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PubMed1986

Serum aspartate aminotransferase storage and the effect of pyridoxal phosphate.

The storage stability of the catalytic activity of aspartate aminotransferase (AST) remains the subject of conflicting reports. We reevaluated this issue for total AST activity and for the cytosolic and mitochondrial isoenzymes from human sera stored at room, refrigerator, and freezer temperatures for up to 28 days. We found that these enzymes were stable for catalytic activity and immunologic (mass) measurements up to 24 hours at ambient temperatures and for at least 28 days at 4 degrees, -20 degrees, and -80 degrees C. The addition of exogenous pyridoxal phosphate (0.1 mmol/L) to serum improved stability of AST storage at ambient temperature (22 degrees C) from 1 day to 7 days.

Alkaline Phosphatase↗
PubMed1975

Urinary 4-pyridoxic acid, plasma pyridoxal phosphate, and erythrocyte aminotransferase levels in oral contraceptive users receiving controlled intakes of vitamin B6.

Fifteen women who had used combination type oral contraceptives (estrogen plus progestogen) and 9 control women who had never used these agents were given a diet deficient in vitamin B6. After 1 month, this diet was supplemented daily with 0.8, 2.0 or 20.0 mg of pyridoxine hydrocholride for an additional month. At weekly intervals, measurements were made of urinary 4-pyridoxic acid, plasma pyridoxal phosphate, and erythocyte alanine and aspartate aminotransterases. No significan differences were observed between oral contraceptive users and controls in any of the above measured indices. The data suggest that if the use of oral contraceptives of the combined estrogen-progestogen type does alter the requirement for vitamin B6, the effect is a mild one and of doubtful clinical significance to the majority of women taking these steroid preparations. The amount of vitamin B6 (as pyridoxine) needed to maintain normal levels of the above indices of vitamin B6 nutrition in these subjects were between 0.8 and 2.0 mg/day.

Adult↗
PubMed2001

Pyridoxal phosphate binding sites are similar in human heme-dependent and yeast heme-independent cystathionine beta-synthases. Evidence from 31P NMR and pulsed EPR spectroscopy that heme and PLP cofactors are not proximal in the human enzyme.

Two classes of cystathionine beta-synthases have been identified in eukaryotes, the heme-independent enzyme found in yeast and the heme-dependent form found in mammals. Both classes of enzymes catalyze a pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine to produce cystathionine. The role of the heme in the human enzyme and its location relative to the PLP in the active site are unknown. (31)P NMR spectroscopy revealed that spin-lattice relaxation rates of the phosphorus nucleus in PLP are similar in both the paramagnetic ferric (T(1) = 6.34 +/- 0.01 s) and the diamagnetic ferrous (T(1) = 5.04 +/- 0.06 s) enzyme, suggesting that the two cofactors are not proximal to each other. This is also supported by pulsed EPR studies that do not provide any evidence for strong or weak coupling between the phosphorus nucleus and the ferric iron. However, the (31)P signal in the reduced enzyme moved from 5.4 to 2.2 ppm, and the line width decreased from 73 to 16 Hz, providing the first structural evidence for transmission to the active site of an oxidation state change in the heme pocket. These results are consistent with a regulatory role for the heme as suggested by previous biochemical studies from our laboratory. The (31)P chemical shifts of the resting forms of the yeast and human enzymes are similar, suggesting that despite the difference in their heme content, the microenvironment of the PLP is similar in the two enzymes. The addition of the substrate, serine, resulted in an upfield shift of the phosphorus resonance in both enzymes, signaling formation of reaction intermediates. The resting enzyme spectra were recovered following addition of excess homocysteine, indicating that both enzymes retained catalytic activity during the course of the NMR experiment.

Amino Acid Sequence↗
PubMed1991

Effect of pyridoxal phosphate on the formation of cytochrome c1-c and cytochrome c-cytochrome oxidase complexes.

The cytochrome c-cytochrome oxidase complex is formed when c reacts with cytochrome oxidase (Kuboyama et al. (1962) Biochem. Biophys. Res. Commun. 9, 534) and the cytochrome c1-cytochrome c complex is formed when c reacts with cytochrome c1 in the presence of the hinge protein (Kim, C.H. and King, T.E. (1981) Biochem. Biophys. Res. Commun. 101, 607). Both complexes are considered to be possible intermediates in electron transfer reaction between these cytochromes. Triply substituted modified cytochrome c by pyridoxal phosphate at lysine residues (Lys-79, 86 and one to be identified) abolishes both complex formations and electron transfer activity with succinate cytochrome c reductase or cytochrome oxidase.

