The relative protective effect of pyridoxine, p-aminobenzoic acid, and various cysteamine derivatives on the x-ray induced degradation of pyridoxal-5-phosphate.
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4-Aminobenzoic Acid↗
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Results for “PYRIDOXAL PHOSPHATE”
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The effect of castrix (crimidin) on pyridoxal-5'-phosphate and the activity of pyridoxal kinase.
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Animals↗
[Novel coenzymatic function of pyridoxal 5'-phosphate: the phosphate group as catalyst].
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Amino Acids↗
The role of pyridoxal 5'-phosphate in plant phosphorylase.
The removal of pyridoxal 5'-phosphate from potato phosphorylase [EC 2.4.1.1] was achieved by incubation in an acidic ammonium sulfate solution containing hydroxylamine. Potato apophosphorylase is catalytically inactive, but reactivated by incubation with pyridoxal phosphate. Upon titration, the degree of recovery of activity agreed well with the degree of incorporation of pyridoxal phosphate. Therefore, it can be concluded that pyridoxal phosphate in the plant enzyme is the cofactor required for the enzyme activity, as it has been shown to be in the animal enzyme. The reconstitution of the apoenzyme using six pyridoxal phosphate analogues modified at the 5' position indicates that the structural properties of the cofactor binding site is similar in potato and rabbit muscle phosphorylases. This suggests that the plant enzyme has also a substrate binding site neighboring with the 5'-phosphate moiety of the bound cofactor similar to that shown to exist in the animal enzyme. On the other hand, the analysis of the far-ultraviolet circular dichroism spectra of these two enzymes shows that the secondary structures are rather different from each other; the potato enzyme is estimated to have 16% alpha-helix and 37% beta-structure, and the muscle enzyme b 41% alpha-helix and 21% beta-structure. This indicates that the cofactor binding locus must have been more strictly conserved than other regions.
Ammonium Sulfate↗
Assessment of vitamin B 6 status. Studies on pregnant women and oral contraceptive users.
The vitamin B6 status of 10 pregnant women (third trimester), 9 oral contraceptive agent users, and 12 notnpregnant women (controls) was investigated over a 10-week period by means of the erythrocyte glutamate-oxaloacetate transaminase (E-GOT) activation test and by measurements of whole blood pyridoxal phosphate levels. Blood pyridoxal phosphate levels in oral contraceptive agent users (7.6 plus or minus 1.1 ng/ml) were significantly lower than in the control group (9.6 plus or minus 1.7 mg/ml)indicating a relative vitamin B6 deficiency. Pyridoxal phosphate levels in pregnant women(5.1 plus or minus 1.3ng/ml) were even lower; in fact, no overlap was found in individual mean values between the pregnant and control groups. The E-GOT activation test did not indicate a vitamin deficiency in the pregnant women or in oral contraceptive agent users and the E-GOT activation factor did not correlate with blood pyridoxal phosphate levels unless results obtained after pyridoxine hydrochloride (20 mg/day) administration were included. The E-GOT activation test appears to be a poor indicator of vitamin B6 status, except in pronounced deficiency as it is less responsive to vitamin depletion than blood pyridoxal phosphate levels, and suffers from relatively large variations in individual control values. This may be a result of factors unrelated to vitamin B6 as blood pyridoxal phosphate levels remained fairly constant in the individuals investigated.
Adolescent↗
Vitamin B6 deficiency in chronic liver disease--evidence for increased degradation of pyridoxal-5'-phosphate.
Plasma levels of pyridoxal-5'-phosphate (PLP), the active coenzyme form of vitamin B6, were found to be significantly lower than normal in 22 out of 31 patients with decompensated cirrhosis or subacute hepatic necrosis. There was no significant difference in plasma PLP levels between those with liver disease due to alcohol and those with other varieties. When intravenous supplements with pyridoxine hydrochloride were given only 33% responded with an increase in plasma PLP. In contrast, all patients given PLP responded, although peak plasma levels were variable, the response being significantly less than that found in normal control subjects. After supplementation with pyridoxine hydrochloride, and with PLP, the urinary excretion of 4-pyridoxic acid, which is derived from the degradation of PLP, was higher in patients who showed the least increase in plasma PLP levels. Although impaired phosphorylation of pyridoxine hydrochloride may be one factor, the most likely explanation for these findings is an increased rate of PLP degradation which may be important in the pathogenesis of vitamin B6 deficiency in patients with severe liver disease.
