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PubMed1992

Alkaline phosphatase activity and pyridoxal phosphate concentrations in the milk of various species.

Because pyridoxal phosphate does not normally cross membranes, it was intriguing that the concentration of pyridoxal phosphate is much higher in goat milk than in human milk. We also noted that, although the total vitamin B-6 concentration of bovine milk was similar to that of caprine milk, the bovine milk had lower pyridoxal phosphate. Preliminary data from five Alpine goats, five Brown Swiss cows, five Holstein cows and three humans suggested that there was an inverse relationship between pyridoxal phosphate concentration and phosphatase activity in the goats and cows but not in the humans. This was confirmed with additional data from Nubian goats, Jersey and Guernsey cows, and crossbred sows. Combining the animal data yielded the following relationship between pyridoxal phosphate (PLP, mumol/L) and alkaline phosphatase (P'ase) activity (mmol/(min.L): PLP = 2.03e(-2.26 P'ase) + 0.03. The human milk samples were low in both pyridoxal phosphate and alkaline phosphatase. We conclude that in goats, cows and pigs a significant fraction of the vitamin B-6 appearing in the milk is secreted as pyridoxal phosphate, probably bound to protein, and varying amounts may then be hydrolyzed back to pyridoxal depending on the alkaline phosphatase activity. Human mammary tissue apparently secretes very little pyridoxal phosphate.

Alkaline Phosphatase↗
PubMed1986

X-ray studies of nucleotide binding and pyridoxal phosphate labeling of the gene 5 DNA unwinding protein.

X-ray diffraction studies have been carried out using difference Fourier methods to evaluate the reaction or interaction of an affinity label and 5'-phosphate nucleotides with the gene 5 DNA binding protein in the crystalline state. In the first case the crystalline protein was reacted with pyridoxal phosphate. Pyridoxal phosphate, which has served as an affinity label for nucleotide binding sites on other enzymes, demonstrated a major site of substitution at the center of the protein's DNA binding cleft adjacent to lysine 46 as well as two other reaction sites near residues implicated in DNA binding. Difference Fourier maps of crystals exposed to 5'-dAMP, 5'-dCMP and 5'-dTMP indicated that phosphate groups were associated with most lysine and arginine side-chains on the surface of the protein but that the nucleoside portion of the ligands were generally disordered. In several cases, however, more specific binding of the nucleotides appeared to have occurred and these sites were primarily within the proposed DNA binding cleft of the protein. In particular, binding was observed near tyrosine 34, phenylalanine 73 and within the curl of the DNA binding loop containing tyrosine 26.

Adamantane↗
PubMed1983

Pyridoxal phosphate as a probe in the active site of ribulose bisphosphate carboxylase/oxygenase.

Ribulose bisphosphate (RuBP) carboxylase is rapidly and irreversibly inactivated by photooxidation sensitized by pyridoxal phosphate. Both pyridoxal and pyridoxamine phosphate were much less effective in sensitizing the photooxidation even when used at twice the concentration of pyridoxal phosphate. These results imply that pyridoxal phosphate binds at the active site not only through a Schiff base, but also through ionic interaction with the phosphate binding region. Spectral analysis of the photooxidized enzyme showed a new absorption maximum at 325 nm due to reduction of the Schiff base between pyridoxal phosphate and a lysyl residue with concomitant oxidation of a histidine residue. The stoichiometry of photooxidative [3H]pyridoxal phosphate incorporation was 0.87 mol/mol of a 70,000-dalton large subunit-small subunit combination. Studies with 3H-labeled diethyl pyrocarbonate showed that both photooxidation and carbethoxylation occur at the same histidine residue. However, photooxidation by pyridoxal phosphate is very specific for an active site histidine residue due to the high specificity of this affinity label. Several competitive inhibitors with respect to ribulose bisphosphate offered appreciable protection against pyridoxal phosphate-induced photooxidation of the enzyme. The photooxidized enzyme showed an increase in the net negative charge on the protein which was evident from the higher mobility of the photooxidized enzyme toward the anode in polyacrylamide gel electrophoresis.

