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PubMed1991

Purification and properties of an iron-sulfur-containing and pyridoxal-phosphate-independent L-serine dehydratase from Peptostreptococcus asaccharolyticus.

L-Serine dehydratase with a specific activity of 15 nkat/mg protein was present in the anaerobic eubacterium Peptostreptococcus asaccharolyticus grown either on L-glutamate or L-serine. The enzyme was highly specific for L-serine with the lowest Km = 0.8 mM ever reported for an L-serine dehydratase. L-Threonine (Km = 22 mM) was the only other substrate. V/Km for L-serine was 500 times higher than that for L-threonine. L-Cysteine was the best inhibitor (Ki = 0.3 mM, competitive towards L-serine). The enzyme was purified 400-fold to homogeneity under anaerobic conditions (specific activity 6 mukat/mg). PAGE in the presence of SDS revealed two subunits with similar intensities (alpha, 30 kDa; beta, 25 kDa). The molecular mass of the native enzyme was estimated as 200 +/- 20 kDa (gel filtration) and 180 kDa (gradient PAGE). In the absence of oxygen the enzyme was moderately stable even in the presence of sodium borohydride or phenylhydrazine (5 mM each). However, by exposure to air the activity was lost, especially when the latter agent was added. The enzyme was reactivated by ferrous ion under anaerobic conditions. The inability of several nucleophilic agents to inactivate the enzyme indicated the absence of pyridoxal phosphate. This was confirmed by a microbiological determination of pyridoxal phosphate. However, the enzyme contained 3.8 +/- 0.2 mol Fe and 5.6 +/- 0.3 mol inorganic sulfur/mol heterodimer (55 kDa) indicating the presence of an [Fe-S] center. The enzyme was successfully applied to measure L-serine concentrations in bacterial media and in human sera.

Amino Acid Sequence↗

PubMed1978

Modification of Rhodospirillum rubrum ribulose bisphosphate carboxylase with pyridoxal phosphate. 1. Identification of a lysyl residue at the active site.

Ribulose 1,5-bisphosphate carboxylase isolated from Rhodospirillum rubrum was strongly inhibited by low concentrations of pyridoxal 5'-phosphate. Activity was protected by the substrate ribulose bisphosphate and to a lesser extent by other phosphorylated compounds. Pyridoxal phosphate inhibition was enhanced in the presence of magnesium and bicarbonate, but not in the presence of either compound alone. Concomitant with inhibition of enzyme activity, pyridoxal phosphate forms a Schiff base with the enzyme which is reversible upon dialysis and reducible with sodium borohydride. Subsequent to reduction of the Schiff base with tritiated sodium borohydride, tritiated N6-pyridoxyllysine could be identified in the acid hydrolysate of the enzyme. Only small amounts of this compound were present when the reduction was performed in the presence of carboxyribitol bisphosphate, an analogue of the intermediate formed during the carboxylation reaction. Therefore, it is concluded that pyridoxal phosphate modifies a lysyl residue close to or at the active site of ribulose bisphosphate carboxylase.

Bicarbonates↗

PubMed2000

Pyridoxal phosphate de-activation by pyrroline-5-carboxylic acid. Increased risk of vitamin B6 deficiency and seizures in hyperprolinemia type II.

We previously identified vitamin B6 deficiency in a child presenting with seizures whose primary diagnosis was the inherited disorder hyperprolinemia type II. This is an unrecognized association, which was not explained by diet or medication. We hypothesized that pyridoxal phosphate (vitamin B6 coenzyme) was de-activated by L-Delta(1)-pyrroline-5-carboxylic acid, the major intermediate that accumulates endogenously in hyperprolinemia type II. The proposed interaction has now been investigated in vitro with high resolution 1H nuclear magnetic resonance spectroscopy and mass spectrometry at a pH of 7.4 and temperature of 310 K. Three novel adducts were identified. These were the result of a Claisen condensation (or Knoevenagel type of reaction) of the activated C-4 carbon of the pyrroline ring with the aldehyde carbon of pyridoxal phosphate. The structures of the adducts were confirmed by a combination of high performance liquid chromatography, nuclear magnetic resonance, and mass spectrometry. This interaction has not been reported before. From preliminary observations, pyrroline-5-carboxylic acid also condenses with other aromatic and aliphatic aldehydes and ketones, and this may be a previously unsuspected generic addition reaction. Pyrroline-5-carboxylic acid is thus found to be a unique endogenous vitamin antagonist. Vitamin B6 de-activation may contribute to seizures in hyperprolinemia type II, which are so far unexplained, but they may be preventable with long term vitamin B6 supplementation.