Ascorbic Acid↗
PubMed2004

A successful treatment with pyridoxal phosphate for West syndrome in hypophosphatasia.

We report a 2-month-old male with West syndrome associated with infantile hypophosphatasia. The male infant was born at term to a healthy mother after an uneventful pregnancy. He was born by cesarean section because of breech presentation. He was observed to have short extremities, and radiographs were consistent with achondroplasia. The serum alkaline phosphatase level was 2 IU/dL. Intractable tonic seizures developed 2 days after birth, and an electroencephalogram revealed a burst-suppression pattern for the first 2 months of life. The seizures were uncontrollable with conventional antiepileptic drugs. At the age of 2 months, he had a series of infantile spasms, and the electroencephalogram indicated hypsarrhythmia. Treatment with high-dose pyridoxal phosphate eliminated his seizures.

Chromatography, High Pressure Liquid↗
PubMed1989

The peptide sequences near the bound pyridoxal phosphate are conserved in serine dehydratase from rat liver, and threonine dehydratases from yeast and Escherichia coli.

The blocked amino-terminal residue of rat liver serine dehydratase was shown to be acetylalanine by analysis of an isolated amino-terminal peptide after digestion with acylamino acid-releasing enzyme. Digestion of the borohydride-reduced, carboxymethylated enzyme with lysyl endopeptidase yielded a single epsilon-N-pyridoxyllysine-containing peptide, whose sequence is Met-Asp-Ser-Ser-Gln-Pro-Ser-Gly-Ser-Phe-Lys(Pxy)-Ile-Arg-Gly- His-Leu-Cys(Cm)-Lys. This peptide comprises residues 30-49 of the cDNA-deduced amino acid sequence. The sequence of seven amino acids around the bound pyridoxal phosphate is highly conserved in serine dehydratase from rat liver, and threonine dehydratases from yeast and Escherichia coli.

Amino Acid Sequence↗
PubMed1997

Substitutions of alanine for cysteine at a reactive thiol site and for lysine at a pyridoxal phosphate binding site of 1-aminocyclopropane-1-carboxylate deaminase.

1-Aminocyclopropane-1-carboxylate (ACC) deaminase catalyzes the cyclopropane ring fragmentation and deamination of ACC. Replacement of cysteine with alanine at a reactive thiol site, Cys-162, of ACC deaminase did not affect the enzyme activity, in spite of the previous result that modification of Cys-162 caused complete loss of the enzyme activity. Substitution of glycine or valine for the cysteine residue gave a higher Km for ACC without a significant change of the K0, indicating that changes of the amino acid side chain had structural effects on substrate binding. Replacement of lysine with alanine at the pyridoxal phosphate (PLP) binding site of the ACC deaminase caused a lower content of PLP and loss of detectable activity of ACC deamination. This mutant enzyme, K51A, showed absorption peaks at 330 nm and 405 nm. The peak at 405 nm was shifted to about 425 nm by the addition of ACC, D-, L-alanine, and D-, L-serine. The formation of aldimine complexes indicated by the spectral shift was reversible. It is suggested that lysine 51 affects the formation of holoenzyme and is important in catalysis.

Absorption↗
PubMedDate not supplied

[Effect of pyridoxal phosphate and pantothenate derivatives on gamma-aminobutyric shunt in the brain of mice].

Repeated administration of D-pantothenate Ca, D-homopantothenate Ca and pantethine at a dose of 150 mmole/kg of body weight (9 injections within 3 days) led to alterations in activity of GABA-shunt enzymes as well as in content of free amino acids in mouse brain. These alterations suggest that B3-vitamin preparations potentiate the regulating effect on gamma-aminobutyrate pathway, which shunts the alpha-ketoglutarate dehydrogenase reaction, depending on CoA content. The most distinct but short-term intesification of the GABA-shung was observed in treatment with homopantothenate containing GABA molecule. Pantothenate and pantethine caused similar alterations, consisting in activation of GABA production and in inhibition of its utilization; in this case pantethine was less effective. Pyridoxal phosphate activated GABA-shunt; this resulted in accelerated decrease in content of dicarboxylic amino acids and their amides.