Binding Sites↗
PubMed1996

Lens proteins changes induced by sugars and pyridoxal phosphate.

Glucose, galactose and pyridoxal phosphate (PLP) bind to lens protein amino groups causing changes in absorbance and fluorescence spectra and inducing aggregation. Sugars and PLP simultaneously cause an increase in fluorophore and chromophore formation, but a decreased aggregation, compared to PLP alone. PLP binds to lens protein amino groups decreasing the sugar binding, but in preventing glycation by PLP attention should be paid to the consequences of its own binding to proteins in diabetes.

Animals↗
PubMed1979

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. Binding of pyridoxal phosphate to 5-aminolaevulinate synthetase.

1. Pyridoxal 5'-phosphate is a cofactor essential for the enzymic activity of aminolaevulinate synthetase from Rhodopseudomonas spheroides. It also aids activation of the low-activity enzyme by trisulphides such as cystine trisulphide, whereas inactivation of enzyme is facilitated by its absence. 2. The fluorescence spectrum of purified high-activity enzyme is that expected for a pyridoxal phosphate--Schiff base, but the firmly bound cofactor does not appear to be at the active centre. In dilute solutions of enzyme this grouping is inaccessible to nucleophiles such as glycine, hydroxylamine, borohydride and cyanide, at pH 7.4. 3. An active-centre Schiff base is formed between enzyne and added pyridoxal phosphate, which is accessible to nucleophiles. Concentrated solutions of this enzyme--Schiff base on treatment with glycine yield apo- and semi-apoenzyme, which can re-bind pyridoxal phosphate. 4. Two types of binding of pyridoxal phosphate are distinguishable in dilute solution of enzyme, but these become indistinguishable when concentrated solutions are treated with cofactor. A change occurs in the susceptibility towards borohydride of the fluorescence of the "structural" pyridoxal phosphate. 5. One or two molecules of cofactor are bound per subunit of mol. wt. 50 000 in semiapo- or holo-enzyme. The fluorescence of pyridoxamine phosphate covalently bound to enzyme also indicates one to two nmol of reducible Schiff base per 7000 units of activity in purified and partially purified samples of enzyme. 6. Cyanide does not convert high-activity into low-activity enzyme, but with the enzyme-pyridoxal phosphate complex it forms a yellow fluorescent derivative that is enzymically active.

5-Aminolevulinate Synthetase↗
PubMed2003

Crystal structure of Escherichia coli PdxA, an enzyme involved in the pyridoxal phosphate biosynthesis pathway.

Pyridoxal 5'-phosphate is an essential cofactor for many enzymes responsible for the metabolic conversions of amino acids. Two pathways for its de novo synthesis are known. The pathway utilized by Escherichia coli consists of six enzymatic steps catalyzed by six different enzymes. The fourth step is catalyzed by 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA, E.C. 1.1.1.262), which converts 4-hydroxy-l-threonine phosphate (HTP) to 3-amino-2-oxopropyl phosphate. This divalent metal ion-dependent enzyme has a strict requirement for the phosphate ester form of the substrate HTP, but can utilize either NADP+ or NAD+ as redox cofactor. We report the crystal structure of E. coli PdxA and its complex with HTP and Zn2+. The protein forms tightly bound dimers. Each monomer has an alpha/beta/alpha-fold and can be divided into two subdomains. The active site is located at the dimer interface, within a cleft between the two subdomains and involves residues from both monomers. A Zn2+ ion is bound within each active site, coordinated by three conserved histidine residues from both monomers. In addition two conserved amino acids, Asp247 and Asp267, play a role in maintaining integrity of the active site. The substrate is anchored to the enzyme by the interactions of its phospho group and by coordination of the amino and hydroxyl groups by the Zn2+ ion. PdxA is structurally similar to, but limited in sequence similarity with isocitrate dehydrogenase and isopropylmalate dehydrogenase. These structural similarities and the comparison with a NADP-bound isocitrate dehydrogenase suggest that the cofactor binding mode of PdxA is very similar to that of the other two enzymes and that PdxA catalyzes a stepwise oxidative decarboxylation of the substrate HTP.

