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Complexes of vitamin B6XX: equilibrium and mechanistic studies of the reaction of pyridoxal-5'-phosphate with pyridoxamine-5'-phosphate in the presence of copper(II).

The interaction of Cu(II) with pyridoxamine-5'-phosphate (PMP) and pyridoxal-5'-phosphate (PLP) was studied potentiometrically. The titration data were assessed by MINIQUAD program. Several protonated and nonprotonated complexes have been found to exist in solution. The reaction of PLP with Cu(II)-PMP has been studied kinetically, using the stopped-flow technique. Two rate steps have been observed. The first step has been attributed to the formation of a Schiff's base metal complex. The second step may be due to the formation of a ternary complex formation. A mechanism was suggested.

Chemical Phenomena↗

Pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate concentrations in blood and tissues of mice fed ethanol-containing liquid diets.

The effects of chronic ethanol administration on vitamin B-6 metabolism were studied in female Long-Sleep (LS) and Short-Sleep (SS) mice. Animals were fed an ethanol containing liquid diet (AIN-76) for four weeks. Concentration of ethanol in the diet increased from 10 to 25% ethanol-derived calories (EDC) during weeks 1-3 and was maintained at 30% EDC for 1 additional week. We measured concentrations of pyridoxal 5'-phosphate (PLP) in plasma, erythrocytes and whole blood, and liver and brain PLP and pyridoxamine 5'-phosphate (PMP) in ethanol-fed and pair-fed control mice. Chronic ethanol administration significantly increased PMP and total (PLP + PMP) levels in the liver of SS mice. In LS mice ethanol feeding significantly decreased PMP and total (PLP + PMP) levels in the brain, but these values were still within normal limits. These results suggest that both control and ethanol-containing liquid diets are nutritionally adequate with respect to vitamin B-6, and that chronic ethanol administration does not adversely affect vitamin B-6 metabolism in adult mice.

Animals↗

Sphingosine-1-phosphate lyase SPL is an endoplasmic reticulum-resident, integral membrane protein with the pyridoxal 5'-phosphate binding domain exposed to the cytosol.

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that functions as a bioactive lipid molecule. S1P is degraded either by S1P lyase or by S1P phosphohydrolase. The gene encoding mammalian S1P lyase, SPL, has been identified. Here, we characterize the SPL protein in its expression, localization, and topology. The expression levels of the SPL protein correlated well with the dihydrosphingosine-1-phosphate (DHS1P) lyase activity in most tissues. However, liver and heart exhibited high DHS1P lyase activities compared to their SPL protein levels. The SPL mRNA expression was temporally regulated during mouse embryonal development. Immunofluorescence microscopy demonstrated that SPL is localized at the endoplasmic reticulum. Proteinase K digestion studies revealed that the large hydrophilic domain, containing the active site, faces the cytosol. This active site orientation is opposite to that of S1P phosphohydrolase, indicating that the degradation of S1P by two S1P-degrading enzymes occurs in spatially separated sides of the endoplasmic reticulum.

Aldehyde-Lyases↗

Calcium phosphate and fluorinated calcium phosphate coatings on titanium deposited by Nd:YAG laser at a high fluence.

Calcium phosphate coatings are known to enhance long-term fixation, reliability and promote osteointegration of cementless titanium-based implant devices. This study was aimed at the pulsed laser deposition of calcium phosphate coatings onto titanium using hydroxyapatite and hydroxyapatite-fluorapatite targets. The deposition was carried out at the high laser beam fluence conditions, about 12 J/cm(2). The coatings were characterized with respect to their morphology, phase composition and hardness. X-ray energy dispersive analysis revealed the coatings retain their elemental composition, and fluoride content within the film is the same as in the initial target. However, unlike sintered targets, the deposited films contain no apatite-like phases. The hardness of the films, about 18 GPa, is surprisingly high compared to that of hydroxyapatite and hydroxyapatite-fluorapatite ceramic targets. The deposited coatings of 2.7-2.9 microm thickness have uniform and dense microstructure, containing the solidified droplets of the expulsed from the target phase. The uncommon structure and hardness of the films can be attributed to the melting and phase decomposition of the initial material in the laser plasma.

Calcium Phosphates↗

Stereospecific deuteration of 2-deoxyerythrose 4-phosphate using 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.