Alcoholism↗
Phosphonate and alpha-fluorophosphonate analogue probes of the ionization state of pyridoxal 5'-phosphate (PLP) in glycogen phosphorylase.
To investigate the role of the essential cofactor pyridoxal phosphate in rabbit muscle glycogen phosphorylase catalysis, two phosphonate analogues of pyridoxal phosphate, 5'-deoxypyridoxal 5'-methylenephosphonic acid and 5'-deoxypyridoxal 5'-difluoromethylenephosphonic acid, have been prepared and reconstituted into apophosphorylase b. UV/Vis spectroscopic and 31P and 19F NMR studies confirmed the successful reconstitution and revealed significant changes in phosphate environment upon nucleotide activation. Both such reconstituted enzymes had activities of approximately 25%-30% of that observed in the native enzyme, while K(m) values were similar to those of the native enzyme. Very similar dependences upon pH of Vmax, K(m), and Vmax/K(m) were found for the two reconstituted enzyme derivatives and the native enzyme despite the considerable difference in phosphonic acid pKa values. These results suggest that pyridoxal phosphate does not function as an essential acid/base catalyst in glycogen phosphorylase; rather they suggest that the cofactor phosphate moiety remain dianionic throughout catalysis and functions as an essential dianion. Mechanistic implications of these findings are discussed.
Animals↗
Phosphorus-31 nuclear magnetic resonance study of pyridoxal 5'-phosphate binding to Escherichia coli tryptophan synthase.
The pyridoxal phosphate dependent bienzyme complex tryptophan synthase from Escherichia coli has been investigated using 31P nuclear magnetic resonance (NMR) at 72.86 MHz. In both the isolated beta 2 subunit and the fully assembled alpha 2 holo beta 2 complex, the pyridoxal phosphate 31P chemical shift is pH independent, indicating that the phosphate group of the cofactor is excluded from interaction with water and fixed in its dianionic form to the protein moiety. The line width of the phosphate signal of the coenzyme in the beta 2 subunit is unusually large pointing to different equilibrium conformations of the protein. In the native holo complex, however, the corresponding line width may be interpreted to result from moderate mobility of the cofactor leading to a higher rate of the transaldimination step of the catalytic mechanism.
Bacterial Proteins↗
Inhibitor studies of phage T4 wild-type and mutant DNA polymerases. II. Differential inhibition by pyridoxal 5'-phosphate.
The sensitivities to pyridoxal 5'-phosphate of phage T4 wild-type and two ts mutant DNA polymerases, L98 (mutator) and CB 121 (antimutator), were studied. The wild-type and the mutator enzyme we inhibited to an equal extent, while the antimutator enzyme was six times more sensitive. The mode of inhibition was competitive with the deoxynucleoside triphosphate substrates. The CB121 DNA polymerase had a three times lower affinity to its substrates but a twofold affinity to pyridoxal 5'-phosphate. The L98 enzyme had lower affinities to both the substrates and the inhibitor.
DNA-Directed DNA Polymerase↗
Phosphorus-31 nuclear magnetic resonance study of D-serine dehydratase: pryridoxal phosphate binding site.
The pyridoxal phosphate dependent enzyme D-serine dehydratase has been investigated using 31P nuclear magnetic resonance (NMR) at 72.86 MHz. In the native enzyme, the pyridoxal phosphate 31P chemical shift is pH dependent with pKa = 6.4, indicating exposure of the phosphate group to solvent. Binding of the competitive inhibitor isoserine results in the formation of the isoserine-pyridoxal phosphate complex. This transaldimination complex is fixed to the enzyme via the phosphate group of the cofactor as the dianion, independent of pH. At pH 6.6 the dissociation constant KD for isoserine determined by NMR is 0.43 mM. Reconstitution of the apoenzyme with pyridoxal phosphate monomethyl ester produces an inactive enzyme. NMR and fluorescence measurements show that this enzyme does not form the transaldimination complex, indicating that the fixation of the dianionic phosphate (probably via a salt bridge with an arginine residue) observed in the native enzyme is required for the transaldimination step of the catalytic mechanism.
