phosphoranes derived from o-alkyl-substituted triaryl phosphates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Phosphate binding gels: balancing phosphate adsorption and aluminum toxicity.

The phosphate binding capacity of five commercial aluminum hydroxide phosphate-binding gels and two crystalline forms of aluminum hydroxide was studied by an in vitro procedure which simulated passage through the stomach to the small intestine. The potential for aluminum toxicity was estimated by determining the fraction of the dose which was converted into soluble aluminum species by acid neutralization at pH 3, 37 degrees C. The commercial products varied widely both in phosphate binding capacity and production of soluble aluminum species. The evidence suggests that the ideal phosphate binder will have a surface area small enough to minimize the formation of soluble aluminum species during the gastric residence time but also large enough to adsorb a clinically significant amount of phosphate.

Aluminum↗
PubMed1977

Inter-relationships of carbonate, phosphate, monohydrogen phosphate, calcium, magnesium and sodium in uraemic bone: comparison of dialysed and non-dialysed patients.

1. Chemical and morphological features of uraemic bone disease were studied by comparison of bone composition in 44 patients with uraemia (12 dialysed and 32 non-dialysed) and 36 control subjects. The significant changes included decreased bone mineral carbonate associated with calcium, a concomitant increase in phosphate, and an increase in magnesium. There was also an increase in osteoid and a reduction in the specific gravity of the compact bone. 2. The most marked changes in bone composition were observed in patients with uraemia of more than 1 year's duration, who had been dialysed. Bone mineral sodium concentrations were not significantly altered in any group. 3. The changes in bone mineral composition appeared to be the result of several simultaneous and/or successive mechanisms: (i) loss of fixed base, calcium carbonate; (ii) replacement of carbonate by phosphate; (iii) the addition of immature bone mineral, which contains high concentrations of phosphate and relatively low concentrations of carbonate. 4. These observations are consistent with earlier views of the bone salt as an indefinite calcium/phosphate/carbonate complex. Variations in bone composition may arise from a reciprocal relationship between phosphate and carbonate. The bone mineral analogue that best explains these variations in bone composition is octacalcium phosphate carbonate [Ca4 (PO4)2(HPO4)x(CO3)1-x,zH2O].

Adult↗
PubMed2002

Isoprenoid biosynthesis via the methylerythritol phosphate pathway. Mechanistic investigations of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase.

The 1-deoxyxylulose 5-phosphate reductoisomerase (DXR, EC 1.1.1.267) catalyzes the conversion of 1-deoxy-d-xylulose 5-phosphate (DXP) into 2-C-methyl-d-erythritol 4-phosphate (MEP). This transformation is a two-step process involving a rearrangement of DXP into the putative intermediate 2-C-methyl-d-erythrose 4-phosphate followed by a NADPH-dependent reduction of the latter aldehyde. By using [1-(13)C]DXP as a substrate, the rearrangement of DXP into [5-(13)C]2-C-methyl-d-erythrose 4-phosphate was shown to be NADPH dependent, although it does not involve areduction step. The putative aldehyde intermediate, obtained by chemical synthesis, was converted into MEP by the DXR in the presence of NADPH and into DXP in the presence of NADP(+), indicating the reversibility of the reaction catalyzed by the DXR. This reversibility was confirmed by the conversion of MEP into DXP in the presence of NADP(+). The equilibrium was, however, largely displaced in favour of the formation of MEP. The reduction step required the presence of a divalent cation such as Mg(2+) or Mn(2+).

Aldose-Ketose Isomerases↗
PubMed1997

Phosphatidylinositol-4-phosphate 5-kinase isozymes catalyze the synthesis of 3-phosphate-containing phosphatidylinositol signaling molecules.

Phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) utilize phosphatidylinositols containing D-3-position phosphates as substrates to form phosphatidylinositol 3,4-bisphosphate. In addition, type I PIP5Ks phosphorylate phosphatidylinositol 3, 4-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate, while type II kinases have less activity toward this substrate. Remarkably, these kinases can convert phosphatidylinositol 3-phosphate to phosphatidylinositol 3,4,5-trisphosphate in a concerted reaction. Kinase activities toward the 3-position phosphoinositides are comparable with those seen with phosphatidylinositol 4-phosphate as the substrate. Therefore, the PIP5Ks can synthesize phosphatidylinositol 4,5-bisphosphate and two 3-phosphate-containing polyphosphoinositides. These unexpected activities position the PIP5Ks as potential participants in the generation of all polyphosphoinositide signaling molecules.

Animals↗
PubMed2001

Refixation of phosphates released during bio-P sludge handling as struvite or aluminium phosphate.

Phosphate release and phosphate fixation during sludge treatment of waste activated sludge was investigated with a pilot plant for enhanced biological phosphorus removal, complemented by laboratory investigations of sludge samples from different large enhanced biological phosphorus removal plants. The major part of the eliminated phosphorus in the pilot plant was due to the storage of polyphosphate in the waste activated sludge and was accompanied by an uptake of magnesium and potassium. Stabilising waste activated sludge from the enhanced biological phosphorus removal pilot plant results in a hydrolysis of polyphosphate. As a result of polyphosphate hydrolysis in stabilising systems, phosphate, magnesium and potassium are released, but only potassium remains in solution whereas magnesium and a part of the released phosphate was precipitated as struvite. Another large fraction of the released phosphate was fixed by participation of aluminium.

Aluminum Compounds↗
PubMed1992

Iron absorption from bread in humans: inhibiting effects of cereal fiber, phytate and inositol phosphates with different numbers of phosphate groups.

Iron absorption was measured from five kinds of bread made from various types of flour and fermented in different ways in order to obtain a wide variation in the content of fiber, phytate (inositol hexaphosphate) and its degradation products, inorganic phosphate and inositol phosphates with fewer numbers of phosphate groups (inositol pentaphosphate through monophosphate). Each experiment had 9-10 subjects and, in each subject, iron absorption was measured from control rolls made from low extraction wheat flour and one kind of test roll using two different radioiron tracers: 55Fe and 59Fe. The inhibition of iron absorption was closely related to the content of phytate-phosphorous as determined using the AOAC method, and to the sum of the tri- through hexaphosphate groups as determined using the HPLC method. As an example, prolonged fermentation of whole-rye bread reduced total inositol phosphates to the same amount as in the control rolls and increased fractional iron absorption to the same high level, in spite of a fiber content five times as great. The results strongly suggest that the inhibitory effect of bran on iron absorption is due to its content of phytate and other inositol phosphates present after fermentation, rather than to its content of fiber or other constituents. Thus, effective fermentation will increase the bioavailability of iron in whole-meal bread.

Absorption↗