Racemic 2-deoxyerythrose 4-phosphate was synthesized and one enantiomer of this compound was found to be a substrate for Escherichia coli 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway. When the reaction was carried out in deuterium oxide, an enzyme-catalyzed regio- and stereoselective incorporation of deuterium into the product was observed.

3-Deoxy-7-Phosphoheptulonate Synthase↗

Characterization and comparative analysis of Arabidopsis phosphatidylinositol phosphate 5-kinase 10 reveals differences in Arabidopsis and human phosphatidylinositol phosphate kinases.

Arabidopsis phosphatidylinositol phosphate (PtdInsP) kinase 10 (AtPIPK10; At4g01190) is shown to be a functional enzyme of the subfamily A, type I AtPtdInsP kinases. It is biochemically distinct from AtPIPK1 (At1g21980), the only other previously characterized AtPtdInsP kinase which is of the B subfamily. AtPIPK10 has the same K(m), but a 10-fold lower V(max) than AtPIPK1 and it is insensitive to phosphatidic acid. AtPIPK10 transcript is most abundant in inflorescence stalks and flowers, whereas AtPIPK1 transcript is present in all tissues. Comparative analysis of recombinant AtPIPK10 and AtPIPK1 with recombinant HsPIPKIalpha reveals that the Arabidopsis enzymes have roughly 200- and 20-fold lower V(max)/K(m), respectively. These data reveal one explanation for the longstanding mystery of the relatively low phosphatidylinositol-(4,5)-bisphosphate:phosphatidylinositol-4-phosphate ratio in terrestrial plants.

Amino Acid Sequence↗

Phosphate release and force generation in cardiac myocytes investigated with caged phosphate and caged calcium.

The phosphate (P(i)) dissociation step of the cross-bridge cycle was investigated in skinned rat ventricular myocytes to examine its role in force generation and Ca(2+) regulation in cardiac muscle. Pulse photolysis of caged P(i) (alpha-carboxyl-2-nitrobenzyl phosphate) produced up to 3 mM P(i) within the filament lattice, resulting in an approximately exponential decline in steady-state tension. The apparent rate constant, k (rho i), increased linearly with total P(i) concentration (initial plus photoreleased), giving an apparent second-order rate constant for P(i) binding of 3100 M(-1) s(-1), which is intermediate in value between fast and slow skeletal muscles. A decrease in the level of Ca(2+) activation to 20% of maximum tension reduced k (rho i) by twofold and increased the relative amplitude by threefold, consistent with modulation of P(i) release by Ca2+. A three-state model, with separate but coupled transitions for force generation and P(i) dissociation, and a Ca(2+)-sensitive forward rate constant for force generation, was compatible with the data. There was no evidence for a slow phase of tension decline observed previously in fast skeletal fibers at low Ca(2+), suggesting differences in cooperative mechanisms in cardiac and skeletal muscle. In separate experiments, tension development was initiated from a relaxed state by photolysis of caged Ca(2+). The apparent rate constant, k(Ca), was accelerated in the presence of high P(i) consistent with close coupling between force generation and P(i) dissociation, even when force development was initiated from a relaxed state. k(Ca) was also dependent on the level of Ca(2+) activation. However, significant quantitative differences between k (rho i) and k(Ca), including different sensitivities to Ca(2+) and P(i) indicate that caged Ca(2+) tension transients are influenced by additional Ca(2+)-dependent but P i-independent steps that occur before P(i) release. Data from both types of measurements suggest that kinetic transitions associated with P(i) dissociation are modulated by the Ca(2+) regulatory system and partially limit the physiological rate of tension development in cardiac muscle.

Animals↗

Carbamate kinase can replace in vivo carbamoyl phosphate synthetase. Implications for the evolution of carbamoyl phosphate biosynthesis.

The exclusive involvement of carbamate kinase (CK) in fermentative ATP production and of carbamoyl phosphate synthetase (CPS) in the production of carbamoyl phosphate (CP) for pyrimidines and arginine biosynthesis was challenged by the finding of CK as the only activity synthesising CP in the archaea Pyrococcus furiosus and Pyrococcus abyssi. We now show that CK can replace CPS in vivo: transformation of Escherichia coli devoid of the CPS gene with plasmids encoding the CK from P. furiosus or from Enterococcus faecalis (which uses CK for making ATP) restores the ability of CPS-deficient E. coli to grow in the absence of arginine and uracil if ammonia and bicarbonate are present.

