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PubMed2005

Age-dependent changes of pyridoxal phosphate synthesizing enzymes immunoreactivities and activities in the gerbil hippocampal CA1 region.

In the present study, age-related changes of pyridoxal 5'-phosphate (PLP) synthesizing enzymes, pyridoxal kinase (PLK) and pyridoxine 5'-phosphate oxidase (PNPO), their protein contents and activities were examined in the gerbil hippocampus proper. Significant age-dependent changes in PLK and PNPO immunoreactivities were found in the CA1 region, but not in the CA2/3 region. In the postnatal month 1 (PM 1) group, PLK and PNPO immunoreactivities were detected mainly in the stratum pyramidale of the CA1 region. PLK and PNPO immunoreactivities and their protein contents were highest in the PM 6 group, showing that many CA1 pyramidal cells had strong PLK and PNPO immunoreactivities. Thereafter, PLK and PNPO immunoreactivities started to decrease and were very low at PM 24. Alterations in the change patterns in protein contents and total activities of PLK and PNPO corresponded to the immunohistochemical data, but their specific activities were not altered in any experimental group. Based on double immunofluorescence study, PLK and PNPO immunoreactive cells in the strata oriens and radiatum were identified as GABAergic cells. Therefore, decreases of PLK and PNPO in the hippocampal CA1 region of aged brains may be involved in aging processes related with gamma-aminobutyric acid (GABA) function.

Age Factors↗
PubMed1991

High-performance liquid chromatography of thiazolidinic compounds obtained by condensation of pyridoxal 5'-phosphate or pyridoxal with aminothiols (L- or D-cysteine, cysteamine, L-cysteine ethyl ester).

We investigated six thiazolidine 4-carboxylic acids of biological interest, obtained by condensation of pyridoxal 5'-phosphate or pyridoxal with L- or D-cysteine, cysteamine or L-cysteine ethyl ester. A reversed-phase high-performance liquid chromatographic method, using a C18 column for their separation, was developed by sequential optimization of the pH and the gradient of the mobile phase. Resolution of the compounds was obtained with an analysis time of less than 20 min.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

[Modification of the spectrophotometric method of determination of pyridoxal-5'-phosphate and pyridoxal in enzymatic preparations].

In quantitative measurements of pyridoxal-5'-phosphate and pyridoxal in enzymes routinely used phenylhydrasine was substituted for 4-nitrophenylhydrasine. This increased the sensitivity of the method by 70%. The modified procedure had another advantage: it allowed measurements of the optic density of resulting 4-nitrophenylhydrasones at 430 nm for acid solutions and at 550 nm for alkaline solutions.

Animals↗
PubMed1977

Phosphoribulokinase from Nitrobacter winogradskyi: activation by reduced nicotinamide adenine dinucleotide and inhibition by pyridoxal phosphate.

CO2 fixation by particle-free extracts from Nitrobacter winogradskyi increased by addition of reduced nicotinamide adenine dinucleotide (NADH). Ribulose-1,5-diphosphate, however, increased CO2 fixation, even in the absence of NADH. Phosphoribulokinase (EC 2.7.1.19) was the enzyme of Nitrobacter extracts that was activated specifically by NADH. Pyridoxal-5-phosphate inhibited both CO2 fixation and NADH-activated phosphoribulokinase from Nitrobacter. However, it did not affect phosphoribulokinase from spinach leaves. Since the spinach enzyme had also no requirement for reduced pyridine nucleotides, it appears that pyridoxal phosphate interferes only with the binding of NADH and not with the binding of ribulose-5-phosphate and adenosine-5'-triphosphate. The regulation of phosphoribulokinase activity by NADH provided Nitrobacter with an energy-dependent control mechanism of CO2 assimilation.

Carbon Dioxide↗
PubMed1983

Immunoblot detection of pyridoxal phosphate binding proteins in liver and hepatoma cytosolic extracts.