Aldehydes↗

PubMed1985

Alteration of amino acid metabolism in epileptogenic mice by elevation of brain pyridoxal phosphate.

A single intraperitoneal injection of pyridoxal-5'-phosphate (PLP) in a species of mouse, DBA/2J, that is normally susceptible to sound-induced convulsion exacerbated its epileptic condition. The effect of injection was most pronounced about 30 min after the administration and subsided gradually within the following 4 h. Correlated with this increased seizure susceptibility were enhanced levels of synaptosomal aspartate and glutamate, and a diminished gamma-aminobutyric acid (GABA) level. The concentrations of nonneuroactive amino acids remained unchanged. When stimulated with veratrine, synaptosomes prepared from PLP-injected mice showed an increased release of aspartate and glutamate and a decreased release of GABA compared to those prepared from control mice. The activity of glutamate decarboxylase in the brains of PLP-treated mice was lowered, whereas the activity of GABA-transaminase was enhanced. Finally, the epileptic condition of DBA mice could be ameliorated by maintenance on a diet composed of vitamin B6-deficient feed and cellulose.

Acoustic Stimulation↗

PubMed1990

Pyridoxal phosphate inhibits the DNA-binding activity of the ecdysteroid receptor.

The effect of pyridoxal 5'-phosphate on the binding of the ecdysteroid receptor from a nuclear extract of Drosophila melanogaster to DNA-cellulose was studied. The binding of hormone-receptor complexes to DNA-cellulose was completely blocked after a 30-min incubation with 3 mM pyridoxal 5'-phosphate at 0-4 degree C. The effect was specific for pyridoxal 5'-phosphate since related compounds (pyridoxal, pyridoxamine 5'-phosphate and pyridoxamine) were not effective or gave only 17% inhibition (pyridoxal). Under standard conditions, none of the compounds tested exerted a significant effect on the stability of [3H](20R,22R)-2 beta,3 beta, 14 alpha,20,22-pentahydroxy-5 beta-cholest-7-en-6-one ([3H]ponasterone A)-receptor complexes. The loss of DNA-binding activity caused by pyridoxal 5'-phosphate is accompanied by changes in the molecular properties of [3H]ponasterone-A-receptor complexes. A shift of [3H]ponasterone-A binding was observed from the 8.0-8.5 S to the 4.5-5.0 S region, when [3H]ponasterone-A-receptor complexes were exposed to pyridoxal 5'-phosphate during sucrose-gradient centrifugation. The inhibition of DNA-cellulose binding by pyridoxal 5'-phosphate can be reversed. Probably, pyridoxal 5'-phosphate forms a Schiff base with a critical lysine group of the ecdysteroid receptor, presumably at its DNA-binding site. The hormone-receptor complexes obtained after removal of pyridoxal 5'-phosphate had the same affinity for DNA-cellulose as 'native' complexes. DNA-cellulose-bound [3H]ponasterone-A complexes were efficiently eluted from DNA-cellulose with pyridoxal 5'-phosphate in 0.1 M KCl resulting in a 104-fold purification of the ecdysteroid receptor. The results reflect possible structural similarities between ecdysteroid and vertebrate steroid receptors.

Cellulose↗

PubMedDate not supplied

[Influence of pyridoxal phosphate in measuring aminotransferases activities in patients with viral hepatitis].

