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PubMed1988

Isotope effect studies of the pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii.

The pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii shows a nitrogen isotope effect k14/k15 = 0.9770 +/- 0.0021, a carbon isotope effect k12/k13 = 1.0308 +/- 0.0006, and a carbon isotope effect for L-[alpha-2H]histidine of 1.0333 +/- 0.0001 at pH 6.3, 37 degrees C. These results indicate that the overall decarboxylation rate is limited jointly by the rate of Schiff base interchange and by the rate of decarboxylation. Although the observed isotope effects are quite different from those for the analogous glutamate decarboxylase from Escherichia coli [Abell, L. M., & O'Leary, M. H. (1988) Biochemistry 27, 3325], the intrinsic isotope effects for the two enzymes are essentially the same. The difference in observed isotope effects occurs because of a roughly twofold difference in the partitioning of the pyridoxal 5'-phosphate-substrate Schiff base between decarboxylation and Schiff base interchange. The observed nitrogen isotope effect requires that the imine nitrogen in this Schiff base is protonated. Comparison of carbon isotope effects for deuteriated and undeuteriated substrates reveals that the deuterium isotope effect on the decarboxylation step is about 1.20; thus, in the transition state for the decarboxylation step, the carbon-carbon bond is about two-thirds broken.

Carbon Isotopes↗

PubMed1981

Effect of pyridoxal 5'-phosphate on oligodeoxynucleotide binding of mouse uterine cytosol estradiol-receptor complexes.

Pyridoxal-5'-phosphate, an inhibitor of DNA binding of glucocorticoid-, progesterone-, and estrogen-receptor complexes, also blocks the interaction of mouse uterine cytosol estradiol-receptor complexes with oligodeoxynucleotides covalently linked to celluloses. Other derivatives of pyridoxine were ineffective. The inhibition was irreversible only after borohydride reduction. Experiments with three oligodeoxynucleotides showed a 4-fold range of differences in the pyridoxal-5'-phosphate concentrations needed to inhibit 50% of the binding: oligo(dG)-cellulose, 3.1 mM; oligo(dT)-cellulose, 1.5 mM; and oligo(dC)-cellulose, 0.8 mM. Inhibition of binding occurred more rapidly with the oligodeoxypyrimidines than with oligo(dG)-cellulose. Pyridoxal-5'-phosphate was competitive with the oligodeoxynucleotides in its mode of inhibition, suggesting the polynucleotide-binding domain as a site of action. These data indicate a microheterogeneity of oligodeoxynucleotide-binding sites in the estradiol-receptor complex which is reflected in sensitivity to pyridoxal-5'-phosphate.

Amino Acids↗

PubMed1983

Effect of pyridoxal 5'-phosphate on Ca2+-stimulated adenosine triphosphatase activity in microsomes of rat submandibular gland in vitro.

1. The Ca2+-ATPase activity in microsomes of rat submandibular gland was inhibited by pyridoxal 5'-phosphate in vitro. 2. The dissociation constant of the enzyme-pyridoxal 5'-phosphate complex was estimated to be 6.5 mM. 3. The inhibition of pyridoxal 5'-phosphate for both ATP and Ca2+ was competitive. 4. The order of inhibitory effectiveness of pyridoxal 5'-phosphate analogs was pyridoxal 5'-phosphate greater than pyridoxal HCl greater than pyridoxamine 5'-phosphate greater than pyridoxamine HCl. 5. The enzyme-pyridoxal 5'-phosphate complex was nonreducible with sodium borohydride.

Adenosine Triphosphate↗

PubMed1992

Phosphorylation-induced conformational changes in the phosphorylase ab hybrid as revealed by resolution of pyridoxal 5'-phosphate with imidazole citrate and cysteine.

