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PubMed1983

Pyridoxal 5'-phosphate deficiency in uremic undialyzed, hemodialyzed, and non-uremic kidney transplant patients.

In this study, we have investigated plasma pyridoxal 5'-phosphate (PLP) concentrations in undialyzed and dialyzed uremic patients and in kidney transplant subjects, using an enzymatic technique with thermal deproteinization to liberate PLP from plasma proteins. The specificity of the reaction indicates no interference with pyridoxal and only 3% interference with pyridoxamine phosphate. In 17 hemodialyzed patients, a deficiency of about 50% of plasma PLP concentration is found as compared to 25 healthy subjects (22.2 +/- 2.47 vs. 48.8 +/- 3.00 nmol . l-1), as mean +/- SEM). In seven undialyzed uremic patients with end-stage renal failure, the plasma PLP concentration is also decreased (29.3 +/- 1.74 nmol . l-1). The absence of PLP in plasma ultrafiltrates demonstrates that no loss of PLP occurs due to hemodialysis. The daily oral supplementation with 250-750 mg pyridoxal induces a supraphysiological increase in plasma PLP concentration in hemodialyzed as well as in undialyzed patients. In 116 non-uremic kidney transplant subjects, the mean plasma PLP concentration was 33.8 +/- 3.50 nmol . l-1). In 65% of these patients, a marked deficit (below 20 nmol . l-1) was observed. In conclusion, uremic patients have a deficient vitamin B6 state. Its correction with pyridoxal to restore physiological plasma PLP concentration necessitates oral supplementation with lower doses that those widely used at present. In kidney transplant patients a similar plasma PLP deficiency is observed in the absence of chronic renal failure.

Adult↗
PubMed1988

Glutamate decarboxylase inhibition and vitamin B6 metabolism in brain of cirrhotic rats chronically treated with carbon tetrachloride.

In a previous work we found that the activity of glutamate decarboxylase (GAD), the enzyme responsible for the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), is decreased in comatose cirrhotic rats after chronic treatment with CCl4. In the present report we studied the participation of pyridoxal phosphate in the inhibition of GAD, as well as the concentration of this coenzyme and the activity of its synthesizing enzyme, pyridoxal kinase, in the brain of the cirrhotic rats. Furthermore, cirrhotic animals were treated with three inhibitors of GAD, and the effects of such treatment were compared to those of ammonium. Liver failure resulted in a 25% inhibition of GAD activity when measured in the absence of added pyridoxal phosphate. Treatment with the GAD inhibitors thiosemicarbazide or 3-mercaptopropionic acid enhanced this inhibition and produced convulsions at a dose that had no behavioral effects in control rats. Treatment with ammonia resulted in a comatose state and in a 25-40% inhibition of GAD. Both pyridoxal kinase activity and pyridoxal phosphate levels were found to be decreased by 15-20% in the brain of the cirrhotic rats. We concluded that chronic liver failure results in a decreased pyridoxal phosphate and GABA synthesis in brain, with a consequent diminished efficiency of GABAergic neurotransmission; these effects are probably related to the manifestations of neuronal hyperexcitability that are frequently seen in human hepatic encephalopathy.

Acetates↗
PubMed2002

Affinity labeling of the guanine nucleotide binding site of transducin by pyridoxal 5'-phosphate.

Transducin (T), a guanine nucleotide binding regulatory protein composed of alpha-, beta-, and gamma-subunits, serves as an intermediary between rhodopsin and cGMP phosphodiesterase during signaling in the visual process. Pyridoxal 5'-phosphate (PLP), a reagent that has been used to modify enzymes that bind phosphorylated substrates, was probed here as an affinity label for T. PLP inhibited the guanine nucleotide binding activity of T in a concentration dependent manner, and was covalently incorporated into the protein in the presence of [3H]NaBH4. Approximately 1 mol of 3H was bound per mol of T. GTP and GTP analogs appreciably hindered the incorporation of 3H to T, suggesting that PLP specifically modified the protein active site. Interestingly, PLP modified both the alpha- and beta-subunits of T. Moreover, PLP in the presence of GDP behaved as a GTP analog, since this mixture was capable of dissociating T from T:photoactivated rhodopsin complexes.

Animals↗
PubMed1978

The changes in activation of intracellular aspartate aminotransferase by pyridoxal 5-phosphate after cell death.