A monoclonal antibody, highly selective for the 5'-phosphopyridoxyl group, can be used to detect cytosolic pyridoxal-5'-phosphate binding proteins by an immunoblot procedure. This technique, when applied to sodium borohydride-treated cytosolic extracts obtained from normal rat liver at various stages of development as well as several liver-derived Morris hepatomas, reveals patterns of pyridoxal-5'-phosphate binding proteins that are characteristic of the various sources of cytosol. These findings suggest that there are developmental and tumor-specific requirements for pyridoxal-5'-phosphate, the coenzymatically active form of vitamin B-6.

Aging↗
PubMed1995

In vivo modification of GABAA receptor with a high dose of pyridoxal phosphate induces tonic-clonic convulsion in immature mice.

The biologic cofactor, pyridoxal-5'-phosphate (PLP), is responsible for tonic-clonic convulsion in immature mice. The mechanisms underlying such convulsive fits induced by administration of a single high dose of PLP were studied. The administration of PLP resulted in a 13% increase of PLP in the P2 fraction compared to control P2, and the calculated data suggested that membrane bound PLP increased over 31% (approximately 1 microM). The P2 fraction of administered mice was treated with [3H]NaBH4 and analyzed by SDS-polyacrylamide gel electrophoresis. The radioactivity was mainly incorporated into a 52 kDa protein which corresponded to a GABAA receptor subunit. The addition of PLP in vitro competitively inhibited [3H]GABA binding as well as [3H]flunitrazepam binding to synaptic membranes in a concentration-dependent manner, and 50% inhibition was achieved with 1 mM PLP. The results obtained in the present study demonstrate that PLP was rapidly permeable into the brain through the immature blood-brain barrier and then bound directly to GABAA receptor. It is probable that specific amino groups of lysine residues on the GABAA receptor react in vivo with PLP to form Schiff bases, and that the in vivo modification of the receptor produces a degeneration of GABAergic neurotransmission leading to the onset of a convulsive fit.

Animals↗
PubMed1988

Vitamin B6 nutritional status in asthma: the effect of theophylline therapy on plasma pyridoxal-5'-phosphate and pyridoxal levels.

Plasma pyridoxal-5'-phosphate concentrations were significantly lower (p less than 0.001) in a group of 28 asthmatic women when compared to 33 controls. Plasma pyridoxal levels in the two groups were not different. Theophylline was administered to a group of 17 volunteers and resulted in large reductions in plasma pyridoxal-5'-phosphate levels, while plasma pyridoxal levels and urinary 4-pyridoxic acid excretion were unaffected by theophylline therapy. An in vitro study showed that theophylline did not interfere with the high performance liquid chromatography assay for pyridoxal-5'-phosphate, indicating that theophylline could affect liver metabolism of vitamin B6.

Adolescent↗
PubMed1995

Aminolevulinate synthase: functionally important residues at a glycine loop, a putative pyridoxal phosphate cofactor-binding site.

5-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway in nonplant higher eukaryotes. The enzyme functions as a homodimer and requires pyridoxal 5'-phosphate as its cofactor. Lysine-313 in murine erythroid aminolevulinate synthase has been identified as the residue involved in the Schiff base linkage with pyridoxal 5'-phosphate [Ferreira, G. C., Neame, P. J., & Dailey, H. A. (1993) Protein Sci. 2, 1959-1965]. However, other residues involved in binding and orienting the cofactor remain unknown. We studied the informational content of each residue within an 11 amino acid glycine-rich region, which we propose to be part of the phosphate-binding motif, based on amino acid sequence comparison with other pyridoxal 5'-phosphate-dependent enzymes and nucleotide-binding proteins. Partial random mutagenesis of this region in murine erythroid aminolevulinate synthase gene was followed by an efficient biological selection, using a hemA- Escherichia coli strain to recover functional unnatural enzymes. Among the total of 5444 variants produced, 283 were found to be functional. DNA sequencing results of 226 functional mutants indicated that most residues in this region contained a low informational content, being able to tolerate several other amino acid substitutions. However, three residues, namely, Arg-149, Gly-142, and Gly-144, were found to contain high informational content; Arg-149 was conserved in all of the functional mutants sequenced, while Gly-142 and Gly-144 could only tolerate alanine replacement. Two codon-specific random libraries of Arg-149, and Gly-142 and -144, respectively, were constructed to test further the stringency of these three positions.(ABSTRACT TRUNCATED AT 250 WORDS)

