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PubMed1980

Pyridoxal phosphate concentration in blood in newborn infants and their mothers compared with the amount of extra pyridoxol taken during pregnancy and breast feeding.

The concentrations of pyridoxal phosphate have been estimated in cord blood and capillary blood samples taken at 3 hours, 2 days, 4 days, 7 days and 6 weeks of age, from eleven full-term infants. Pyridoxal phosphate concentrations were also determined in venous blood samples taken from the mothers at delivery. A highly significant correlation between pyridoxal phosphate in cord whole blood and venous whole blood taken from the mothers at delivery was found. Infants whose mothers had taken extra pyridoxol during pregnancy had a higher concentration of pyridoxal phosphate at 3 hours of age compared with infants whose mothers had not taken extra pyridoxol. During the first week of life the concentrations of pyridoxal phosphate in capillary blood decreases strikingly. At 6 weeks of age the concentration of pyridoxal phosphate is in the same range as that of normal adults. Findings are also discussed which indicates that: 1) Vitamin B6 is transported in breast milk; 2) The giving of supplemental pyridoxol during pregnancy in ordinary doses (2-6 mg/day) does not have an antilactogenic effect. No correlation between the erythrocyte aspartate aminotransferase activation with pyridoxal phosphate in vitro and pyridoxal phosphate concentration in plasma was found during the first 6 weeks of life.

Adolescent↗
PubMed1985

Effect of vanadate and pyridoxal phosphate on S-adenosylmethionine.

Vanadate in the presence of pyridoxal phosphate promotes the decarboxylation of S-adenosylmethionine. Pyridoxal has a lower effect; pyridoxine none. The rate of decarboxylation depends on pyridoxal phosphate and vanadate concentration. Vanadate as low as 10(-7) M gives significant decarboxylation. The reaction seems to occur through the formation of a Schiff base. The spectral shift elicited by S-adenosylmethionine on pyridoxal phosphate due to the presence of the sulfonium function is influenced by vanadate. Orthovanadate is a little less effective then metavanadate; vanadyl sulfate is even less efficient, and the effect of Cu2+ at the same concentration is still lower. Bleomycin partially prevents the vanadium effect. In vivo, vanadate promotes a marked increase in chicken liver S-adenosylmethionine and S-adenosylhomocysteine concentration, whereas the polyamine concentration is unaffected.

Animals↗
PubMed1975

Effects of buffers on aspartate aminotransferase activity and association of the enzyme with pyridoxal phosphate.

Using purified enzymes of human origin and patients' sera, we examined factors influencing the in vitro association of pyridoxal phosphate with aspartate aminotransferase (EC 2.6.1.1). The rate of association was markedly retarded by phosphate buffer in comparison with tris(hydroxymethyl)aminomethane or six other buffers. Pyridoxal phosphate at an incubation concentration of 130 mumol/liter reactivated the entire apoenzyme portion of an apoenzyme/holoenzyme mixture within 5 min in tris(hydroxymethyl)aminomethane; in contrast, less than 20% was associated during 15 min in phosphate. Activity measured in tris(hydroxymethyl)aminomethane-buffer without exogenous pyridoxal phosphate was 4% greater than that in phosphate and was slightly increased by increasing the pH of the assay mixture from 7.5 to 8.0. Aspartate in the incubation medium did not retard the stimulation in tris(hydroxymethyl)aminomethane buffer. While the magnitude of stimulation varied greatly among sera, a consistent mean stimulation of 30% for groups of sera with normal activities was found when asparate at 125 mmol/liter, 2-oxoglutarate at 6.7 mmol/liter and tris(hydroxymethyl)aminomethane at 90 mmol/liter were used, an increase over the 16% with phosphate buffer [Clin. Chem. 19, 92 (1973)]. Absorbance spectra suggest pyridoxal phosphate exists as the Schiff base of tris(hydroxymethyl)aminomethane or aspartate, or both, under conditions of assay incubation (without addition of 2-oxoglutarate). Nonenzymatic catalysis of the reaction by pyridoxal phosphate alone or a formation of a protein/pyridoxal phosphate adduct was discounted with use of a D-asparate substrates.