Effect of a pyridoxal phosphate (PP) supplementation of reagents used for ALT and AST measurement was studied in serum of 20 patients suffering from viral hepatitis. Measurements of enzyme activities were carried out at 37 degrees C, using an automate (AU 600, Olympus). Significant differences (p < 0.0001) were observed both for ALT and AST, meanwhile they were more marked for ALT than for AST. This difference was associated with a strong interindividual variability regarding PP activation effect on ALT. In conclusion, aminotransferase measurements should be carried out with a reagent supplemented with PP, when the enzyme activity is used to evaluate a cytolysis. The same recommendation applies when ALT results are integrated into various combinations developed for the evaluation of liver status.

Adult↗

PubMed1997

Cysteine-S-conjugate beta-lyase activity and pyridoxal phosphate binding site of onion alliin lyase.

Purification of onion alliin lyase gave two fractions by cation exchange chromatography. Both fractions showed the comparable high catalytic activity of cysteine-S-conjugate beta-lyase with that of alliin lyase using S-(2-chloro-6-nitrophenyl)-L-cysteine and alliin, S-allyl-L-cysteine sulfoxide as substrates. All the active substrates tested with onion alliin lyase were also active to the cysteine-S-conjugate beta-lyase of Mucor javanicus, but the catalytic activity of the Mucor enzyme was lower for all the substrates. The pyridoxal phosphate binding site of the onion alliin lyase was identified as Lys 285 in the amino acid sequence deduced from cDNA which has been reported. This lysine was conserved in all the sequences from the alliin lyase cDNAs, while similarity was not found between the sequences around pyridoxal phosphate binding sites of both the onion alliin lyase and the Mucor cysteine-S-conjugate beta-lyase.

Allium↗

PubMed1980

Inhibition of nucleic acid and chromatin binding of the rat prostate androgen receptor by pyridoxal phosphate, heparin and Cibacron blue.

The androgen receptor from rat prostate binds 5 alpha-dihydrotestosterone and related androgenic steroids at a steroid binding site and in addition shows selective binding to structures related to nucleic acids (chromatin binding site). Cytoplasmic androgen receptor, labeled with the synthetic androgen methyltrienolone (R 1881) was readily bound at 40c by 2',5'-ADP-agarose, DNA-cellulose and phosphocellulose. The binding to ADP-agarose and DNA-cellulose was used as a model for study of the nucleic acid binding site of the receptor. Complete elution of androgen receptors from these matrices could be obtained with low concentrations of pyridoxal phosphate (10 mM), heparin (0.2 mg/ml) and Cibacron blue (0.4 mM). Sodium molybdate (10 mM) did not interfere significantly with binding of the androgen receptor to ADP-agarose and had little effect on elution of receptors from the gel. Pyridoxal phosphate, heparin and Cibacron blue in low ionic strength buffers were also shown to be very effective for the extraction of androgen receptors from nuclei of rat prostatic tissue. These results suggest gross similarities in the structure of the androgen receptor with activated forms of receptors for corticoids, estrogens and progestins with respect to nucleic acid binding.

Animals↗

PubMed1975

Plasma pyridoxal phosphate levels and tryptophan metabolism in children with burns and scalds.

Depression of the pyridoxal phosphate concentration in the plasma was observed in eight children after burning or scalding. The concentrations remained low for several weeks with the usual dietary supplements but could be restored rapidly with large doses of pyridoxine; this does not seem to alter the excretion of xanthurenic acid but causes an increased loss of kynurenic acid and kynurenine.

Burns↗

PubMed1989

Plasma pyridoxal phosphate levels in newborn infants, their mothers and in the mothers' breast milk.

Plasma pyridoxal phosphate (PLP) was determined in 10 newborn infants (two pairs of twins), their 8 mothers, and in addition in the mothers' breast milk. Mothers who did not take extra vitamin B-6 during pregnancy had lower levels of PLP both in plasma and in their milk. The infants' PLP decreased during the first two weeks of life when receiving only their mothers' milk. None of the mothers nor the infants showed any clinical signs of vitamin B-6 deficiency.

Female↗

PubMed1984

Effects of pyridoxal phosphate treatment on the (Na + K)-ATPase.