Ammonia↗

Studies on phosphate-solubilizing bacteria in soil and rhizosphere of different plants. II. Selection of the most efficient phosphate-dissolvers and their morphological grouping.

Two hundred colonies which showed positive reaction on the plates prepared for the phosphate-dissolving bacteria from control soil rhizosphere soils and rhizoplane samples of maize, peas, or cotton were isolated at random. Fifty isolates were selected as the most efficient isolates according to their capability for increasing the amounts of available phosphorus in the media with corresponding decreases in pH values. The percentage of the most efficient isolates differed according to type of plant and location of isolation. Not only the morphological types of the phosphate-dissolving bacteria differed in soil and in rhizosphere, but they also differed in the rhizosphere soil of each special plant. Morphological differences in the isolates from rhizosphere soil and from rhizoplane samples of the same plant were also occurring. The abundance of mycelial-forming bacteria and of aerobic sporeformers in Egyptian soil is important as they are well known to resist adverse conditions, such as high temperature and dryness to which our soils are subjected most time of the year.

Bacteria↗

Alpha-tricalcium phosphate hydrolysis to octacalcium phosphate: effect of sodium polyacrylate.

Alpha-Tricalcium phosphate (alpha-TCP) hydrolysis into octacalcium phosphate (OCP) has been investigated in phosphoric acid solution at different concentrations of sodium polyacrylate (NaPA). The hydrolysis process has been followed by powder X-ray diffraction, infrared absorption and scanning electron microscopy analyses. In the absence of the polyelectrolyte, alpha-TCP undergoes a complete transformation into OCP in 24 h. The presence of polyacrylate in solution inhibits the hydrolysis so that a NaPA concentration of 0.5 microm is sufficient to lengthen the time required to complete the hydrolysis to 4 days. The variation of Ca2+ concentration in the soaking solution suggests that the transformation occurs through alpha-TCP dissolution followed by OCP precipitation. The delayed OCP nucleation and growth in the presence of polyacrylate implies a preferential adsorption of the polyelectrolyte on the growing OCP crystals, which induces an anisotropic reduction of the coherence lengths of the perfect crystalline domains.

Acrylic Resins↗

Biomimetic calcium phosphate coatings on Ti6AI4V: a crystal growth study of octacalcium phosphate and inhibition by Mg2+ and HCO3-.

The biomimetic approach for coating metal implants allows the deposition of new calcium phosphate (Ca-P) phases. Films elaborated at physiological conditions might have structures closer to bone mineral than hydroxylapatite (HA) plasma-sprayed coatings. In this study, different Ca-P coatings have been deposited through a two-step procedure. After cleaning and etching, Ti6Al4V plates were pretreated by soaking in a simulated body fluid (SBF), i.e., a solution containing inorganic components in concentration more or less similar to body fluids: a thin amorphous carbonated Ca-P layer precipitated on the metal substrate. Second, by soaking these thinly coated metal substrates in another SBF, with different concentrations, the thin amorphous carbonated Ca-P layer led to the fast precipitation of a second and thick Ca-P layer. Different SBF solutions were used in order to investigate the influence of magnesium and carbonate ions. From SBF containing only Ca2+ and HPO4(2-) ions, an octacalcium phosphate layer grew epitaxially on the substrate. When Mg2+ was added into this SBF, the coating was composed of Ca-deficient apatite crystals, while the addition of HCO3- in SBF led to the formation of a B-carbonated apatite layer. Magnesium and carbonate acted as inhibitors of crystal growth. The three phases obtained by our biomimetic process are closer to bone mineral structure than plasma-sprayed HA. Therefore, the obtained results may be particularly relevant for the development of biomimetic Ca-P coatings with optimal bioactivity.

Alloys↗

Phosphate absorption and efflux of three ectomycorrhizal fungi as affected by external phosphate, cation and carbohydrate concentrations.

