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PubMed1983

Determination of pyridoxal phosphate levels in the brains of audiogenic and normal mice.

The concentrations of pyridoxal-5'-phosphate in the brains of audiogenic and normal mice were measured fluorimetrically. The brain of the audiogenic mouse (DBA/2J) contains 25% more pyridoxal-5'-phosphate than the brain of a control mouse. Intraperitoneal injection of this substance causes a transient increase of its concentration in the brain, lasting a few hours. The substance thereafter is degraded to pyridoxal and pyridoxic acid.

Animals↗
PubMed1978

Affinity labeling of the pyridoxal phosphate binding site of the beta2 subunit of Escherichia coli tryptophan synthase.

We have synthesized bromoacetylpyridoxamine phosphate and bromoacetylpyridoxamine and have shown that they meet three criteria for affinity labels of the beta2 subunit of tryptophan synthase: (i) the kinetic data of inactivation indicate that a binary complex is formed prior to covalent attachment; (ii) inactivation is largely prevented by the presence of pyridoxal phosphate; and (iii) inactivation is stoichiometric with incorporation of 0.7 to 0.8 mol of chromophore/mol of beta monomer. Our conclusion that inactivation of the apo beta2 subunit by bromoacetylpyridoxamine phosphate is due to the modification of cysteine is based on the disappearance of 1 mol of -SH/beta monomer and on the finding that [14C]carboxymethyl derivative in the acid hydrolysate of the protein modified by bromo[14C]acetylpyridixamine phosphate. A 39-residue tryptic peptide containing this essential cysteine has been isolated and purified from the bromo[14C]acetylpyridoxamine phosphate-labeled beta2 subunit.

Affinity Labels↗
PubMed1977

Plasma pyridoxal phosphate levels in fulminant hepatic failure and the effects of parenteral supplementation.

Plasma concentrations of pyridoxal-5'-phosphate (PLP), the active coenzyme form of vitamin B6, were found to be markedly raised in patients with fulminant hepatic failure, when estimated within one week of the onset of symptoms. In parallel with the rise in plasma PLP, there was an increase in serum aminotransferase activity, suggesting that as a result of the severe hepatocyte injury, vitamin is released from theliver in the form of transaminase holoenzymes. There was no correlation between plasma levels of PLP and the urinary excretion of 4-pyridoxic acid, its main metabolite, either in the patients or normal control subjects. There was a progressive decline in plasma PLP levels after the initial period, which was not prevented by administration of high doses of pyridoxine hydrochloride (100 mg intravenously daily), suggesting that thseepatients are either unable to convert pyridoxine to PLP, or that degradation of PLP occurs at a pathologically incrased rate in this condition.

Hepatic Encephalopathy↗
PubMed2005

Inhibition of acetyl-CoA carboxylase isoforms by pyridoxal phosphate.

Mammalian isoforms of acetyl-CoA carboxylase (ACC-1 and ACC-2) play important roles in synthesis, elongation, and oxidation of long-chain fatty acids, and the possible significance of ACC in the development of obesity has led to interest in the development of inhibitors. Here, we demonstrate that pyridoxal phosphate (PLP) is a linear and reversible inhibitor of ACC-1 and ACC-2. ACC from rat liver and white adipose tissue (largely ACC-1) exhibited an IC50 of approximately 200 microm, whereas ACC-2 from heart or skeletal muscle exhibited an IC50 exceeding 500 microm. ACC from rat liver was equally sensitive to PLP following extensive purification by avidin affinity chromatography. When added before citrate, PLP inhibited ACC with a Ki of approximately 100 microm, reducing maximal activity >90% and increasing the Ka for citrate approximately 5-fold but having little effect on substrate Km values. Pre-treatment with citrate increased the apparent Ki for ACC inhibition by PLP by approximately 4-fold. Inhibition of ACC was reversed by removal of PLP, either by washing or by reaction with hydroxylamine or amino-oxyacetate. ACC was irreversibly inhibited and radiolabeled, to a stoichiometry of approximately 0.4 mol[H]/mol subunit, in the presence of PLP plus [3H]borohydride. Studies with structurally related compounds demonstrated that the reactive aldehyde and negatively charged substituents of PLP contribute importantly to ACC inhibition. The studies reported here suggest a rationale to develop ACC inhibitors that are not structurally related to the substrates or products of the reaction and an approach to probe the citrate-binding site of the enzyme.

Acetyl-CoA Carboxylase↗
PubMed1990

Hippocampal zinc thionein and pyridoxal phosphate modulate synaptic functions.