Reaction of a dog kidney (Na + K)-ATPase with pyridoxal phosphate, followed by borohydride reduction, reduced the catalytic activity when measured subsequently. The time course of inactivation did not follow a first-order process, and certain characteristics of the residual enzymatic activity were modified. Moreover, various catalytic activities were diminished differently: Na-ATPase activity was largely spared, K-phosphatase activity was diminished only by half that of the (Na + K)-ATPase, whereas (Na + K)-CTPase and Na-CTPase activities were diminished more. ATP, ADP, CTP, nitrophenyl phosphate, and Pi all protected against inactivation. Increasing salt concentrations increased inactivation, but KCl slowed and NaCl hastened inactivation when compared with choline chloride. Occupancy of certain substrate or cation sites seemed more crucial than selection of conformational states. For the residual (Na + K)-ATPase activity the K0.5 for K+ was lower and the K0.5 for Na+ higher, while the sensitivities to ouabain, oligomycin, and dimethylsulfoxide were diminished; for the residual K-phosphatase activity the K0.5 for K+ was unchanged, the sensitivity to ouabain and oligomycin diminished, but the stimulation by dimethylsulfoxide increased. These properties cannot be wholly accommodated by assuming merely shifts toward either of the two major enzyme conformations.

Adenosine Triphosphatases↗

PubMed1985

Depressed plasma pyridoxal phosphate concentrations in adult asthmatics.

In 15 adult patients with bronchial asthma, plasma and erythrocyte pyridoxal phosphate (PLP) concentrations were significantly lower than in 16 controls (P less than 0.0001 and P less than 0.005, respectively). Oral supplementation of seven asthmatics with 50 mg pyridoxine as pyridoxine X HC1 twice daily failed to produce a sustained elevation of PLP in either the plasma or erythrocytes. However, all subjects reported a dramatic decrease in frequency and severity of wheezing or asthmatic attacks while taking the supplement. The reasons for the failure of a uniform elevation in plasma and erythrocyte PLP concentration and for the apparent beneficial effects of pyridoxine supplementation on the asthmatic symptoms of the patients are unknown at present.

Adult↗

PubMed1993

Interaction of pyridoxal phosphate with thymidylate synthase: spectral and equilibrium dialysis studies.

1. Changes in the spectrum of pyridoxal phosphate (PLP) were produced by adding an equimolar amount of native thymidylate synthase, but not by adding denatured enzyme or enzyme modified by sulfhydryl-blocking reagents. 2. The dissociation constant of the thymidylate synthase-PLP complex determined by equilibrium dialysis was 9 +/- 1.6 microM, the maximum number of PLP molecules bound per molecule of native thymidylate synthase was 2.5 +/- 0.4, and the Hill coefficient was 0.97. 3. No evidence of PLP binding was found with denatured thymidylate synthase, and only slight binding was observed when enzyme SH groups were blocked or when the active site was blocked with 5-fluorodeoxyuridylate (FdUMP) and methylene tetrahydrofolate. 4. The presence of dUMP, dTMP, or FdUMP interfered with the binding of PLP to thymidylate synthase, and the presence of equimolar amounts of PLP interfered with the binding of dUMP.

Binding Sites↗

PubMed1977

The quantitative histologic distribution of pyridoxal phosphate in the rat stomach, effects of drugs influencing secretion.

The quantitative histologic distribution of pyridoxal phosphate (PLP), the coenzyme of histidine decarboxylase in the biosynthesis of histamine as well as the coenzyme of other enzyme systems, was established in the rat glandular stomach in different secretory states. This was accomplished by analyzing PLP with an improved fluorometric microprocedure in histologically identified fresh-frozen microtome tissue sections cut serially through the stomach wall of fed and 24-hr fasted rats, and of the latter injected with drugs that stimulate (histamine, theophylline and urecholine) or inhibit (atropine) secretion. In fasted compared with fed rats, and in fasted rats injected with histamine (low dose) or theophylline, compared with saline injected controls, the PLP level was reduced not only in the chief cell region where PLP is utilized in histamine production, but also in the parietal and mucous neck cell region, indicating PLP function in other enzyme systems in these sites. No change in PLP level occurred when histamine was injected at a high dose known to suppress histidine decarboxylase activity. Administration of urecholine or atropine had negligible effect on the PLP levels, indicating a lack of vagal stimulatory or inhibitory influence.