Binding Sites↗
Inactivation of yeast fatty acid synthetase by modifying the beta-ketoacyl reductase active lysine residue with pyridoxal 5'-phosphate.
Treatment of yeast fatty acid synthetase with pyridoxal 5'-phosphate inhibited the enzyme. Assays of the partial activities of the pyridoxal phosphate-treated synthetase showed that only the beta-ketoacyl reductase was significantly inhibited. NADPH prevented inactivation of the enzyme by pyridoxal phosphate, indicating that pyridoxal modifies a residue near or in the beta-ketoacyl reductase site. The pyridoxal-treated synthetase shows a fluorescence spectrum with a maximum of 426 nm after uv irradiation at 325 nm. Binding of the pyridoxal phosphate to the synthetase is reversible as shown by the disappearance of the fluorescence band after dialysis of pyridoxal-treated enzyme. Reduction with NaBH4 of the pyridoxal-treated enzyme eliminates this fluorescence maximum and causes the appearance of a new band at 393 nm. These observations suggest that pyridoxal phosphate interacts with the synthetase by forming a Schiff base with lysine residue at the beta-ketoacyl reductase site. Amino acid analyses of the HCl hydrolysates of the borohydride-reduced, pyridoxal-treated synthetase showed the presence of 6 mol of N6-pyridoxal derivative of lysine per mole of fatty acid synthetase, indicating the presence of six sites of beta-ketoacyl reductase in the native enzyme. Autoradiography of sodium dodecyl sulfate-polyacrylamide gels of the pyridoxal phosphate enzyme reduced with NaB3H4 indicates that the alpha subunit contains the beta-ketoacyl reductase domain. These findings are consistent with the proposed structure of the alpha 6 beta 6 complex required for palmitoyl-CoA synthesis.
Alcohol Oxidoreductases↗
P2 purinoceptor antagonist properties of pyridoxal-5-phosphate.
The antagonist properties of pyridoxal-5-phosphate, a synthesis precursor of pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, were investigated on P2 purinoceptor-mediated responses of the rat isolated vagus nerve and vas deferens. In addition, the effect of this agent was studied on high affinity tritiated alpha,beta-methylene adenosine triphosphate (alpha,beta-meATP) binding to rat vas deferens membranes, thought to represent binding to functional P2x purinoceptors. In the rat vagus nerve, pyridoxal-5-phosphate (10(-5)-10(-4) M) produced concentration-related antagonism of depolarisation responses induced by alpha,beta-meATP, measured using an extracellular recording technique. In contrast, depolarisation responses to 5-hydroxytryptamine (5-HT) were unaffected by pyridoxal-5-phosphate. In the rat vas deferens, pyridoxal-5-phosphate (10(-5)-10(-4) M) antagonised contractile responses produced by alpha,beta-meATP while contractions to phenylephrine were unaffected. However, responses of the vagus nerve and the vas deferens to alpha,beta-meATP were not antagonised by pyridoxal hydrochloride (10(-4) M). Pyridoxal-5-phosphate competed for high affinity binding of [3H]alpha,beta-meATP to homogenised membranes of the rat vas deferens with a pKi estimate of 4.91 +/- 0.12 and a Hill slope of 0.80 +/- 0.03. Pyridoxal hydrochloride only competed for binding at concentrations in excess of 10(-4) M, yielding a pKi estimate of 3.21 +/- 0.04 and a Hill slope of 1.82 +/- 0.12. These findings indicate that pyridoxal-5-phosphate acts as a specific antagonist of P2 purinoceptors in the vagus nerve and vas deferens of the rat and that the phosphate moiety is required for activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Triphosphate↗
Pyridine analogs inhibit the glucosyltransferase of Streptococcus mutans.