A prerequisite for symbiotic phosphate transfer in an ectomycorrhizal (ECM) association is hypothesized to be conditions in the interface between both symbiotic partners, that either promote the release of inorganic phosphate (P) from the Hartig net into the interfacial apoplast and/or decrease the fungal reabsorption from this location. To get more information about conditions, which might be involved in the regulation of P efflux or P reabsorption, the effect of various external conditions on 33P-orthophosphate (33P) uptake or efflux by axenic cultures of the ECM basidiomycetes Hebeloma crustliniforme, Amanita muscaria and Laccaria laccata was analysed. In short-time experiments the following external conditions were analysed: an external supply of (1) P in the preculture, (2) cations (0.1-100 mM K, 0.1-50 mM Na, Mg and Ca), and (3) carbohydrates (0.5-50 mM glucose, fructose or sucrose). The P absorption was generally reduced in cultures previously supplied with an abundant P supply and with increased P concentrations in their tissues. The P uptake was also affected by an external supply of cations, whereas carbohydrates had only a slight effect. Compared to Na, Mg and Ca, the P absorption by H. crustuliniforme and L. laccata was increased by 0.1 mM K in the labelling solution but decreased after a supply of 100 mM K and then did not differ from the other cation treatments. Compared to other cations, an addition of 50 mM Ca led to a decrease of P absorption by A. muscaria, whereas 50 mM Mg increased the P uptake by H. crustuliniforme. The P efflux from the fungi was affected by both the cation and carbohydrate concentration of the bathing solution. High concentrations of the monovalent cations K and Na (5 mM or 50 mM) in the bathing solution increased the P efflux by H. crustuliniforme (only Na) and L. laccata (K and Na), but had little effects on A. muscaria. By contrast, the same concentrations of the divalent cation Mg reduced the P efflux from all fungal basidiomycetes. The P efflux from hyphae of H. crustuliniforme and A. muscaria was also affected by the carbohydrate concentration in the bathing solution. Compared to the hexoses, glucose and fructose, the P efflux by both basidiomycetes was stimulated even by low concentrations of sucrose (0.5 mM) in the bathing solution. The effect of carbohydrates on P efflux especially from hyphae of H. crustuliniforme was clearly concentration-dependent and the maximal efflux rates were observed after a supply of 2.5 mM sucrose or fructose or 5 mM glucose in the bathing solution. A further rise of the carbohydrate concentration led to a progressive decrease of P efflux from hyphae of H. crustuliniforme. The results are discussed with respect to the transfer processes between both symbiotic partners in a mycorrhizal association and a possible regulation of these exchange processes.

Basidiomycota↗

Novel polyamine-dialkyl phosphate conjugates for gene carriers. Facile synthetic route via an unprecedented dialkyl phosphate.

To develop a novel nonviral gene carrier, three types of polyamine-dialkyl phosphates conjugates were synthesized via an unprecedented synthetic intermediate, dimerized dicetyl phosphate (DCP) anhydride, and the transfection efficiency and the complexation properties of the conjugate-DNA were evaluated. Condensation of DCP by 1,3,5-triisopropylbenzenesulfonyl chloride, TPSCl, gives the dimerized anhydride, which is stable enough to isolate by column chromatography in approximately 90% yield. The anhydride is reactive with various amines, i.e., spermidine, spermine, and polyethylenimine (PEI(1800)), providing corresponding polyamine-DCP conjugates via phosphoramidate linkage. The polyamine-DCP conjugates exhibited moderate transfection efficacy evaluated by beta-galactosidase assay. The conjugate-DNA complex was observed by using an atomic force microscope (AFM), revealing that the PEI(1800)-DCP conjugate, which showed the most efficient transfection, enables the formation of the more compact complex with DNA.

Animals↗

Bound adenosine 5'-triphosphate formation, bound adenosine 5'-diphosphate and inorganic phosphate retention, and inorganic phosphate oxygen exchange by chloroplast adenosinetriphosphatase in the presence of Ca2+ or Mg2+.

When the heat-activated chloroplast F1 ATPase hydrolyzes [3H, gamma-32P]ATP, followed by the removal of medium ATP, ADP, and Pi, the enzyme has labeled ATP, ADP, and Pi bound to it in about equal amounts. The total of the bound [3H]ADP and [3H]ATP approaches 1 mol/mol of enzyme. Over a 30-min period, most of the bound [32P]Pi falls off, and the bound [3H]ATP is converted to bound [3H]ADP. Enzyme with such remaining tightly bound ADP will form bound ATP from relatively high concentrations of medium Pi with either Mg2+ or Ca2+ present. The tightly bound ADP is thus at a site that retains a catalytic capacity for slow single-site ATP hydrolysis (or synthesis) and is likely the site that participates in cooperative rapid net ATP hydrolysis. During hydrolysis of 50 microM [3H]ATP in the presence of either Mg2+ or Ca2+, the enzyme has a steady-state level of about one bound [3H]ADP per mole of enzyme. Because bound [3H]ATP is also present, the [3H]ADP is regarded as being present on two cooperating catalytic sites. The formation and levels of bound ATP, ADP, and Pi show that reversal of bound ATP hydrolysis can occur with either Ca2+ or Mg2+ present. They do not reveal why no phosphate oxygen exchange accompanies cleavage of low ATP concentrations with Ca2+ in contrast to Mg2+ with the heat-activated enzyme. Phosphate oxygen exchange does occur with either Mg2+ or Ca2+ present when low ATP concentrations are hydrolyzed with the octyl glucoside activated ATPase. Ligand binding properties of Ca2+ at the catalytic site rather than lack of reversible cleavage of bound ATP may underlie lack of oxygen exchange under some conditions.