Animals↗
PubMed1985

Effect of deficient and excess dietary vitamin B-6 on amino transaminase and glycogen phosphorylase activity and pyridoxal phosphate content in two muscles from postpubertal gilts.

Postpubertal gilts averaging 111 kg and gaining 2.7 kg/wk were fed daily 1.9 kg/d of a diet providing 0.45, 2.1 or 83 mg of vitamin B-6/d. An additional group of animals were fed the high vitamin B-6 diet providing 83 mg of vitamin B-6/d for the initial 57 d of the experiment and then switched to 0.45 mg of vitamin B-6/d for the remainder of the 121-d experiment (61 gilts total). On d 0, 57 and 121, animals from each treatment were killed, and samples of the semitendinosus (ST) and semimembranosus (SM) were removed. Glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), glycogen phosphorylase and pyridoxal phosphate (PLP) were measured in muscle tissues. The erythrocyte GOT activity coefficient indicated that gilts consuming 0.45 or 2.1 mg of vitamin B-6/d developed a vitamin B-6 deficiency. A vitamin B-6 deficiency resulted in the loss of whole-muscle transaminase activity (enzyme activity X muscle weight) with little effect on whole-muscle total phosphorylase or total PLP content. Excess dietary vitamin B-6 increased whole-muscle total PLP and total phosphorylase content with small decreases in whole-muscle transaminase. Under these conditions, muscle tissue acts as a nonmobile reservoir of PLP. Sixty to 95% of muscle PLP was bound to muscle glycogen phosphorylase.

Alanine Transaminase↗
PubMed2004

Characterization of two kinases involved in thiamine pyrophosphate and pyridoxal phosphate biosynthesis in Bacillus subtilis: 4-amino-5-hydroxymethyl-2methylpyrimidine kinase and pyridoxal kinase.

Two Bacillus subtilis genes encoding two proteins (currently annotated ThiD and YjbV) were overexpressed and characterized. YjbV has 4-amino-5-hydroxymethyl-2-methylpyrimidine and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate kinase activity and should be reannotated ThiD, and B. subtilis ThiD has pyridoxine, pyridoxal, and pyridoxamine kinase activity and should be reannotated PdxK.

Bacillus subtilis↗
PubMed1984

Interactions of tryptophan synthase, tryptophanase, and pyridoxal phosphate with oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan: support for an indolenine intermediate in tryptophan metabolism.

We have examined the interaction of tryptophan synthase and tryptophanase with the tryptophan analogues oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan. Since these analogues have tetrahedral geometry at carbon 3 of the heterocyclic ring, they are structurally similar to the indolenine tautomer of L-tryptophan, a proposed intermediate in reactions of L-tryptophan. Oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan are potent competitive inhibitors of both tryptophan synthase and tryptophanase, with KI values (3-17 microM) 10-100-fold lower than the corresponding Km or KI values for L-tryptophan. Addition of oxindolyl-L-alanine or 2,3-dihydro-L-tryptophan to solutions of the alpha 2 beta 2 complex of tryptophan synthase results in new absorption bands at 480 or 494 nm, respectively, which are ascribed to a quinonoid or alpha-carbanion intermediate. Spectrophotometric titration data give half-saturation values of 5 and 25 microM, which are comparable to the KI values obtained in kinetic experiments. Our finding that both enzymes catalyze incorporation of tritium from 3H2O into oxindolyl-L-alanine is evidence that both enzymes form alpha-carbanion intermediates with oxindolyl-L-alanine. These results support the proposal that the indolenine tautomer of L-tryptophan is an intermediate in reactions catalyzed by both tryptophanase and tryptophan synthase. In addition, we have found that oxindolyl-L-alanine reacts irreversibly with free pyridoxal phosphate to form a covalent adduct.

Alanine↗
PubMed1999

Influence of folic acid, pyridoxal phosphate and cobalamin on plasma homocyst(e)ine levels and the susceptibility of low-density lipoprotein to ex-vivo oxidation.