The accessibility of pyridoxal 5'-phosphates of the phosphorylase ab hybrid to resolution by imidazole citrate and cysteine was studied and compared with that of the b and a forms. Promotion of resolution of phosphorylated forms by raising the temperature or in the presence of glycogen indicates that the resistance of phosphorylase a and ab to resolution at 0 degrees C is due rather to their tetrameric state than their phosphorylation-related active conformation. The pattern of resolution of the ab hybrid was similar to that of the a and differed from that of the b forms in that it occurred at 30 degrees C and 37 degrees C but not at 0 degrees C, moreover, it did not show first-order kinetics. On the other hand, inhibition of resolution by ligands binding to the nucleotide site of phosphorylase reflected an intermediate sensitivity of the ab form between that of the b and a forms. We conclude that partial phosphorylation of phosphorylase b elicits conformational change(s) in both subunits which influence the monomer-monomer interactions and resolution of pyridoxal 5'-phosphates. Resistance of ab hybrid to monomerizing agents as imidazole citrate, comparable to that of other forms, argues for its stability, ruling out its reshuffling into mixtures of phosphorylase b and a.

Adenosine Monophosphate↗

PubMed1989

Effect of pyridoxal 5'-phosphate and valproic acid on phospholipid synthesis in neuroblastoma NA.

Phospholipid metabolism in neuroblastoma cells in monolayer culture after acute exposure to pyridoxal phosphate (PLP) has been studied. (a) A strong depression of the rate of biosynthesis of cellular phospholipids from labeled choline and ethanolamine, is demonstrated in neuroblastoma cells grown in culture media containing PLP. (b) Valproic acid reverses the effect of PLP on ethanolamine and choline incorporation into cell lipid. Other anticonvulsants (clonazepam, diazepam, carbamazepine, diphenylhydantoin and ethosuximide) have little or no effect on reversing the inhibition of lipid synthesis produced by PLP. (c) PLP decreases the cellular uptake of choline. This effect might be responsible for the decreased lipid synthesis and is partially reversed by valproic acid. (d) The energy charge of the cell is not affected by either PLP or valproic acid, but it is diminished by the two compounds together. (e) The degradation of choline lipids is decreased by PLP and valproic acid. The hydrolysis of phosphocholine and the outflow of choline from cultured cells is also affected by the drugs. Variations of ethanolamine and choline transport should not be due to any effects of PLP or valproic acid on the lipid phase of the membranes since these molecules have no effect on the permeability of liposomes. (f) It is concluded that ethanolamine and choline lipid metabolism in cultured neuroblastoma cells is influenced by PLP and/or valproic acid, probably through a mechanism involving the transport of precursors across the membrane, although other mechanisms cannot be ruled out.

Energy Metabolism↗

PubMed1997

Modulation of the catalytic activity of brain succinic semialdehyde reductase by reaction with pyridoxal 5'-phosphate.

An NADPH-dependent succinic semialdehyde reductase from bovine brain was inactivated by pyridoxal 5'-phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After sodium borohydride reduction of the inactivated enzyme, it was observed that 1 mol phosphopyridoxyl residue was incorporated/mol enzyme monomer. The coenzyme, NADPH, protected the enzyme against inactivation by pyridoxal 5'-phosphate. After tryptic digestion of the enzyme modified with pyridoxal 5'-phosphate in the presence and absence of NADPH followed by [1H]NaBH4 reduction, a radioactive peptide absorbing at 310 nm was isolated by reverse-phase HPLC. The amino acid sequence of the peptide identified a portion of the pyridoxal-5'-phosphate-binding site as the region containing the sequence I-L-E-N-I-Q-V-F-X-K, where X indicates that the phenylthiohydantoin amino acid could not be assigned. The missing residue, however, can be designated as a phosphopyridoxyl lysine as interpreted from the result of amino acid composition of the peptide. It is suggested that the catalytic function of succinic semialdehyde reductase is modulated by binding of pyridoxal 5'-phosphate to a specific lysyl residue at or near the coenzyme-binding site of the protein.

Amino Acids↗

PubMed1982

Changes in activation of aspartate aminotransferase by pyridoxal 5'-phosphate after experimental liver damage in rabbits.