Aspartate aminotransferase activity was measured in plasma, in liver and in heart mitochondrial and cytoplasmic preparations from rats immediately after death and after a post-mortem interval of 15 h. No significant stimulation of activity on addition of pyridoxal 5-phosphate to the assay medium could be demonstrated in any preparations obtained immediately after death. Significant stimulation occurred in both cytoplasmic and mitochondrial preparations of liver and myocardium after a 15-h post-mortem interval, but not in plasma stored for the same period. It appears, therefore, that variations in the intracellular saturation of apoenzyme with coenzyme cannot account for the observed differences in activation of aspartate aminotransferase by pyridoxal 5-phosphate in sera from patients with myocardial infarction and liver disease. Changes in degree of saturation of apoenzyme seem to occur intracellularly after cell death or injury and before release into the circulation.

Animals↗
PubMed2006

APHO1 from the yeast Arxula adeninivorans encodes an acid phosphatase of broad substrate specificity.

The extracellular acid phosphatase-encoding Arxula adeninivorans APHO1 gene was isolated using degenerated specific oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1449 bp encoding a protein of 483 amino acids with a calculated molecular mass of 52.4 kDa. The sequence includes an N-terminal secretion sequence of 17 amino acids. The deduced amino acid sequence exhibits 54% identity to phytases from Aspergillus awamori, Asp. niger and Asp. ficuum and a more distant relationship to phytases of the yeasts Candida albicans and Debaryomyces hansenii (36-39% identity). The sequence contains the phosphohistidine signature and the conserved active site sequence of acid phosphatases. APHO1 expression is induced under conditions of phosphate limitation. Enzyme isolates from wild and recombinant strains with the APHO1 gene expressed under control of the strong A. adeninivorans-derived TEF1 promoter were characterized. For both proteins, a molecular mass of approx. 350 kDa, corresponding to a hexameric structure, a pH optimum of pH 4.8 and a temperature optimum of 60 degrees C were determined. The preferred substrates include p-nitrophenyl-phosphate, pyridoxal-5-phosphate, 3-indoxyl-phosphate, 1-naphthylphosphate, ADP, glucose-6-phosphate, sodium-pyrophosphate, and phytic acid. Thus the enzyme is a secretory acid phosphatase with phytase activity and not a phytase as suggested by strong homology to such enzymes.

Acid Phosphatase↗
PubMed1978

Tyrosine aminotransferase from chick liver. Heat activation and cold inactivation of the enzyme.

The reversible heat activation and cold inactivation of tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) of chick liver were investigated. When the enzyme obtained by gel filtration was preincubated at 37 degrees C for 10 min with 50 micrometer pyridoxal 5'-phosphate (pyridoxal-5'-P), a 7-fold increase in enzyme activity was detected. When the preincubated enzyme was cooled to 0 degrees C, it lost its activity. Furthermore, the dramatic cyclical changes in enzyme activity occurred by sequential heating at 37 degrees C and cooling to 0 degrees C of the enzyme, in the presence of pyridoxal-5'-P, over shorter periods of time without loss of enzyme activity. However, when alpha-ketoglutarate was added to the enzyme during cold exposure, no further decrease in activity was observed. This protective effect was seen at a concentration of 5 muM.

Animals↗
PubMed1976

The derivatization of oxidized polysaccharides for protein immobilization and affinity chromatography.

The present report describes the preparation of modified polysaccharides matrices useful for the synthesis of affinity adsorbents and immobilized proteins. Hydrazido-matrices were synthesized by condensing an excess of the bifunctional reagent, adipic acid dihydrazide, with periodate oxidized cellulose paper, Sephadex, or Sepharose matrices. Ribonucleotide dialdehyde cofactors, glyceraldehyde 3-phosphate, pyridoxal 5'-phosphate and oxidized DNAase B were separately bound to the hydrazido-polymers. Azido-matrices obtained by modification of the hydrazido-derivatives were coupled to specific amino ligands such as amino acids and proteins. Several adsorbents were prepared and used as models for affinity chromatography.

Aldehydes↗
PubMed1991

Plasma B6 vitamer and plasma and urinary 4-pyridoxic acid concentrations of middle-aged obese black women.