Mild hyperhomocyst(e)inaemia is a risk factor for atherosclerotic vascular disease. In-vitro studies have shown that autooxidation of homocyst(e)ine is accompanied by the generation of oxygen radicals. This may lead to oxidative modification of low-density lipoproteins (LDL) and promote atherosclerotic vascular lesions. In male patients with peripheral arterial occlusive disease we determined fasting and post methionine load homocyst(e)ine levels by high performance liquid chromatography and the susceptibility of their LDL particles to ex-vivo oxidation by continously measuring the conjugated diene production induced by incubation with copper ions. Oxidation resistance (expressed as lag time), maximal oxidation rate, and extent of oxidation (expressed of total diene production) of LDL from patients with normal or mildly elevated homocyst(e)ine levels did not differ significantly. Folic acid, pyridoxal phosphate and cobalamin supplementation significantly decreased plasma homocyst(e)ine levels in hyperhomocyst(e)inaemic patients. This went along with a significant decrease in the extent of LDL oxidation and additionally increased HDL-cholesterol levels. The clinical relevance of these findings for the long-term course of atherosclerotic vascular disorders has to be determined by intervention studies.

Adult↗
PubMed1994

Binding of glucose, galactose and pyridoxal phosphate to lens crystallins.

Glycation of proteins plays an important role in diabetic complications. Both glucose and galactose were shown to bind progressively to lens crystallins with decreased binding in the presence of increasing concentrations of pyridoxal 5-phosphate (PLP). In longer term incubations (10 mM sugar for 21 days) glucose produced no significant yellowing of the protein, that is no detectable advanced glycation products, but pyridoxal phosphate (15 mM) caused an increased absorbance at 325 nm. This increase was greater in the presence of glucose. It appears that PLP becomes firmly attached to the protein and that this binding is enhanced in the presence of glucose or galactose. Changes produced by sodium borohydride indicate that the PLP is attached to protein amino groups as a Schiff base. Incubation of lens crystallins with PLP also led to increased fluorescence which was greater when sugar was present. However, borohydride experiments indicated that glucose and galactose may decrease the formation of non-reducible adducts of PLP. The decreased glycation in the presence of PLP supports the notion that it might be useful in prevention of diabetic complications, but the reaction of PLP itself with protein is less encouraging.

Animals↗
PubMed1982

Inactivation of pyridoxal phosphate enzymes by gabaculine. Correlation with enzymic exchange of beta-protons.

Gabaculine, 5-amino-1,3-cyclohexadienylcarboxylate, is a very efficient enzyme-activated inhibitor of gamma-aminobutyrate transaminase (Rando, R. R. (1977) Biochemistry 16, 4604-4610). However, enzymes for which gamma-aminobutyrate is not a substrate are also inactivated by gabaculine. Thus, purified D-amino acid transaminase, L-alanine transaminase, and L-aspartate transaminase are also inactivated (Ki values of 0.1 mM, 1 mM, and 55 mM, respectively). The effects of this inhibitor on such a diverse group of enzymes appear to be related to the enzymic exchange of beta-protons of their normal substrates. L-Alanine transaminase and L-aspartate transaminase are known to catalyze such an exchange (Walter, U., Luthe, H., Gerhart, F., and Söling, H.-D. (1975) Eur. J. Biochem. 59, 395-403). D-Amino acid transaminase and gamma-aminobutyrate transaminase, which are inactivated by gabaculine, also catalyze exchange of the beta-protons of their substrates. Alanine racemase and tryptophanase, which are known not to catalyze an analogous exchange, were found to be insensitive to gabaculine. We postulate that aromatization of gabaculine, in which the beta-proton is removed, is an enzyme-catalyzed event for those pyridoxal phosphate enzymes that have a nucleophilic group at the active site to catalyze this process.

4-Aminobutyrate Transaminase↗
PubMed1983

Comparison of pyridoxal phosphate and 0.4 M KCl-extracted nuclear glucocorticoid receptors in HeLa S3 cells.