The aspartate aminotransferase activity with and without pyridoxal 5'-phosphate supplementation was examined in mitochondrial and cytoplasmic preparations from fresh human heart and liver samples. The apoenzyme was fully saturated in all cases. Liver cell damage was produced by ischaemia and carbon tetrachloride poisoning in two groups of rabbits. The activity of aspartate aminotransferase with and without pyridoxal 5'-phosphate was measured in the plasma and in cytoplasmic and mitochondrial preparations from both groups. After carbon tetrachloride poisoning the enzyme activity in the plasma increased within 2 h but was not enhanced by pyridoxal 5'-phosphate. Following ischaemia, plasma enzyme activity only increased between 4 and 8 h and was progressively stimulated by pyridoxal 5'-phosphate. Up to 15 h after carbon tetrachloride poisoning the liver cytoplasmic and mitochondrial apo-enzyme remained fully saturated with co-enzyme. In contrast, a pronounced loss of co-enzyme occurred in both fractions of the ischaemic group. These result suggest that the type of injury and not necessarily the organ affected could determine the degree of activation of aspartate aminotransferase by pyridoxal 5'-phosphate observed in human myocardial infarction and liver disease.

Animals↗

PubMed1986

Depressed plasma pyridoxal-5'-phosphate levels in tobacco-smoking men.

Tobacco-smoking men (n = 106) showed considerably lower plasma pyridoxal-5'-phosphate levels (P less than 0.001) than non-smoking controls (n = 143). A previously reported inverse relationship between plasma pyridoxal-5'-phosphate levels and age is confirmed by using new and sensitive high-performance liquid chromatography methodology for pyridoxal-5'-phosphate determinations. Plasma pyridoxal levels were neither lower in tobacco-smoking men nor could any relationship between pyridoxal levels and age be demonstrated. Two major risk factors for coronary artery disease, tobacco-smoking and increasing age, are thus associated with lower plasma pyridoxal-5'-phosphate levels.

Adult↗

PubMed2006

Aminophospholipid glycation and its inhibitor screening system: a new role of pyridoxal 5'-phosphate as the inhibitor.

Peroxidized phospholipid-mediated cytotoxity is involved in the pathophysiology of a number of diseases [i.e., the abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) found in the plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (deoxy-D-fructosyl PE, or Amadori-PE), because Amadori-PE causes oxidative stress. However, lipid glycation inhibitor has not been discovered yet because of the lack of a lipid glycation model useful for inhibitor screening. We optimized and developed a lipid glycation model considering various reaction conditions (glucose concentration, temperature, buffer type, and pH) between PE and glucose. Using the developed model, various protein glycation inhibitors (aminoguanidine, pyridoxamine, and carnosine), antioxidants (ascorbic acid, alpha-tocopherol, quercetin, and rutin), and other food compounds (L-lysine, L-cysteine, pyridoxine, pyridoxal, and pyridoxal 5'-phosphate) were evaluated for their antiglycative properties. Pyridoxal 5'-phosphate and pyridoxal (vitamin B(6) derivatives) were the most effective antiglycative compounds. These pyridoxals could easily be condensed with PE before the glucose/PE reaction occurred. Because PE-pyridoxal 5'-phosphate adduct was detectable in human red blood cells and the increased plasma Amadori-PE concentration in streptozotocin-induced diabetic rats was decreased by dietary supplementation of pyridoxal 5'-phosphate, it is likely that pyridoxal 5'-phosphate acts as a lipid glycation inhibitor in vivo, which possibly contributes to diabetes prevention.

Adult↗

PubMed2000

Folic acid and pyridoxal-5'-phosphate losses during high-efficiency hemodialysis in patients without hydrosoluble vitamin supplementation.