Soluble glucan synthesis catalyzed by dextransucrase preparations from Streptococcus mutans 6715 were inhibited by pyridoxal-5-phosphate and several other pyridine analogs, including pyridoxine, pyridoxamine, pyridoxamine-5-phosphate, pyridoxal, and 4-pyridoxic acid. Pyridine and pyridine-4-carboxaldehyde were not effective inhibitors of the enzyme. Kinetic analyses suggested that pyridoxal-5-phosphate is a noncompetitive inhibitor of dextransucrase. The inactivation was dependent on time, pyridoxal-5-phosphate concentration, and hydrogen ion concentration. Apparent Ki values were 4.9 mM at pH 7.0 and 4.2 mM at pH 5.5. Dextransucrase activity could be restored by dialysis to remove the inhibitors. Maximum inhibition was observed after a 120-min incubation of the enzyme with pyridoxal-5-phosphate. The pH optima for inhibition by pyridoxal-5-phosphate were 4 and 7. The sucrose-dependent adherence of S. mutans cells to saliva-coated hydroxylapatite beads was also inhibited by pyridoxal-5-phosphate but only marginally by the other pyridine anatogs. In addition, pyridoxal-5-phosphate markedly reduced the rate of acid production by intact S. mutans cells from sucrose or glucose substrates. Another pyridoxal-5-phosphate analog, 2-methyl-5-hydroxypyridine, was also effective in preventing the production of acid by S. mutans from sucrose or glucose. When S. mutans cells were preincubated with pyridoxal-5-phosphate or pyridine analogs, significant reductions in the rate of D-glucose uptake were observed. It is suggested that the inhibition of dextransucrase occurs because of a change iun enzyme conformation which results from the binding of the pyridine derivatives. The results suggest that pyridoxal-5-phosphate or structural analogs may ultimately be useful in reducing the incidence of dental caries.
Adhesiveness↗
Homocysteine, vitamin B(6), and lipid in cardiovascular disease.
OBJECTIVES: Elevated homocysteine (Hcy) is considered an independent risk factor for cardiovascular disease (CVD). An elevated plasma Hcy level may interact with conventional CVD risk factors to further increase vascular disease risk. Therefore, we investigated the plasma levels of Hcy, vitamin B(6) status (pyridoxal phosphate and pyridoxal), and lipid profile in patients with CVD. METHODS: Possible associations between sex, age, body mass index (BMI), and waist-to-hip ratio (WHR) to levels of plasma Hcy and plasma Hcy to vitamin B(6) status and lipid profile were examined. RESULTS: Plasma Hcy level, body mass index, and waist-to-hip ratio were significantly higher in patients with CVD than in controls. Male CVD patients had significantly higher plasma Hcy levels than did female patients. Plasma levels of pyridoxal phosphate and total B(6) aldehyde were significantly higher in male than in female patients. Plasma Hcy levels of patients did not correlate to their plasma vitamin B(6) status or to their lipid profiles. Plasma Hcy level correlated positively with age, body mass index, and waist-to-hip ratio (P < 0.0001). CONCLUSIONS: This suggested that patients with CVD have higher levels of plasma Hcy that are influenced by sex, age, body mass index, and waist-to-hip ratio and not by their plasma vitamin B(6) status and lipid profiles.
Adult↗
The role of acetaldehyde in mediating the deleterious effect of ethanol on pyridoxal 5'-phosphate metabolism.
Previous studies in vivo and with isolated perfused rat livers have suggested that the deleterious effect of ethanol on hepatic pyridoxal 5'-phosphate metabolism is mediated by acetaldehyde. Inasmuch as acetaldehyde has no effect on the synthesis of pyridoxal phosphate, it has also been postulated that acetaldehyde accelerates pyridoxal phosphate degradation by displacing this coenzyme from binding proteins, which protect it against hydrolysis. To test these hypotheses, studies have been performed with isolated rat hepatocytes, subcellular fractions of rat liver, and human erythrocytes. Ethanol oxidation lowered the pyridoxal phosphate content of isolated liver cells when acetaldehyde oxidation was inhibited by either disulfiram or prior treatment of rats with cyanamide. Additions of 7.5 mM acetaldehyde alone at 40-min intervals to cell suspensions decreased hepatic pyridoxal phosphate content only slightly because acetaldehyde was rapidly metabolized. However, when acetaldehyde oxidation and reduction were inhibited by cyanamide treatment and by 4-methyl-pyrazole and isobutyramide, respectively, a 40% decrease in hepatic pyridoxal phosphate content was observed in 80 min of incubation. In equilibrium dialysis experiments, acetaldehyde, 7.5 and 15 mM, displaced protein-bound pyridoxal phosphate in undialyzed hepatic cytosol and in hemolysate supernate containing added pyridoxal phosphate. In the presence of alkaline phosphatase, acetaldehyde accelerated the degradation of pyridoxal phosphate in dialyzed hemolysate supernate and hepatic cytosol with added pyridoxal phosphate. Acetaldehyde also inhibits tyrosine aminotransferase. The kinetics of inhibition were mixed competitive-noncompetitive with respect to pyridoxal phosphate. These observations support the hypothesis that the deleterious effect of ethanol oxidation on pyridoxal phosphate metabolism is mediated at least in part by acetaldehyde which displaces this coenzyme from protein binding, thereby enhancing its degradation.