A comparison of the physicochemical properties between pyridoxal 5'-phosphate- and 0.4 M KCl-extracted nuclear glucocorticoid receptors has been made utilizing HeLa S3 cells as a source of receptor. Both pyridoxal 5'-phosphate/NaBH4-reduced and 0.4 M KCl-extracted receptors sedimented as approximately 3.5-4.5 S species in 5-20% sucrose gradients containing 0, 0.15, and 0.4 M KCl. Under low-ionic-strength buffer conditions, pyridoxal 5'-phosphate-extracted receptor elutes close to the void volume of a Sephacryl S-300 gel-exclusion column. Increasing the [KCl] of the column to 0.4 M resulted in the elution of receptor with a Stokes radius of 58 A and calculated Mr = 96,000. Nuclear receptors extracted with 0.4 M KCl also formed a large-molecular-weight complex which eluted close to the void volume of the gel-exclusion column. Increasing the [KCl] to 0.4 M had the effect of shifting this receptor form to a species which had a Stokes radius of 62 A and calculated Mr = 89,700. Ion-exchange analysis of nuclear-extracted receptors revealed that 0.4 M KCl-extracted receptors exhibited considerable charge heterogeneity, whereas pyridoxal 5'-phosphate-extracted receptors did not. Pyridoxal 5'-phosphate-extracted receptors (approximately 86%) eluted from DEAE-cellulose at a [KCl] greater than 0.15 M; approximately 14% of the receptors had little affinity for DEAE-cellulose. Pyridoxal phosphate-treated receptors had little affinity for hydroxylapatite, phosphocellulose, and DNA-cellulose. The predominant form of 0.4 M KCl-extracted nuclear receptors (approximately 78%) eluted from DEAE-cellulose between 0.05 and 0.15 M KCl, a position coincident with "activated" glucocorticoid receptors. The remaining receptor fraction (approximately 22%) eluted from DEAE-cellulose at a [KCl] greater than 0.15 M, a position coincident with "unactivated" glucocorticoid receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Nucleus↗
PubMed1998

In vivo formation of a Schiff base of aminoguanidine with pyridoxal phosphate.

Aminoguanidine (AG) is considered to be a promising compound for the treatment of diabetic complications. We examined the in vitro and in vivo formation of Schiff bases of AG with pyridoxal 5'-phosphate (PLP) and pyridoxal (PL). AG reacted in vitro far more rapidly with PLP to form a Schiff base (PLP-AG) than with PL to form another Schiff base (PL-AG). Administration of AG at 7 mM in drinking water for 18 weeks caused the formation of PLP-AG in the liver and kidney of mice (12.1 +/- 1.6 and 3.8 +/- 0.64 nmol/g of tissue, respectively, mean +/- SD, N = 6). The amount of PLP in the liver of mice AG administered was significantly lower than that of control mice (4.0 +/- 1.4 vs 17.4 +/- 1.3 nmol/g of wet tissue, mean +/- SD, N = 6). Simultaneous administration of pyridoxine (1 mM in drinking water) with AG (7 mM in drinking water) did not ameliorate the decrease in tissue PLP and caused the excess formation of PLP-AG. The results suggest that attention should be paid to the deficiency of tissue PLP in the clinical use of AG.

Animals↗
PubMed2001

Structure-activity relationships of pyridoxal phosphate derivatives as potent and selective antagonists of P2X1 receptors.

Novel analogues of the P2 receptor antagonist pyridoxal-5'-phosphate 6-azophenyl-2',5'-disulfonate (2) were synthesized and studied as antagonists in functional assays at recombinant rat P2X1, P2X2, and P2X3 receptors expressed in Xenopus oocytes (ion flux stimulation) and at turkey erythrocyte P2Y1 receptors (phospholipase C activation). Selected compounds were also evaluated as antagonists of ion flux and the opening of a large pore at the recombinant human P2X7 receptor. Modifications were made in the 4-aldehyde and 5'-phosphate groups of the pyridoxal moiety: i.e. a CH2OH group at the 4-position in pyridoxine was either condensed as a cyclic phosphate or phosphorylated separately to form a bisphosphate, which reduced potency at P2 receptors. 5-Methylphosphonate substitution, anticipated to increase stability to hydrolysis, preserved P2 receptor potency. At the 6-position, halo, carboxylate, sulfonate, and phosphonate variations made on the phenylazo ring modulated potency at P2 receptors. The p-carboxyphenylazo analogue, 4, of phosphate 2 displayed an IC50 value of 9 nM at recombinant P2X1 receptors and was 1300-, 16-, and > 10,000-fold selective for P2X1 versus P2X2, P2X3, and P2Y1 subtypes, respectively. The corresponding 5-methylphosphonate was equipotent at P2X1 receptors. The 5-methylphosphonate analogue containing a 6-[3,5-bis(methylphosphonate)]phenylazo moiety, 9, had IC50 values of 11 and 25 nM at recombinant P2X1 and P2X3 receptors, respectively. The analogue containing a phenylazo 4-phosphonate group, 11, was also very potent at both P2X1 and P2X3 receptors. However, the corresponding 2,5-disulfonate analogue, 10, was 28-fold selective for P2X1 versus P2X3 receptors. None of the analogues were more potent at P2X7 and P2Y1 receptors than 2, which acted in the micromolar range at these two subtypes.

Adenosine Triphosphate↗