5-Aminolevulinate Synthetase↗
PubMed2001

Computational studies on nonenzymatic and enzymatic pyridoxal phosphate catalyzed decarboxylations of 2-aminoisobutyrate.

A computational study of nonenzymatic and enzymatic pyridoxal phosphate-catalyzed decarboxylation of 2-aminoisobutyrate (AIB) is presented. Four prototropic isomers of a model aldimine between AIB and 5'-deoxypyridoxal, with acetate interacting with the pyridine nitrogen, were employed in calculations of both gas phase and water model (PM3 and PM3-SM3) decarboxylation reaction paths. Calculations employing the transition state structures obtained for the four isomers allow the demonstration of stereoelectronic effects in transition state stabilization as well as a separation of the contributions of the Schiff base and pyridine ring moieties to this stabilization. The unprotonated Schiff base contribution (approximately 16 kcal/mol) is larger than that of the pyridine ring even when it is protonated (approximately 10 kcal/mol), providing an explanation of the catalytic power of pyruvoyl-dependent amino acid decarboxylases. An active site model of dialkylglycine decarboxylase was constructed and validated, and enzymatic decarboxylation reaction paths were calculated. The reaction coordinate is shown to be complex, with proton transfer from Lys272 to the coenzyme C4' likely simultaneous with C alpha--CO(2)(-) bond cleavage. The proposed concerted decarboxylation/proton-transfer mechanism provides a simple explanation for the observed specificity of this enzyme toward oxidative decarboxylation.

Aminoisobutyric Acids↗
PubMed1995

Aminolevulinate synthase: lysine 313 is not essential for binding the pyridoxal phosphate cofactor but is essential for catalysis.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in animals and some bacteria. Lysine-313 of the mouse erythroid aminolevulinate synthase was recently identified to be linked covalently to the pyridoxal 5'-phosphate cofactor (Ferreira GC, Neame PJ, Dailey HA, 1993, Protein Sci 2:1959-1965). Here we report on the effect of replacement of aminolevulinate synthase lysine-313 by alanine, histidine, and glycine, using site-directed mutagenesis. Mutant enzymes were purified to homogeneity, and the purification yields were similar to those of the wild-type enzyme. Although their absorption spectra indicate that the mutant enzymes bind pyridoxal 5'-phosphate, they bind noncovalently. However, addition of glycine to the mutant enzymes led to the formation of external aldimines. The formation of an external aldimine between the pyridoxal 5'-phosphate cofactor and the glycine substrate is the first step in the mechanism of the aminolevulinate synthase-catalyzed reaction. In contrast, lysine-313 is an essential catalytic residue, because the K313-directed mutant enzymes have no measurable activity. In summary, site-directed mutagenesis of the aminolevulinate synthase active-site lysine-313, to alanine (K313A), histidine (K313H), or glycine (K313G) yields enzymes that bind the pyridoxal 5'-phosphate cofactor and the glycine substrate to produce external aldimines, but which are inactive. This suggests that lysine-313 has a functional role in catalysis.

5-Aminolevulinate Synthetase↗
PubMed1989

Changes in pyridoxal phosphate and pyridoxamine phosphate in blood, liver and brain in the pregnant mouse.