Animals↗

PubMed1994

Inhibition of gp120-CD4 interaction and human immunodeficiency virus type 1 infection in vitro by pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate and related compounds were tested for their ability to inhibit gp120-CD4 interaction and human immunodeficiency virus infection in vitro. The results show that pyridoxal 5'-phosphate is a unique CD4 antagonist whose antiviral potency derives from the presence of both lysine-reactive and anionic substituents.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1989

Reactions of phosphonate analogs of pyridoxal phosphate with apo-aspartate aminotransferase.

We have investigated reactions of the 5-phosphonoethyl and 5-phosphonoethenyl analogs of pyridoxal 5'-phosphate in the coenzyme site of cytosolic aspartate aminotransferase. Acid dissociation constants and equilibrium constants for hydration and for tautomerization have been evaluated for these compounds. In confirmation of previous results, both compounds are partially active. They bind to apoenzyme well and undergo conversion in the presence of glutamate to amine forms which show induced circular dichroism comparable to that of native enzyme. A normal "external" Schiff base is evidently formed with 2-methylaspartate, but the amounts of quinonoid intermediate formed with erythro-3-hydroxyaspartate are less than those formed with pyridoxal phosphate. The pKa of the imine group of the enzyme reconstituted with the phosphonoethyl analog is more than two units lower than that in the native enzyme. Binding of the dicarboxylates glutarate, 2-oxoglutarate, and succinate shifts the pKa upward. The absorption spectra of the resulting complexes indicate the existence of at least three low pH species. A shift of 2.3 to 2.9 ppm to a lower frequency was observed for the 31P NMR signal upon binding of these dicarboxylates or of 2-methylaspartate. Enzyme containing the analogs crystallizes. Polarized absorption spectra suggest that the coenzyme has an orientation similar to that of pyridoxal phosphate in the native enzyme.

Amines↗

PubMed1976

Band-shape analysis and resolution of electronic spectra of pyridoxal phosphate and other 3-hydroxypyridine-4-aldehydes.

The electronic absorption spectra of individual ionic forms of pyridoxal phosphate and of a series of related aldehydes have been evaluated together with pKa values. Spectral resolution with lognormal curves has permitted the quantitative description of equilibria for hydration and tautomerization. Precise values of peak positions for both aldehyde and hydrate forms have been obtained. Measurements of temperature-induced changes in the spectra have provided additional information. Knowing the hydration ratios and stepwide acid dissociation constants, it is possible to evaluate microscopic acid dissociation constants for both the aldehyde and hydrate forms of the compounds.

Molecular Conformation↗

PubMed1986

Simultaneous and direct determination of pyridoxal, pyridoxal-5'-phosphate, and pyridoxic acid in serum by derivative synchronous fluorescence spectroscopy.

Simultaneous and direct assays of pyridoxal, pyridoxal-5'-phosphate, and pyridoxic acid in human serum are described. The method applied is based on the reaction of these compounds with beryllium in an ammoniacal medium to yield highly fluorescent derivatives. Overlapping of conventional fluorescence spectra is resolved by using second-derivative fluorescence spectroscopy, thus making the use of separation techniques unnecessary. The proposed method is simple (only beryllium and an ammoniacal buffer are needed to develop fluorescence), rapid (the derivative formation is instantaneous and serum treatment only requires deproteinization), and inexpensive (no sophisticated detection equipment is necessary, any conventional modern spectrofluorimeter being adequate for use). The analytical recovery achieved was of about 96% for pyridoxal, 97% for pyridoxal-5'-phosphate, and 100% for pyridoxic acid. Measurements were carried out in a single scan.

Beryllium↗