Animals↗
PubMed1981

Subcellular localization and properties of pyridoxal phosphate phosphatases of human polymorphonuclear leukocytes and their relationship to acid and alkaline phosphatase.

Using a novel fluorimetric assay for pyridoxal phosphate phosphatase, human polymorphonuclear leucocytes were found to exhibit both acid an alkaline activities. The neutrophils were homogenised in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrigfugation. The alkaline pyridoxal phosphate phosphatase showed a very similar distribution to alkaline phosphatase an was located solely to the phosphasome granules. Fractionation experiments on neutrophils treated with isotonic sucrose containing digitonin and inhibitor studies with diazotised sulphanilic acid and levamisole further confirmed that both enzyme activities had similar locations and properties. Acid pyridoxal phosphate phosphatase activity was located primarily to the tertiary granule with a partial azurophil distribution. Fractionation studies on neutrophils homogenised in isotonic sucrose containing digitonin and specific inhibitor studies showed that acid pyridoxal phosphate phosphatase and acid phosphatase were not the result of a single enzyme activity, Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (munits/mg protein) of alkaline pyridoxal phosphate phosphatase an alkaline phosphatase varied widely in the three groups and the alterations occurred in a parallel manner. The specific activities of acid pyridoxal phosphate phosphatase and of acid phosphatase were similar in the three groups. These results, together with the fractionation experiments and inhibition studies strongly suggest that pyridoxal phosphate is a physiological substrate for neutrophil alkaline phosphatase.

Acid Phosphatase↗
PubMed1988

Neurospora tryptophan synthase. Characterization of the pyridoxal phosphate binding site.

Tryptophan synthase, which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein that requires pyridoxal phosphate for two of its three distinct enzyme activities. Tryptophan synthase from Neurospora crassa, a homodimer of two 75-kDa subunits, was shown to bind 1 mol of pyridoxal phosphate/mol of subunit with a calculated dissociation constant for pyridoxal phosphate of 1.1 microM. The spectral properties of the holoenzyme, apoenzyme, and reconstituted holoenzyme were characterized and compared to those previously established for the heterotetrameric (alpha 2 beta 2) enzyme from Escherichia coli. The Schiff base formed between pyridoxal phosphate and the enzyme was readily reduced by sodium borohydride, but not sodium cyanoborohydride. The active site residue that binds pyridoxal phosphate, labeled by reduction of the Schiff base with tritium-labeled sodium borohydride, was determined to be lysine by high performance liquid chromatography analysis of the protein hydrolysate. A 5400-dalton peptide containing the reduced pyridoxal phosphate moiety was generated by cyanogen bromide treatment, purified and sequenced. The sequence is 85% homologous with the corresponding sequence obtained for yeast tryptophan synthase (Zalkin, H., and Yanofsky, C. (1982) J. Biol. Chem. 257, 1491-1500); the lysine derivatized by pyridoxal phosphate is located at the same relative position as that in the yeast and E. coli enzymes.

Amino Acid Sequence↗
PubMed1976

Inactivation of porcine heart cytoplasmic malate dehydrogenase by pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate (pyridoxal-5'-P) has been found to act as a bifunctional reagent during the inactivation of porcine heart cytoplasmic malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37). The biphasic kinetics and X-azolidine-like structure formed were similar to those observed for mitochondrial malate dehydrogenase (Wimmer, M.J., Mo, T., Sawyers, D.L., and Harrison, J.H. (1975) J. Biol. Chem. 250, 710-715). In the cytoplasmic enzyme, however, irreversible inactivation representing X-azolidine formation was found to be the dominant characteristic of the interaction with pyridoxal-5'-P. Spectral evidence indicated that at total inactivation 2 mol of pyridoxal-5'-P were incorporated per mol of enzyme or one pyridoxal-5'-P per enzymatic active site. The presence of NADH protected the enzyme from inactivation suggesting interaction of pyridoxal-5'-P at or near the enzymatic active centers of this enzyme. Fluorometric titrations indicated that pyridoxal-5'-P-inactivated enzyme failed to bind NADH or at least failed to bind NADH in the same fashion as native enzyme.