Plasma B6 vitamer and plasma and urinary 4-pyridoxic acid (4-PA) concentrations of fifteen middle-aged obese black women were determined by high-performance liquid chromatography (HPLC). Estimated protein and vitamin B6 intakes of the subjects, aged 27-52 years, were 64.5 +/- 15.6 g and 1.21 +/- 0.68 mg (mean +/- S.D.), respectively. Mean HPLC-derived plasma B6 vitamer and 4-PA concentrations for these subjects were 68.9, 3.1, 1.2, 4.1, 3.4, 7.2 and 2.0 nmol/l for pyridoxal 5'-phosphate (PLP), pyridoxine 5'-phosphate, pyridoxamine 5'-phosphate, pyridoxal, pyridoxine, pyridoxamine and 4-PA, respectively. The mean urinary 4-PA/creatinine ratio of the women was 0.88 mumol/mmol. All subjects had plasma PLP levels indicative of adequate vitamin B6 status. Vitamin B6 status parameters of the middle-aged obese black women were similar to those previously reported for white nonobese women having adequate vitamin B6 status.

Adult↗
PubMed1999

An appraisal of vitamin B6 status indices and associated confounders, in young people aged 4-18 years and in people aged 65 years and over, in two national British surveys.

OBJECTIVE: To compare vitamin B6 status indices with each other and with potential confounding factors, in the datasets from two national British surveys and associated studies. DESIGN: Vitamin B6 status was measured by plasma pyridoxal phosphate (PLP) and plasma pyridoxic acid (PA) in both surveys, and also by erythrocyte aspartate aminotransferase activation coefficient (EAATAC) in one of the surveys. Plasma alpha1-antichymotrypsin was measured as an index of acute phase status; plasma creatinine was measured as an index of renal function; and plasma total alkaline phosphatase activity was measured as a proxy for PLP hydrolase activity. SETTING: The survey of people aged 65 years and over was carried out in 80 postcode sectors across mainland Britain during 1994-95 and the survey of young people was carried out in 132 postcode sectors across mainland Britain during 1997. SUBJECTS: Blood samples from c. 1,000 subjects of both sexes in each survey permitted measurements of plasma PLP and PA. There were also measurements of EAATAC in the young people's survey. RESULTS: According to published limits of normality, only 5% or less of the young people had unacceptable vitamin B6 status as measured by plasma PLP. About half had apparently unacceptable status by EAATAC, but this observation is difficult to interpret. The young people had considerably higher plasma concentrations of PLP and lower concentrations of PA than the older people. In both surveys, plasma PLP was strongly correlated with plasma PA and in the young persons' survey it was also correlated, although much less strongly, with the basal activity and activation coefficient of aspartate aminotransferase. Both plasma PLP and EAATAC (but not PA nor basal aspartate aminotransferase activity) were influenced by acute phase status in young people, as indicated by significant correlations with alpha1-antichymotrypsin. In people aged 65 years and over, PA (but not PLP) was correlated with renal function, as indicated by its relation with plasma creatinine; however PLP (but not PA) was correlated with plasma alkaline phosphatase activity. CONCLUSIONS: Several potential confounders - acute phase reaction, kidney malfunction and hydrolase activity - may influence vitamin B6 status indices, although differently for different indices and different age groups. Since older people have relatively poor vitamin B6 status, which may have important health implications for them, more reliable vitamin B6 status indices are needed.

Adolescent↗
PubMed1991

Structure-function studies of substrate oxidation by bovine serum amine oxidase: relationship to cofactor structure and mechanism.

The chemical mechanism of substrate oxidation, catalyzed by bovine serum amine oxidase, has been explored by a detailed investigation of structure-reactivity correlations. Past mechanistic studies, involving the reductive trapping of substrate to cofactor [Hartmann, C., & Klinman, J. P. (1987) J. Biol. Chem. 262, 962], implied the intermediacy of a substrate imine complex in the catalytic redox mechanism. These studies led to the proposal of a transamination mechanism for substrate oxidation, analogous to pyridoxal phosphate dependent enzymes. In pyridoxal phosphate catalyzed reactions, the transamination process involves the transient formation of a resonance-stabilized carbanion intermediate. Although evidence has been presented describing the participation of an active site base in bovine serum amine oxidase catalysis [Farnum, M. F., Palcic, M. M., & Klinman, J. P. (1986) Biochemistry 25, 1898], the nature of the intermediate derived from C-H bond cleavage has not been directly addressed. To examine this question, a structure-reactivity study was performed using a series of para-substituted benzylamines. Having prior knowledge of the intrinsic isotope effect for an enzymatic reaction permits calculation of microscopic rate constants from steady-state data [Palcic, M. M., & Klinman, J. P. (1983) Biochemistry 22, 5957]. Deuterium isotope effects on kcat and kcat/Km parameters were determined for all substrates, allowing for the calculation of rate constants for C-H bond cleavage (k3) and substrate dissociation constants (Kd). Pre-steady-state constants obtained for p-acetylbenzylamine, p-(trifluoromethyl)benzylamine, and unsubstituted benzylamine exhibited excellent agreement with values calculated from steady-state isotope effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Amine Oxidase (Copper-Containing)↗
PubMed1987

Purification and properties of Escherichia coli 4'-phosphopantothenoylcysteine decarboxylase: presence of covalently bound pyruvate.