Adenosine Diphosphate↗

Substrate deactivation of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase by erythrose 4-phosphate.

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS, EC 4.1.2.15) catalyzes the condensation of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to give DAH7P via an ordered sequential mechanism. In the absence of PEP (the first substrate to bind), E4P binds covalently to the phenylalanine-sensitive DAH7PS of Escherichia coli, DAH7PS(Phe), deactivating the enzyme. Activity is restored on addition of excess PEP but not if deactivation was carried out in the presence of sodium cyanoborohydride. Electrospray mass spectrometry indicates that a single E4P is bound to the protein. These data are consistent with a slow, reversible Schiff base reaction of the aldehydic functionality of E4P with a buried lysine. Molecular modeling indicates that Lys186, a residue at the base of the substrate-binding cavity involved in hydrogen bonding with PEP, is well placed to react with E4P forming an imine linkage that is substantially protected from solvent water.

3-Deoxy-7-Phosphoheptulonate Synthase↗

Enantiodivergence in small-molecule catalysis of asymmetric phosphorylation: concise total syntheses of the enantiomeric D-myo-inositol-1-phosphate and D-myo-inositol-3-phosphate.

Peptide-based catalysts have been found that catalyze the enantiodivergent phosphorylation of a meso myo-inositol-derived triol (1). The sequential screening of random peptide libraries, followed by the evaluation of a focused library, led to the identification of two peptides (2 and 24) that are complementary in producing enantiomeric D-myo-inositol-1-phosphate and D-myo-inositol-3-phosphate derivatives. The catalysts were then used to complete efficient total syntheses of both D-I-1P and D-I-3P in optically pure form. Additional information is gleaned from relative rate experiments that unambiguously show the catalysts to afford enantioselection through rate accelerative pathways with respect to simple achiral alkylimidazole catalysts. Furthermore, solvent effect studies show that the two enantiodivergent catalysts exhibit different tolerances of polar media. The systematic discovery of site-selective catalysts establishes a basis for future studies of chiral catalysts that differentiate unique functional groups in polyfunctional molecules.

Catalysis↗

Pyridoxal phosphate. 5. 2-Formylethynylphosphonic acid and 2-formylethylphosphonic acid, potent inhibitors of pyridoxal phosphate binding and probes of enzyme topography.

The title compounds (17 and 21) were prepared in good yield by synthesis of the phosphonate diester acetals (14 and 19), deesterification with chloro- or bromotrimethylsilane, hydrolysis of the acetal group, and formation of the characterized barium salts. The 3-carbon aldehydrophosphonic acids (17 and 21) were potent inhibitors (Ki = 2 X 10(-6)) of pyridoxal phosphate (PPal) binding to apoaspartate aminotransferase (AAT) and are believed to span between and bind to the enzymic functional groups which bind the aldehyde and phosphate groups of PPal.

Apoenzymes↗

Novel phosphate anthelmintics. 3. Alkyl and aryl 1-methyleneallyl phosphates, phosphonates, and phosphinates.

A series of new highly chlorinated 1-methyleneallyl ("butadienyl") dialkyl phosphates and related phosphonates and phosphinates has been synthesized and assessed for anthelmintic activity in mice against the tapeworm Hymenolepis nana and the pinworm Syphacia obvelata. Highest activity was observed with diethyl 2,3,3-trichloro-1-dichloromethyleneallyl ("perchlorobutadienyl") phosphate (14), while replacement of both ethoxy groups by methoxy and larger alkoxy or phenyl groups gave less efficacious compounds. In general, chlorine depletion of the 1,3-butadien-2-yl moiety or saturation of one double bond reduced anthelmintic responses.

Animals↗