Acetaldehyde↗
Pyridoxal 5'-phosphate: a possible physiological substrate for alkaline phosphatase in human neutrophils.
The intracellular localization of pyridoxal phosphatase activity was demonstrated in human neutrophils by electron microscope cytochemistry. Under alkaline conditions, an enzyme active against pyridoxal phosphate was localized to a cytoplasmic granule population, the phosphasome. These granules have previously been shown by electron microscope cytochemical techniques and by subcellular fractionation to be rich in alkaline phosphatase. Under acidic conditions, a phosphatase activity against pyridoxal phosphate was localized to intracellular multilamellar bodies resembling secondary lysosomes. These were quite distinct from the primary, secondary and phosphasome granules and this unique localization corresponds to that previously demonstrated (tertiary granules) by subcellular fractionation studies of these cells. The similarity in the enzyme reaction requirements of alkaline pyridoxal phosphatase and alkaline phosphatase, and their localization to the same subcellular organelle, suggests that pyridoxal phosphate may be a physiological substrate for human neutrophil alkaline phosphatase.
Alkaline Phosphatase↗
Vitamin B6 metabolism in chronic alcohol abuse The effect of ethanol oxidation on hepatic pyridoxal 5'-phosphate metabolism.
Individuals with chronic alcohol abuse frequently exhibit lowered plasma levels of pyridoxal 5'-phosphate, the coenzyme form of vitamin B6. Because the liver is the primary source of this coenzyme in plasma and also the principal organ that oxidizes ethanol, the effect of ethanol on hepatic pyridoxal phosphate metabolism was studied in the rat. The chronic feeding of ethanol (36 percent of the total dietary calories) for 6 wk significantly decreased the hepatic pyridoxal phosphate content both in animals given a sufficient amount of vitamin B6 in their diet and in those rendered vitamin B6 deficient. In isolated perfused livers, the addition of 18 mM ethanol lowered the pyridoxal phosphate content of livers from vitamin B6-sufficient animals and deceased the net synthesis of pyridoxal phosphate from pyridoxine by the livers of vitamin B6-deficient animals. Ethanol also diminished the rate of release of pyridoxal phosphate into the perfusate by the livers of vitamin B6-deficient rats. These effects of ethanol, in vitro, were abolished by 4-methyl pyrazole, an inhibitor of alcohol dehydrogenase. Thus the derangement of pyridoxal phosphate metabolism produced by ethanol is dependt upon its oxidation. These data support previous findings whic indicate that acetaldehyde is the responsible agent which acts by accelerating the degradation of intracellular pyridoxal phosphate.
Acute Disease↗
Inhibition of the recBC enzyme of Escherichia coli by specific binding of pyridoxal 5'-phosphate to DNA binding site.
Pyridoxal 5'-phosphate rapidly abolished the DNA-hydrolyzing activities as well as DNA-dependent ATP-ase activity of the recBC enzyme of Escherichia coli. Pyridoxal also had an inhibitory effect on the enzyme but less effective than that of pyridoxal 5'-phosphate. Pyridoxamine 5'-phosphate, pyridoxamine, or pyridoxine had no effect on the activities of the enzyme. The inhibition was rapidly reversed by dilution but could be made irreversible by reduction with sodium borohydride prior to dilution. This suggests the formation of Schiff base between pyridoxal 5'-phosphate and an epsilon-amino group of a lysine residue which is essential for the enzyme activity. Pyridoxal 5'-phosphate is a competitive inhibitor of DNA substrate but not of ATP. Furthermore, the presence of DNA substrate protected the enzyme from inactivation by the reduction but the presence of ATP showed no effect. Thus, the recBC enzyme appears to have an essential lysine residue at or near the DNA binding site of the enzyme, and the enzyme possesses two independent catalytic sites, such as a DNA binding site and an ATP binding site.
DNA, Bacterial↗