Cytoplasm↗
PubMed1983

Levels and subcellular localisation of pyridoxal and pyridoxal phosphate in human polymorphonuclear leukocytes and their relationship to alkaline phosphatase activity.

Neutrophil leukocytes, isolated from normal subjects and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation, showed very similar cytosol distributions of pyridoxal and pyridoxal phosphate and lactate dehydrogenase. The small amounts of pyridoxal and pyridoxal phosphate associated with the dense granule fractions were not associated with the alkaline phosphatase containing granules. The levels of pyridoxal and pyridoxal phosphate were determined in neutrophils from control subjects, women in the third trimester of pregnancy and patients with chronic granulocytic leukaemia. Neutrophil pyridoxal phosphate was increased in women in the third trimester of pregnancy compared to controls, but there was little variation in the level of pyridoxal between the groups. There was no consistent correlation between the pyridoxal phosphate and the neutrophil alkaline phosphatase activity in the patient groups. Although in vitro neutrophil alkaline phosphatase rapidly hydrolyses pyridoxal phosphate, it is suggested that in vivo this is unlikely to be the principal function of the enzyme.

Alkaline Phosphatase↗
PubMed1983

A fluorimetric method for the measurement of pyridoxal and pyridoxal phosphate in human plasma and leucocytes, and its application to patients with sideroblastic marrows.

A highly sensitive fluorimetric assay for the measurement of pyridoxal and pyridoxal phosphate in biological tissues is described. The method involves the enzymic hydrolysis of pyridoxal phosphate to pyridoxal. The pyridoxal (free or total) is separated on an anion-exchange column, concentrated by cation-exchange chromatography and reacted with potassium cyanide under slightly alkaline conditions to form 4-pyridoxolactone, a highly fluorescent compound. The method is applied to the measurement of pyridoxal, pyridoxal phosphate and total pyridoxal in plasma and neutrophils from control subjects and patients with sideroblastic marrow and identified the patient with pyridoxine-responsive sideroblastic anaemia.

Anemia, Sideroblastic↗
PubMed1982

Pyridoxal phosphate blocks aggregation of molybdate treated glucocorticoid receptors in HeLa S3 cells.

We have compared the effects of sodium molybdate and pyridoxal phosphate on the sedimentation properties of cytoplasmic glucocorticoid receptors. Whole HeLa S3 cells were incubated with [3H]-Dexamethasone (Dex) at 0-4 degrees C to produce unactivated cytoplasmic steroid receptors. When these cells are lysed in 10 mM Tris, 1 mM EDTA, 12 mM alpha-thioglycerol pH 7.4 (buffer A), [3H]-Dex receptor sediment as 7-8S species in sucrose gradients prepared in the same buffer. Sedimentation of receptors on gradients containing buffer A plus 20 mM sodium molybdate (buffer B) results in an approximately 9S species. Treatment of these receptors with pyridoxal phosphate (10 mM) followed by reduction with NaBH4 and sedimentation on sucrose gradients prepared in either buffer A or B results in the production of 3-4S species. Although [3H]-Dex labeled cells lysed in buffer B yield approximately 9S species when analyzed in gradients prepared in buffer B, centrifugation of these receptors in buffer A yield 7-8S species. Similarly when cells are lysed in buffer A labeled receptors sediment in sucrose gradients prepared in buffer B as approximately 9S species. Receptors prepared in buffer B and treated with pyridoxal phosphate and NaBH4 sediment in buffer B gradients as two discrete approximately 7S and approximately 3.5S species. Pyridoxal phosphate and NaBH4 treated receptors prepared in buffer B sediment as disperse 3-6S species when analyzed in buffer A gradients. Based on these observations we conclude that pyridoxal phosphate, NaBH4 reduced receptors are not particularly susceptible to subsequent molybdate action, but molybdate pre-treated receptors can be influenced by pyridoxal phosphate.

Centrifugation, Density Gradient↗
PubMed1986

Experimental diabetes causes mitochondrial loss and cytoplasmic enrichment of pyridoxal phosphate and aspartate aminotransferase activity.