The hippocampus, a component of the limbic system, is a prominent subcortical structure, which not only contains high concentrations of zinc, but also exhibits regional variations in this essential element, with concentrations being highest in the hilar region and lowest in the fimbria. For example, the concentration of zinc in the mossy fiber axons has been estimated to approach 300-350 microM. Both zinc and pyridoxal phosphate (PLP) deficiency and excess have been reported to produce epileptiform seizures, which are blocked by gamma-aminobutyric acid (GABA). The proposed mechanism is that at physiological concentrations zinc stimulates the activity of the hippocampal pyridoxal kinase (50% stimulation at 1.7 x 10(-7) M), enhancing the formation of PLP, whereas in pharmacological doses zinc inhibits the activity of glutamate decarboxylase (GAD) directly (50% inhibition at 6.5 X 10(-4) M) by preventing the binding of PLP to HoloGAD. Furthermore, recent studies have shown that two forms of GAD are found in the rat brain. One form (GAD A) does not require PLP for maximal activity, while another form (GAD B) does. Furthermore, the ratio between GAD A and GAD B is nonuniform throughout brain areas, and the hippocampus contains twice as much GAD B (the PLP-requiring GAD) as GAD A. Although the hippocampus is a common target of exogenous neurotoxic agents, "free" zinc in greater than physiological concentrations should be considered an endogenous central neurotoxin. For example, iontophoretically applied zinc in the frontoparietal cortex enhances and prolongs the firing rate of neurons in urethane-anesthetized rat. In addition, zinc (50-500 microM) significantly depresses the paired-pulse potentation in the hippocampal CA3 subfield. Moreover, zinc selectively blocks the action of N-methyl-D-aspartate on cortical neurons and enhances the quisqualate receptor-mediated injury. Finally zinc competitively inhibits the calcium-dependent release of transmitter by inhibiting the entry of Ca2+ into the nerve terminals. Since zinc in a concentration of 300-350 microM could not possibly remain "unbound" in the hippocampus, we searched for and identified a metallothionein-like protein (MT) in the bovine hippocampus, which produces two isoforms on reverse-phase HPLC and lacks aromatic amino acids, but possesses metallomercaptide bonds. We believe that the hippocampal metallothionein, by donating zinc to an extensive number of zinc-activated, PLP-mediated biochemical reactions, modulates synaptic functions. Furthermore, by virtue of its inducibility, metallothionein binds additional amounts of zinc, maintains its steady-state concentration, prevents inhibition of an extensive number of sulfhydryl-containing enzymes and receptor sites, and hence averts metal-related neurotoxicity.

Animals↗
PubMed1986

Isolation of a 5,300-dalton peptide containing a pyridoxal phosphate binding site from the 38,000-dalton domain of band 3 of human erythrocyte membranes.

A hydrophobic 5,300-dalton peptide was isolated from the 38,000-dalton domain of Band 3 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography. The peptide was affinity labeled with pyridoxal phosphate and sodium [3H]borohydride when erythrocytes were incubated in vitro. The peptide was not labeled with these agents when cells were incubated in the presence of a specific inhibitor of anion transport, suggesting that the peptide contains at least a part of the active center for the anion transport system in the cell membrane. The peptide was eluted from a reversed-phase high-performance liquid chromatography column with a high concentration of acetonitrile (more than 65%), although the elution pattern of the hydrophobic peptide was not as sharp as that of the soluble peptides. However, a satisfactory separation was achieved when this procedure was employed in combination with sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1990

Sequence similarities between tryptophan synthase beta subunit and other pyridoxal-phosphate-dependent enzymes.

On the basis of 8 tryptophan synthase beta subunits (EC 4.2.1.20) consensus patterns were constructed comprising two conserved motifs. Screening of the SWISSPROT protein sequence database with these patterns indicates similarities with O-acetylserine sulfhydrolases (EC 4.2.99.8), threonine synthases (EC 4.2.99.2), L- and D-serine dehydratases (EC 4.2.1.13/EC 4.2.1.14) and threonine dehydratases (EC 4.2.1.16). Using multiple alignment procedures the similar regions could be extended. In connection with their pyridoxal-phosphate-binding-capacity and their positions in biochemical pathways evolutionary relationships among these enzymes are discussed.

Amino Acid Sequence↗
PubMed1985

Lack of a relationship between plasma pyridoxal phosphate levels and ischaemic heart disease.