4'-Phosphopantothenoylcysteine decarboxylase was purified 900-fold from Escherichia coli B with an overall yield of 6%. The enzyme migrates as a single band with a molecular weight of 35,000 +/- 3000 in 10% polyacrylamide gel electrophoresis under denaturing conditions. The native enzyme has an apparent molecular weight of 146,000 +/- 9000 as determined by a gel exclusion column. At pH 7.6 and 25 degrees C, Km = 0.9 mM and Vmax = 600 nmol/(min X mg of protein). The pH optimum for Vmax is between 7.5 and 7.7. Hydroxylamine, phenylhydrazine, potassium cyanide, and sodium borohydride as well as pyridoxal phosphate and pyridoxal inactivated the enzyme. The enzyme contains covalently bound pyruvate as suggested by the isolation of [3H]lactate and pyruvate from [3H]NaBH4-reduced enzyme and native enzyme, respectively. One mole of [3H]lactate was isolated per 39,000 g of [3H]NaBH4-reduced and completely inactivated enzyme, and 1 mol of pyruvate was isolated per 31,000 +/- 4000 g of native enzyme. Mild base treatment released lactate and pyruvate from the reduced and the native enzymes, respectively, suggesting the pyruvate is attached to the enzyme by an ester bond. These findings are in accord with similar results obtained with the horse liver enzyme (R. Scandurra, personal communication). The presence of covalently bound pyruvate in the bacterial and mammalian enzymes suggests that pyruvate plays a major role in the mechanism of action.

Binding Sites↗
PubMed1993

Properties of the pyridoxaldimine form of glutamate semialdehyde aminotransferase (glutamate-1-semialdehyde 2,1-aminomutase) and analysis of its role as an intermediate in the formation of aminolaevulinate.

Glutamate semialdehyde aminotransferase (glutamate-1-semialdehyde 2,1-aminomutase; EC 5.4.3.8) was converted into its pyridoxaldimine form by exhaustive replacement of endogenous pyridoxamine phosphate with pyridoxal phosphate. The isomerization of glutamate 1-semialdehyde to 5-aminolaevulinate by this form of the enzyme followed an accelerating time course which indicated that the enzyme initially had no activity but was converted into the active pyridoxamine phosphate form in an exponential process characterized by a rate constant (k) of 0.027 s-1. The pyridoxaldimine form of the enzyme was converted rapidly into the pyridoxamine form by (S)-4-aminohex-5-enoate and much more slowly by 4-aminobutyrate. The steady-state velocity of the enzyme increased in a markedly non-linear fashion with increasing enzyme concentration, indicating that the extent of dissociation of an intermediate in the reaction to free diaminovalerate and the pyridoxaldimine form of the enzyme depends upon the concentration of the enzyme.

Aminocaproates↗
PubMed1983

The effect of wheat bran on the bioavailability of vitamin B-6 in young men.

The effect of cooked wheat bran on the bioavailability of vitamin B-6 (B-6) was determined in 10 men, aged 20 to 35 years. The subjects consumed a constant diet with and without the addition of 15 g wheat bran during three successive 18-day periods in a switch-back design. Half of the subjects received the additional bran during periods 1 and 3; the other half consumed the bran during period 2. The bran and nonbran diets supplied, respectively, 1.69 and 1.66 mg of B-6 daily. Plasma total B-6 and pyridoxal phosphate (PLP), urinary 4-pyridoxic acid (4-PA), and urinary and fecal B-6 were determined at regular intervals during each period. Bran significantly increased fecal B-6 (P less than 0.05) and decreased urinary 4-PA (P less than 0.01). Bran also significantly depressed plasma B-6 (P less than 0.01) and PLP (P less than 0.05). These results indicate that the addition of 15 g of wheat bran to the diet decreased the bioavailability of B-6. Since this decrease was modest, never exceeding 17% (based on urinary 4PA and B-6), this amount of bran will not adversely affect B-6 status when intake of the vitamin is adequate.

Adult↗
PubMed1991

Insensitivity of the tryptophan-load test to marginal vitamin B-6 intake in rats.