OBJECTIVES: To determine the serum status in folate, pyridoxal-5'-phosphate (the active moiety of pyridoxine), cobalamin, and total homocysteine of chronic dialysis patients not routinely supplemented with B-complex vitamins and to evaluate induced intradialytic losses during high-efficiency hemodialysis. DESIGN: A cross-sectional study. SETTING: A university medical center providing tertiary care. PATIENTS: Thirty-six chronic dialysis patients (23 men and 13 women, mean age 57+/-13 years) treated since 3.8+/-2.2 years by hemodialysis and not supplemented with hydrosoluble vitamins. METHODS: Thrice-weekly hemodialysis was performed using CT-190G (Baxter, IL) or F-20 (Hospal, St-Leonard, Canada) reused dialyzers with a mean blood flow rate of 371+/-40 mL/min, a dialysate flow rate of 500 mL/min, and a mean session time of 3.7+/-0.4 hours. Prehemodialysis serum vitamin B(12) and homocysteine, and predialysis and postdialysis serum folate, pyridoxal-5'-phosphate, and urea were measured. Blood-side folate and pyridoxal-5'-phosphate clearances were calculated. RESULTS: Predialysis serum total homocysteine was above normal in all patients, with values ranging from 14.4 to 158.0 micromol/L (mean 40.2+/-29.6 micromol/L, median 33.5 micromol/L). Whereas the majority, 21 patients, had evidence of coronary, cerebrovascular, and/or peripheral vascular diseases, there was no difference in total homocysteine in patients with or without vascular disease (respectively, 40.8+/-37.0 micromol/L v 39.4+/-15.1 micromol/L, P = NS). Predialysis serum concentrations of pyridoxal-5'-phosphate were reduced in 20 patients (56%) and were in the lower normal range for 14 patients. Predialysis and postdialysis serum folate concentrations were 12.4+/-6.1 nmol/L and 8.6 +/- 3.6 nmol/L, whereas predialysis and postdialysis serum pyridoxal-5'-phosphate concentrations were 11.1+/-7.5 nmol/L and 8.0 +/-5.9 nmol/L. Percent reduction ratios were 68.4% +/- 6.6% for urea, 26.3%+/-16.0% for folates, and 27.9%+/-14.2% for pyridoxal-5'-phosphate. Blood-side clearances reached 134.7+/-22.2 mL/min for folates and 54.4+/-38.2 mL/min for pyridoxal-5'-phosphate. There was no significant difference in predialysis serum folate and pyridoxal-5'-phosphate in patients with or without evidence of vascular disease. CONCLUSION: This study confirms that: (1) total serum homocysteine levels are very high in chronic hemodialysis patients not supplemented with B-complex vitamins; (2) folate is significantly cleared or lost during high-efficiency hemodialysis; and (3) pyridoxal-5'-phosphate, the active moiety of pyridoxine, is depleted in most chronic hemodialysis patients without supplementation and that high-efficiency hemodialysis contributes to its depletion.

Cross-Sectional Studies↗

PubMed1985

Enhancement of brain pyridoxal 5'-phosphate level following chronic ingestion of ethanol.

The concentrations of pyridoxal 5'-phosphate in the brains of DBA/2J mice during the withdrawal period following chronic ethanol ingestion were measured fluorimetrically. The brain of the ethanol-withdrawn mouse contains 22.4 +/- 3.1% more pyridoxal 5'-phosphate than the brain of a control mouse. The enhanced susceptibility to epileptic seizures during the withdrawal phase may in part be due to the increased cerebral content of this coenzyme.

Alcoholism↗

PubMed1988

Inhibition of aromatic L-aminoacid decarboxylase in clonal pheochromocytoma PC12h cells by N-methyl-4-phenylpyridinium ion (MPP+).

N-Methyl-4-phenylpyridinium ion (MPP+), a reaction product of a neurotoxin, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), was found to inhibit aromatic L-aminoacid decarboxylase activity in rat clonal pheochromocytoma PC12h cells. The enzyme activity was enhanced to several folds by addition of a cofactor, pyridoxal phosphate, and MPP+ inhibited the enhancement of the activity by exogenously added pyridoxal phosphate. The inhibition was competitive to pyridoxal phosphate, and the Ki value of MPP+ was 26.7 +/- 0.4 microM, while the Km value of pyridoxal phosphate was 0.645 +/- 0.053 microM. The inhibition was partly irreversible. The enzyme sample was incubated with MPP+ and then dialyzed against phosphate buffer. After dialysis, the inhibited enzyme activity was only partly recovered by addition of pyridoxal phosphate, even though MPP+ was completely removed. Activity of other enzymes, tyrosine hydroxylase and monoamine oxidase could be recovered by dialysis. On the other hand, MPP+ did not affect the binding of the enzyme with the substrate, L-DOPA or 5-hydroxytryptophan.