The 'vitamin B6-homocysteine theory' has been proposed as an alternative to the widely accepted lipid hypothesis in the aetiology of ischaemic heart disease (IHD). In a cross-sectional study of 71 white men with evidence of IHD and 110 male controls (all aged 45-54 years) we have been unable to demonstrate any differences in plasma pyridoxal phosphate (PLP) levels between the groups. It is therefore unlikely that deficiency of vitamin B6 has a primary causal role in development of IHD. However, 31% of the overall study population had low plasma PLP levels, and the possibility that underlying vitamin B6 deficiency may facilitate the actions of the primary risk factors for IHD therefore cannot be excluded.

Coronary Disease↗
PubMed2000

Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), a putative P2Y(1) receptor antagonist, blocks signaling at a site distal to the receptor in Madin-Darby canine kidney-D(1) cells.

Substantial evidence documents the potential importance of P2Y receptor subtypes in the regulation of cellular responses, but few selective antagonists exist for these receptors. In the current study, we assessed the use of pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonate (PPADS) as a putative P2Y(1) receptor-selective blocker in Madin-Darby canine kidney (MDCK-D(1)) cells. We found that the key action of PPADS in MDCK-D(1) cells was blockade of signaling at a postreceptor site. PPADS blocked UTP (P2Y(2))-stimulated accumulation of cAMP [which is dependent on arachidonic acid (AA) metabolism by cyclooxygenase] but not that by 2-methyl thio-adenosine triphosphate (2MeSATP; which is independent of cyclooxygenase and has been attributed to P2Y(1) and P2Y(11) receptors). By contrast, PPADS inhibited AA release mediated by both 2MeSATP and UTP. PPADS displayed uncompetitive antagonism in blockade of AA release in response to 2MeSATP. PPADS also inhibited AA release stimulated by various nucleotides, phenylephrine, and bradykinin, implying that the effect does not involve the inhibition of a specific receptor. Because PPADS also inhibited ionomycin-, thapsigargin-, and phorbol-12-myristate-13-acetate-promoted AA release, it appears to act at a site distal to an increase in intracellular Ca(2+) transients or PKC activation. Inhibition of melittin-stimulated AA release by PPADS suggested that the target of PPADS action may either be a phospholipase A(2) (PLA(2)) or a site distal to PLA(2), but PPADS did not inhibit Ca(2+)-dependent PLA(2) activity in MDCK-D(1) cell homogenate. The data indicate that PPADS blocks AA release in response to multiple compounds and suggest caution in the use of this compound for distinguishing P2Y receptor subtypes.

Adenosine Triphosphate↗
PubMed1985

Use of 6-fluoroderivatives of pyridoxal and pyridoxal phosphate in the study of the coenzyme function in glycogen phosphorylase.

6-Fluoropyridoxal phosphate (6-FPLP) has been synthesized. Its properties were studied, and it was used, along with 6-fluoropyridoxal (6-FPAL), to reconstitute apophosphorylase b. Kinetic studies of the resulting enzymes showed that phosphorylases reconstituted with 6-FPLP and 6-FPAL have characteristics similar to those of native and pyridoxal enzymes, respectively, except that the former two enzymes have lower Vmax values. 19F NMR and UV spectra of 6-FPLP phosphorylase showed that the coenzyme forms a neutral enolimine Schiff base. Because the UV and fluorescence spectra of 6-FPLP phosphorylase are comparable to those obtained with native phosphorylase, it further confirms the postulate that pyridoxal phosphate forms a neutral enolimine Schiff base in phosphorylase. The results suggest that the 3-OH group is protonated and the pyridine nitrogen unprotonated in both 6-FPLP phosphorylase and native enzyme. 19F NMR study of 6-FPLP- and 6-FPAL-reconstituted phosphorylases in the inactive and active states indicates that the protein structure near the coenzyme binding site undergoes certain changes when these enzymes are activated by the substrates and AMP. The comparison of the properties of 6-FPLP-reconstituted and native phosphorylases implies that the ring nitrogen of the coenzyme PLP in phosphorylase may interact with the protein during catalysis, and this interaction is important for efficient catalysis by phosphorylase.

Animals↗
PubMed1982

Effect of pyridoxal phosphate deficiency on aromatic L-amino acid decarboxylase activity with L-DOPA and L-5-hydroxytryptophan as substrates in rats.