The streptozotocin diabetic rat was selected as a model to study how insulin deficiency alters vitamin B6 utilization by focusing on pyridoxal phosphate levels and aspartate aminotransferase activities in liver tissues. Diabetes of 15 weeks' duration lowered plasma pyridoxal phosphate levels by 84%. Normal plasma pyridoxal phosphate was 480 pmole/ml. Fractionation of liver into mitochondrial and extramitochondrial compartments demonstrated that diabetes caused a 43% diminution in mitochondrial pyridoxal phosphate per gram of liver. There was no cytoplasmic change in these diabetic rats. Mitochondrial aspartate aminotransferase activity was decreased 53% per gram of diabetic liver and cytoplasmic aspartate aminotransferase activity was elevated 3.4-fold. Damage to diabetic mitochondria during preparation procedures could not account for the rise in cytoplasmic aspartate aminotransferase activity. Electrophoresis showed that in the diabetic cytoplasm both cathodal and anodal forms of the enzyme were elevated. Speculations concerning mitochondrial loss and cytoplasmic gain of enzyme activity as well as those on the reduction of plasma pyridoxal phosphate in the diabetic rat are presented.

Animals↗
PubMed1978

Modification of Rhodospirillum rubrum ribulose bisphosphate carboxylase with pyridoxal phosphate. 2. Stoichiometry and kinetics of inactivation.

Rhodospirillum rubrum ribulose bisphosphate carboxylase contains two high affinity binding sites for pyridoxal phosphate and two catalytic sites per dimer. However, pyridoxal phosphate binding at only one site is sufficient for inactivation of both catalytic sites. In the presence of 20 mM bicarbonate, 10 mM magnesium, and pyridoxal phosphate, the rates of inactivation and Schiff base formation are pseudo-first-order and show saturation kinetics. These observations provide additional evidence that pyridoxal phosphate binds at the active site of the R. rubrum carboxylase. It is also proposed that the large subunit may contain regulatory as well as catalytic properties.

Allosteric Regulation↗
PubMed1988

Inhibitory effects of pyridoxal phosphate, ascorbate and aminoguanidine on nonenzymatic glycosylation.

Nonenzymatic glycosylation of serum albumin was studied in the presence of naturally occurring metabolites, pyridoxal, pyridoxal phosphate and ascorbate/dehydroascorbate, and a hydrazine compound, aminoguanidine. Pyridoxal, pyridoxal phosphate, ascorbate and dehydroascorbate, at concentrations of 0.1 mM or greater, significantly inhibited the nonenzymatic glycosylation of albumin. Aminoguanidine was the most potent inhibitor of nonenzymatic glycosylation and 54% or 85% inhibition occurred when 5 or 50 mM aminoguanidine, respectively, was present in the incubation mixture containing 20 mM glucose. A major effect of aminoguanidine was to lower the free glucose concentration in the incubation mixture by a direct reaction with glucose as judged by thin layer chromatography. The present studies suggest that vital metabolites such as pyridoxal phosphate and ascorbate may be potentially important in controlling glucose-induced nonenzymatic glycosylation of proteins. Pyridoxal phosphate forms a Schiff base with proteins as does glucose and therefore may be a preferable drug, over aminoguanidine which is a hydrazine, for inhibiting the effects of glucose-induced nonenzymatic glycosylation.

Ascorbic Acid↗
PubMed1976

Studies on the pyridoxal phosphate site in glycogen phosphorylase b.

It is usually accepted that the adduct formed by reaction of pyridoxal phosphate with amines in aprotic solvents is a good model to stimulate some properties of the pyridoxal phosphate site in glycogen phosphorylase. The chemical structure of this adduct was not very well established. An aldimine structure is supported by the infrared, electronic absorption and nuclear magnetic resonance spectra given in this work. Therefore, we conclude that, at neutral pH, the pyridoxal phosphate is bound to the glycogen phosphorylase through a Schiff base structure and embedded in a hydrophobic environment. The polarographic measurements reported in this paper could explain the fact that, at neutral pH, the pyridoxal phosphate can not be reduced onto phosphorylase by NaBH4.

Binding Sites↗