Amino Acid Sequence↗
PubMed1980

Regulation of formation in vivo of pyridoxal phosphate in hydrazine-treated rats.

The formation in vivo of pyridoxal phosphate was studied in hydrazine-treated rats. hydrazine was administered i.p. at a dose of 1.28 mg/day (20% LD50) for each 100 g body weight for 7 days. Hydrazine administered at the present dose did not appear to have an effect on the pyridoxal phosphate level of either liver on kidney tissue. Hydrazine treatment, however, elevated the pyridoxal level in liver, while kidney pyridoxal level remained unaltered under the same condition. The pyridoxine phosphate oxidase activity in liver, unlike that in kidney, was found to be increased after hydrazine treatment. The increase in pyridoxine phosphate oxidase activity in the liver of hydrazine-treated rats was prevented by actinomycin D treatment. It has been suggested that hydrazine treatment at the present dose enhanced the rate of formation of pyridoxal phosphate from pyridoxine-5-phosphate in liver, while the formation of pyridoxal phosphate in kidney appeared to be independent of hydrazine treatment. The increased activity of liver pyridoxine phosphate oxidase in hydrazine-treated rats was ascribed to the induction of the enzyme. It has been further suggested that the unalteration in the liver pyridoxal phosphate level in hydrazine-treated rats inspite of enhanced conversion of pyridoxine-5-phosphate to pyridoxal phosphate, was probably caused by the increased hydrolysis of pyridoxal phosphate.

Animals↗
PubMed1981

Active form of pyridoxal phosphate in glycogen phosphorylase. Phosphorus-31 nuclear magentic resonance investigation.

The substrate analogue alpha-D-glucopyranosyl cyclic 1,2-phosphate has been confirmed to be a good competitive inhibitor of glycogen phosphorylases a and b isolated from rabbit muscle. Effects of tertiary and quaternary structure of the enzyme have been shown to be similar to those induced by the substrate glucose 1-phosphate and different from those of the coincidently binding inhibitor glucose. This information was obtained from study of the ultracentrifugation patterns of the enzyme-inhibitor complex and by determination of its effect on the binding constant for the activator AMP. 31P NMR investigation of the binding of this inhibitor to the enzyme has demonstrated that it both tightens the binding of the nucleotide activator and shifts the resonance of the phosphate group of the pyridoxal phosphate residue to a broad signal around 0 ppm. This situation is further reinforced in the presence of the second substrate, maltopentaose, giving a fully potentiated, but inactive, enzyme-substrate complex. This has not been studied previously by 31 P NMR. The active form of the pyridoxal phosphate (PLP), in the presence of substrates or their analogues, is not therefore a mobile dianionic phosphate as has been previously proposed. It may represent a tightly bound and constrained dianionic phosphate or possible a protonated phosphate in intermediate exchange. The implications of this finding are discussed.

Adenosine Monophosphate↗
PubMed1976

Regulation of blood pyridoxal phosphate in riboflavin deficiency in man.

Synthesis and breakdown of pyridoxal phosphate by erythrocytes were studied in subjects with oral lesions before and after treatment with riboflavin. In vivo conversion of pyridoxine to pyridoxal phosphate as well as in vitro synthesis of pyridoxal phosphate by erythrocytes were lower in subjects with lesions of the mouth and improved markedly after treatment with riboflavin. Erythrocyte phosphatase activity, with pyridoxal phosphate as substrate, was lower in riboflavin-deficient subjects and showed an increase after treatment with riboflavin.

Adult↗
PubMed1987

The organic cofactor in plasma amine oxidase: evidence for pyrroloquinoline quinone and against pyridoxal phosphate.