1-Methyl-4-phenylpyridinium↗

PubMed1988

Compartmentalization of pyridoxal-5'-phosphate during the acute phase of myocardial infarction.

Vitamin B6 nutritional status is assessed by measuring the plasma concentration of one of its vitamers, pyridoxal-phosphate (PLP). Several conditions, e.g., myocardial infarction (MI), can disturb the dynamic equilibrium between the different vitamers resulting in transiently low plasma PLP levels. An important question is whether these low plasma PLP levels observed during MI represent a transient state of deficiency and what the possible clinical consequences of such a fall in plasma PLP could be. Since the main metabolic function of PLP is to act as an intracellular coenzyme, it was decided to monitor the changes not only of PLP but also of PL (transport form of vitamin B6) in both the plasma and red blood cells (RBCs) in patients with myocardial infarctions (MI): 16 patients with proven MI were investigated measuring the aforementioned parameters at regular intervals. It was found that the approximately 40% fall in plasma PLP levels was accompanied by an equivalent increase in RBC PLP levels. Subsequently plasma PLP concentrations returned to normal but RBC PLP values were maintained at the newly elevated steady state (without any vitamin supplementation). Since membranes are impermeable to PLP, the only way in which PLP could have been redistributed to the intracellular compartment was through hydrolysis to PL and rephosphorylation once inside the RBCs. This compartmentalization could be an important adaptive response since it has been shown that PLP reduces O2 affinity of deoxygenated hemoglobin, thereby facilitating O2 delivery to the tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1975

Ox liver glutamate dehydrogenase. The role of lysine-126 reappraised in the light of studies of inhibition and inactivation by pyridoxal 5'-phosphate.

The time-course of inactivation of bovine liver glutamate dehydrogenase by pyridoxal 5'-phosphate was studied in the presence of varied amounts of 2-oxoglutarate or NADH. Pseudo-first-order analysis reveals that the protection by both these compounds is competitive with respect to the chemical modifier. The competition is only partial, however: saturation with either NADH or 2-oxoglutarate decreases the rate constant for inactivation to a finite minimum and not to zero. Similarly, the plot of activity at equilibrium as a function of the concentration of the protecting substrate or coenzyme reveals that neither NADH nor 2-oxoglutarate protects completely against inactivation. In initial-rate experiments, pyridoxal 5'-phosphate, used as an instantaneous inhibitor rather than a long-term inactivator, displayed non-competitive inhibition with respect to both 2-oxoglutarate and NADH. These results clearly indicate that, although there is mutual hindrance between the binding to the enzyme of pyridoxal 5'-phosphate, on the one hand, and 2-oxoglutarate or NADH on the other, binding is not mutually exclusive. These findings are discussed in terms of the two-step mechanism for inactivation by pyridoxal 5'-phosphate. It is concluded that lysine-126 cannot be solely responsible for binding either the substrate or the coenzyme, but could be essential for the catalytic step.

Animals↗

PubMed1976

Effect of pyridoxal 5'-phosphate on the oxygen affinity of human erythrocytes.