This paper describes the distribution of aromatic L-amino acid decarboxylase (AADC) activities in fourteen tissues (eight peripheral tissues and six brain regions) of semicarbazide (SC)-treated rats, using both L-DOPA and L-5-hydroxytryptophan (L-5-HTP) as substrates. The distribution of pyridoxal phosphate (PLP) was also measured in control and SC-treated rats. SC-treatment decreased the PLP concentration in all tissues (about 50-60% of control). AADC activities towards L-DOPA and L-5-HTP as substrates were also decreased significantly in almost all tissues of SC-treated rats. After the addition of exogenous PLP in vitro, AADC activities were recovered only partially in most tissues, but the recovery patterns were not parallel between L-DOPA and L-5-HTP as substrates. L-DOPA decarboxylase activity was more sensitive to PLP-deficiency than L-5-HTP decarboxylase activity in the same tissues. Serum AADC activities were decreased drastically using both L-DOPA and L-5-HTP as substrates. No serum AADC activity was detected in SC-treated rats using L-DOPA as substrate, but low activity was detected in the same sample using L-5-HTP as the substrate; both activities recovered completely after in vitro addition of 10 microM PLP in the incubation mixtures.

5-Hydroxytryptophan↗
PubMed1992

Selenium metabolism and glutathione peroxidase activity in cultured human lymphoblasts. Effects of transsulfuration defects and pyridoxal phosphate.

The metabolism of selenite, selenocysteine (SeCys), and selenomethionine (SeMet) was studied in three human lymphoblast cell lines with defects in the transsulfuration pathway and in control cells without this defect. There were very little differences in the induction of glutathione peroxidase (GPX) activity by selenite and SeCys among these cells. However, markedly higher levels of SeMet were required to induce GPX activity in transsulfuration defective cells than in control cells. Surprisingly, the addition of pyridoxal phosphate (PLP) to the media resulted in elevated GPX activity in all cells regardless of the chemical form of Se used. There is no explanation for this effect of PLP, but it is not through direct reaction with GPX or on the alteration of sulfhydryl groups.

Cell Division↗
PubMed1975

[The effect of pyridoxal phosphate on the tryptophanases of five species of Enterobacteriaceae].

Escherichia coli B and E. aurescens, Shigella alkalescens, and Proteus vulgaris et P. morganii tryptophanases (TPases) were studied for the spectral forms of the enzyme. The pH effect on the absorption spectrum and on the enzyme specific activity revealed that the coli group TPases are identical with but differ from Proteus TPases which differ themselves. The coli group TPases attach 4 mol of pyridoxal phosphate (PLP)/mol of enzyme, independently of the pH in the presence of K(plus) ions, and 9 mol of PLP/mol of enzyme must be reduced to achieve complete inactivation. The Proteus TPases attach 4 mol of PLP/mol of enzyme at PH 6.8, and 3 mol of PLP/mol of enzyme at pH 7.8 in K(plus) buffer. In P. morganii, 7 mol of PLP/mol of enzyme must be reduced to inactivate the enzyme, whereas P. vulgaris TPase cannot be completely inactivated by this method. These five TPases attach only 3 mol of PLP/mol of enzyme in a Na(plus) buffer, independently of the pH.

Calcium↗
PubMedDate not supplied

[Distribution of 1-[14C] GABA and its conjugates with nicotinate, pyridoxal phosphate and biotin in tissues of guinea pigs in closed systems].

Distribution of the total radioactive label in the blood, different parts of the brain (the whole cortex and its lobes, bulbi olfactorii, hippocamp, striopallidum, hypothalamus, thalamus, peduculi cerebri, cerebellum, pons, medulla oblongata), eye (retina, hyaloid and vascular membrane), hypophysis and liver of guinea pig males has been studied after the hypodermic injection of [14C] GABA and products of its conjugation with pyridoxal phosphate, nicotinic acid and biotin in the equimolar dose (50 nmol/g) under normal conditions and in case of action of the closed volume factors. Accumulation of the total label in the hypophysis after injection of [14C] GABA, PLP-[14C] GABA and nicotinoyl-[14C] GABA was 3.6, 3.1 and 2.7 times (respectively) as much as that in the blood. The content of the labelled compounds in the cortical structures of the telencephalon and in the diencephalon was the greatest in comparison with other parts of the brain. The permeability through a hemato-ophthalmic barrier was 3-5 times as much as that through a hemato-encephalic barrier. The closed volume factors lead to higher accumulation of the label as compared with the control after injection of [14C] GABA, PLP-[14C] GABA and biotinyl-[14C] GABA.

Animals↗