Plasma amine oxidases (EC 1.4.3.6) are classified as containing the organic cofactor pyridoxal phosphate. Biochemical and bioassays on the pig plasma amine oxidase fail to reveal the presence of pyridoxal phosphate and 31P n.m.r. evidence is also inconsistent with pyridoxal phosphate in the enzyme. Resonance Raman spectral studies on phenylhydrazone derivatives of the pig and bovine plasma enzymes have been carried out and comparisons made with the corresponding derivatives of pyridoxal phosphate and pyrroloquinoline quinone (PQQ). The resonance Raman evidence indicates that the cofactor in both plasma amine oxidases is PQQ or a closely related species and not pyridoxal phosphate. The results substantiate earlier reports concerning the identity of the organic cofactor.

Amine Oxidase (Copper-Containing)↗
PubMed1983

Dissociation between epileptic seizures induced by convulsant drugs and alteration in the concentrations of pyridoxal phosphate in rat brain regions.

Allylglycine increased the concentration of pyridoxal phosphate in cerebral cortex from 1011.4 +/- 25.0 to 1318.0 +/- 66.3 and decreased it in cerebellum from 1289.0 +/- 49 to 1147.7 +/- 119.4 ng/g wet tissue during the preictal period. Mercaptopropionic acid increased the concentration of pyridoxal phosphate in cerebellum from 1525 +/- 91 to 1985.7 +/- 275 ng/g wet tissue. Similar effects were noted in hippocampus and cerebral cortex. Picrotoxin increased the concentration of pyridoxal phosphate in hippocampus from 938.7 +/- 44 to 1043 +/- 118 but decreased it in cerebral cortex from 1124.52 +/- 124 to 979.4 +/- 15 ng/g wet brain. The effects of strychnine were identical to those of allylglycine. Bicuculline reduced the concentration of pyridoxal phosphate in cerebral cortex from 1184 +/- 61 to 1075.14 +/- 78 ng/g wet brain.

3-Mercaptopropionic Acid↗
PubMed1975

Pyridoxal phosphate and hypertensive disorders of pregnancy.

Pyridoxal phosphate (vitamin B6) concentrations in peripheral and cord blood obtained at the time of delivery were measured in 30 women. The average plasma concentration in nine women with normal pregnancy was 4.3 ng. per milliliter; in 10 women with pre-eclampsia, 3.3 ng. per milliliter; and in nonpregnant women, 17 ng. per milliliter. The average cord blood plasma concentration of normal infants was 28.4 ng. per milliliter, whereas that of infants of pre-eclamptic mothers was 12.2 ng. per milliliter. This twofold difference in the cord plasma concentrations was statistically significant (p smaller than 0.001). Pyridoxal phosphate concentrations in the infants' cord plasma were increased in all pregnancies studied by administration of pyridoxine either orally or intravenously. These findings together with other data, demonstrating (1) that B6 deficiency during pregnancy may lead to abnormal neurologic development in experimental animals and (2) that brain development in infants of toxemic mothers may be retarded, suggest that dietary supplementation with vitamin B6 should be instituted in women at high risk for development of toxemia of pregnancy.

Administration, Oral↗
PubMed1985

Mitochondrial nicotinamide nucleotide transhydrogenase: inhibition by ethoxyformic anhydride, dansyl chloride, and pyridoxal phosphate.

Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH; EC 1.6.1.1) was inactivated by treatment with pyridoxal phosphate, ethoxyformic anhydride (EFA) or dansyl chloride. NADP and NADPH, but not NAD and NADH, protected TH against inhibition by pyridoxal phosphate, and L-lysine reversed this inhibition. The results suggested modification of an essential lysyl residue by pyridoxal phosphate, possibly at the NADP(H) binding site of TH. EFA and dansyl chloride inhibited TH in a similar manner. The effect of pH on the rate of inhibition of TH by EFA and dansyl chloride was the same, and in both cases addition of NADP and particularly NADPH accelerated the rate of inhibition, while addition of NAD or NADH had no effect. Double inhibition studies, using in one experiment dithiothreitol-reversible inhibition by 5,5'-dithiobis(2-nitrobenzoic acid) to protect the thiol groups of TH, and in another experiment lysine-reversible inhibition by pyridoxal phosphate to protect the putative essential lysyl residues of the enzyme, followed in each case by further treatment of the protected TH with EFA or dansyl chloride, suggested that the inhibitions by EFA and dansyl chloride were independent of the inhibitions by 5,5'-dithiobis (2-nitrobenzoic acid) and pyridoxal phosphate. The inhibitors discussed above are interesting, because pyridoxal phosphate is the only reagent known which appears to modify an essential residue in the NADP(H), but not the NAD(H), binding site of TH, and EFA and dansyl chloride are the only inhibitors known which appear to react with essential residues outside the active site of TH. It is possible that EFA and dansyl chloride inhibitions involve modification of essential prototropic residues in the proton translocation domain of the enzyme.