Pyridoxal 5'-phosphate (PLP), an allosteric effector for the oxygenation of haemoglobin, was incorporated readily into erythrocytes and disappeared from them by simple passive diffusion. The disappearance of PLP from the cells was accelerated by the generation of 2,3-DPG in a medium of inosine, pyruvate and phosphate. The oxygen dissociation curve measured at an extracellular pH of 7.4 demonstrated that PLP incorporated into the cells also lowered the oxygen affinity and that PLP functionally compensated for a metabolically reduced 2,3-DPG. However, the dependency of the oxygen affinity on the intracellular PLP concentration showed a different pattern from the observed for 2,3-DPG. On the other hand, the lowering of intracellular pH by organic phosphates accumulated in the cells was much larger with PLP than with 2,3-DPG. The peculiar relationship between the oxygen affinity of erythrocytes and the intracellular PLP concentration is discussed in detail. The present study may offer a new prospect for the preservation of blood with a normal function.

Erythrocytes↗

PubMed1976

The regulation of mouse liver ornithine decarboxylase by metabolites.

The enzyme ornithine decarboxylase (L-Ornithine carboxy-lyase, EC 4.1.1.17), has been partially purified from the livers of mice subjected to partial hepatectomy (6-8 h previously). Mouse liver ornithine decarboxylase requires pyridoxal phosphate, and dithiothreitol for maximal activity. The enzyme has a pH optimum of 7.3, it is inhibited in the presence of 0.3 M phosphate, glycine, Tricine and Tris. It shows no dependence on metal ions and is inhibited by high salt concentrations, particularly ammonium salts. The kinetics of the enzyme have been studied with putrescine (and analogs), spermidine and spermine, in the presence of both high and low levels of pyridoxal phosphate. High concentrations of pyridoxal phosphate inhibit the enzyme. The enzyme is also inhibited by low concentrations of putrescine (1 mM). As the concentration of putrescine increased to 10 mM, non-competitive inhibition was observed, this could be reversed by addition of higher levels of pyridoxal phosphate. Spermidine and spermine inhibit (noncompetitively) only at high concentrations (10 mM). Ornithine inhibits at high concentrations (2 mM). Spectral studies have shown that the observed kinetics of competitive inhibition at low concentrations of polyamine changing to noncompetitive inhibition at high polyamine concentrations are due to competition between enzyme and substrate (or inhibitor) for free (non-enzyme bound) pyridoxal phosphate. Noncompetitive inhibition arises through the formation of transient Schiff base complexes between amines and free pyridoxal phosphate. It also appears that the binding of substrate to the active site takes place through Schiff base formation with enzyme bound pyridoxal phosphate.

Animals↗

PubMed2001

Clinical adaptation of a high-performance liquid chromatographic method for the assay of pyridoxal 5'-phosphate in human plasma.

Vitamin B6, measured as pyridoxal 5'-phosphate (PLP), is a co-enzyme in the transsulfuration pathway of homocysteine metabolism. Since depletion of PLP has been suggested as an independent risk factor for coronary artery disease, PLP is frequently measured to guide patient care. By a change and utilization of an Aquasil C18 column and the addition of an acetonitrile clean-up gradient to the potassium phosphate, with sodium perchlorate and bisulfite buffer between samples we report the modification of a previously described method for analysis of PLP. The result is a more practical, efficient, reliable and robust method for daily clinical use. We also determined and report that it is critical to protect freshly prepared standard PLP samples from light exposure during assay preparation.

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

Full-time course studies of bovine liver glutamate dehydrogenase. Simulation of inhibition by pyridoxal-5'-phosphate.

Inhibition of bovine liver glutamate dehydrogenase by pyridoxal-5'-phosphate was studied by measuring the full time course of the oxidation of NADPH. Progress curves were determined before and after incubation of the enzyme with PLP in the presence of saturating concentrations of alpha-ketoglutarate and ammonium ion, at pH 7.4 and 25 degrees C. The data were fitted to the integrated Michaelis-Menten equation and an inhibition model derived. According to the model, PLP inhibits the enzyme non-competitively, by reversible formation of the complexes E--PLP and E--PLP--NADPH; the oxidation of NADPH is also inhibited by NADP+. After incubation with PLP, the dissociation constants of E--NADPH and E--NADP+ (Km and Kp) show a very definite decrease, while the maximum rate of oxidation (Vm) is increased. The inhibition constants for PLP were also computed.

Animals↗