A decrease in plasma pyridoxal-5'-phosphate (PLP) occurs during pregnancy in humans and experimental animals for reasons that are not known. To determine if mice also develop decreased plasma PLP concentrations during pregnancy, and if plasma PLP levels in pregnancy reflect tissue levels of PLP and pyridoxamine-5'-phosphate (PMP), we measured PLP concentrations in plasma, erythrocytes and whole blood, and liver and brain PLP and PMP in control and pregnant mice. Mice were fed a nonpurified diet containing 8.13 mg pyridoxine-HCl/kg. The PLP analyses were performed in our newly developed apotryptophanase method in which the substrate S-benzyl-L-cysteine is hydrolyzed to benzyl mercaptan, reacted with Ellman's reagent and measured spectrophotometrically. During pregnancy, plasma PLP levels decreased 50% below control levels, but erythrocyte and whole blood PLP levels increased 2.9- and 1.6-fold, respectively. Liver PLP and PMP decreased 25%, in parallel with plasma PLP, but brain PLP and PMP concentrations were unchanged during pregnancy. These results suggest that metabolism or utilization of vitamin B-6 is altered in pregnancy, and that plasma PLP concentrations alone may not be a good indicator of nutritional status in pregnancy.

Animals↗
PubMed1993

Differential leucocyte analysis of samples up to 48 hours old: improved results with citrate pyridoxal phosphate anticoagulant.

Routine haematology screening, including differential leucocyte counts, were performed using a Coulter STKS analyser on blood samples taken into several commonly available anticoagulants at time intervals up to 48 h after venepuncture. An anticoagulant based on citrate phosphate dextrose adenine, with a platelet anti-aggregant/dis-aggregant pyridoxal-5-phosphate (CPP), was found to be superior to tri-potassium ethylene diamine tetra-acetic acid (K3EDTA) in its ability to preserve leucocytes for differential counting in the STKS analyser and preserving leucocyte (particularly neutrophil) morphology for light microscopy. CPP performed as well as K3EDTA for other routine haematology measurements and was found to be stable at room temperature for at least one year. The increased cost of CPP will prevent its widespread use, but compatibility with K3EDTA will allow it to be used along with K3EDTA in situations where transit times to the laboratory regularly exceed 24 h.

Anticoagulants↗
PubMed2005

Synthesis of pyridoxal phosphate derivatives with antagonist activity at the P2Y13 receptor.

We have synthesized a series of derivatives of the known P2 receptor antagonist PPADS (pyridoxal-5'-phosphate-6-azo-phenyl-2,4-disulfonate) and examined their ability to inhibit functional activity of the recombinant human P2Y13 nucleotide receptor expressed in 1321N1 human astrocytoma cells co-expressing G(alpha)16 protein (AG32). Analogues of PPADS modified through substitution of the phenylazo ring, including halo and nitro substitution, and 5'-alkyl phosphonate analogues were synthesized and tested. A 6-benzyl-5'-methyl phosphonate analogue was prepared to examine the effect of stable replacement of the azo linkage. The highest antagonistic potency was observed for 6-(3-nitrophenylazo) derivatives of pyridoxal-5'-phosphate. The 2-chloro-5-nitro analogue (MRS 2211) and 4-chloro-3-nitro analogue (MRS 2603) inhibited ADP (100 nM)-induced inositol trisphosphate (IP3) formation with pIC50 values of 5.97 and 6.18, respectively, being 45- and 74-fold more potent than PPADS. The antagonism of MRS 2211 was competitive with a pA2 value of 6.3. MRS2211 and MRS2603 inhibited phospholipase C (PLC) responses to 30 nM 2-methylthio-ADP in human P2Y1 receptor-mediated 1321N1 astrocytoma cells with IC50 values of >10 and 0.245 microM, respectively. Both analogues were inactive (IC50>10 microM) as antagonists of human P2Y12 receptor-mediated PLC responses in 1321N1 astrocytoma cells. Thus, MRS2211 displayed >20-fold selectivity as antagonist of the P2Y13 receptor in comparison to P2Y1 and P2Y12 receptors, while MRS2603 antagonized both P2Y1 and P2Y13 receptors.

Cell Line, Tumor↗