Animals↗
PubMed1984

Localization of pyridoxal phosphate binding site on the mero-receptor domain of the glucocorticoid receptor.

Previous studies have demonstrated that the vitamin pyridoxal phosphate can alter the physicochemical properties of glucocorticoid receptors. We now report the localization of a pyridoxal phosphate binding site within the mero-receptor domain of this glucocorticoid receptor. Mero-glucocorticoid receptors that are generated by trypsin (10 micrograms/ml) or chymotrypsin (100 micrograms/ml) digestion of intact receptors sediment as 2.6 S species on 5-20% sucrose gradients in the presence or absence of pyridoxal phosphate. Mero-glucocorticoid receptors prepared by exogenous proteinases are hydrophobic and show no affinity for DEAE Bio-Gel A. Treating either trypsin-generated or chymotrypsin-generated mero-receptors with pyridoxal phosphate rapidly converts the proteins (60 and 35%, respectively) into forms that bind to DEAE Bio-Gel A. Induction of DEAE binding is specific to pyridoxal phosphate, for treating mero-receptors with pyridoxal, pyridoxamine or pyridoxine phosphate is ineffective. Furthermore, DEAE binding cannot be induced by adding other pyridoxal phosphate-treated cytosols to untreated mero-receptors. High-resolution polyacrylamide gel isoelectric focussing studies indicated that treating mero-receptor generated by either proteinase with pyridoxal phosphate shifted the isoelectric points of both to lower pH values. The conversion of the mero-receptor to a more acidic form also occurred when the intact glucocorticoid receptor was treated with the vitamin prior to proteolysis. These studies localize at least one pyridoxal phosphate binding site on the mero-receptor domain of the rat thymocyte glucocorticoid receptor.

Animals↗
PubMed1966

The oxidation of Schiff bases of pyridoxal and pyridoxal phosphate with amino acids by manganous ions and peroxidase.

1. Oxygen was taken up rapidly when pyridoxal or pyridoxal phosphate was added to mixtures of pea-seedling extracts and Mn(2+) ions. 2. The increases in total oxygen uptake were proportional to the pyridoxal or pyridoxal phosphate added and were accompanied by the disappearance of these compounds. 3. In addition to Mn(2+) ions, the reactions depended on two factors in the extracts, a thermolabile one in the non-diffusible material and a thermostable one in the diffusate; these factors could be replaced in the reactions by horse-radish peroxidase (donor-hydrogen peroxide oxidoreductase, EC 1.11.1.7) and amino acids respectively. 4. When pyridoxal phosphate was added to mixtures of amino acids and Mn(2+) ions oxygen uptake was rapid after a lag period of 30-90min.; the lag period was shortened to a few minutes by peroxidase, particularly in the presence of traces of p-cresol, or by light. 5. When pyridoxal replaced pyridoxal phosphate relatively high concentrations were required and peroxidase had only a small activating effect. 6. Pyridoxal or pyridoxal phosphate disappeared during the reactions and carbon dioxide and ammonia were formed. 7. With phenylalanine as the amino acid present, benzaldehyde was identified as a reaction product. 8. It is suggested that the reactions are oxidations of the Schiff bases formed between pyridoxal or pyridoxal phosphate and amino acids, mediated by a manganese oxidation-reduction cycle, and resulting in oxidative decarboxylation and deamination of the amino acids.

Aldehydes↗