The tryptophan-load test for vitamin B-6 nutritional status was administered to adult female Long-Evans rats fed graded levels of pyridoxine hydrochloride (PN.HCl) in two experiments, and its sensitivity to marginal vitamin B-6 intake was evaluated. In Experiment 1, rats were 4-h meal-fed an AIN-76A (20% casein) diet devoid of PN.HCl for 3 wk, then repleted (n = 12) for 6 wk with 4-h pair-fed meals of either 0.25, 0.5, 1.0 or 7.0 (control) mg PN.HCl/kg diet. In Experiment 2, rats (n = 16) were pair-fed for 10 wk either 0.0, 0.5, 1.0 or 7.0 (control) mg PN.HCl/kg diet, with 24-h access to food. Vitamin B-6 nutritional status was assessed at the end of each experiment. Except in rats fed 0 mg PN.HCl/kg diet, mean body weights were not significantly different among diet groups of either experiment. Plasma pyridoxal phosphate (PLP), pyridoxal and total vitamin B-6 concentrations, determined by HPLC, were very sensitive to gradations in dietary PN.HCl concentrations (P less than 0.05). Red blood cell endogenous and PLP-stimulated alanine and aspartate aminotransferase activity did not statistically differentiate all levels of dietary vitamin B-6, although the calculated activity coefficient for each enzyme (stimulated/endogenous activity) did. Urinary xanthurenic acid excretion following a tryptophan load [24.5 mumol (5 mg) L-tryptophan/100 g body weight, injected intraperitoneally] was significantly (P less than 0.05) elevated compared with controls only in the group fed 0 mg PN/HCl/kg diet. At the tryptophan dose used here, the tryptophan-load test was not useful in detecting marginal vitamin B-6 intake in rats.

Administration, Oral↗
PubMed1992

Transport Processes and Corresponding Changes in Metabolite Levels in Relation to Starch Synthesis in Barley (Hordeum vulgare L.) Etioplasts.

Intact etioplasts with an intactness of 85% and with a cytosolic and a mitochondrial contamination of less than 10% were isolated from 8-d-old dark-grown barley (Hordeum vulgare) leaves. These plastids contained starch equivalent to 21.5 mumol of glucose per mg protein. From various likely precursors applied to isolated etioplasts, only dihydroxyacetone phosphate (DHAP) had significant effects on metabolite levels and on the internal ATP/ADP ratio. The concentration dependence of DHAP uptake exhibited saturation characteristics with half saturation at 0.36 mm DHAP and a maximal velocity of 6.6 mumol mg(-1) of protein h(-1). The transport was significantly inhibited by inorganic phosphate, pyridoxal-5'-phosphate, and 4,4'-diisothiocyano-2,2'-stilbenedisulfonate. The rate of glucose-6-phosphate uptake was much lower and not saturable up to a concentration of 10 mm. Exogenously applied [(14)C]DHAP was incorporated into starch at a rate of 0.14 mumol of DHAP mg(-1) of protein h(-1). Enzyme activities required to convert DHAP into starch were found to be present in etioplasts. Furthermore, enzymes generating ATP from DHAP for ADPglucose synthesis were also detected. Finally, a scheme is presented suggesting DHAP uptake to serve both as carbon skeleton and as energy source for starch synthesis, mediated by a translocator with properties similar to those of the triose phosphate translocator from chloroplasts.

Journal Article↗
PubMed1989

Interaction of rat liver glucocorticoid receptor with histones.

The activated glucocorticoid-receptor complexes (GRC) from rat liver bind tightly to histone (from calf thymus)-agarose and cannot be eluted with 3 M KCl or 50% ethylene glycol, but can be eluted with 20 mM pyridoxal 5'-phosphate (PALP). Purified activated GRC were found to have much higher affinity for histones H3 and H4 (arginine-rich histones) than for histones H2A and H2B (slightly lysine-rich histones) and to have negligible affinity for histone H1 (lysine-rich histone). The GRC bound to immobilized histones could be eluted with PALP, but not with its related compounds, such as pyridoxamine, pyridoxamine 5'-phosphate, pyridoxal, and pyridoxine, suggesting a specific effect of PALP. Not only activated GRC but also unactivated GRC were found to bind to immobilized histones. However, from their profiles of elution from a histone-agarose column by 20 mM PALP, the activated GRC seemed to have higher affinity than unactivated GRC for histone. Our results suggest that the binding of GRC to histones could be associated with a mechanism for alteration of the chromosome structure by the hormonal signals, before the binding of the GRC to a specific sequence of DNA in regulatory elements of glucocorticoid-responsible genes.

Animals↗