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PubMed1976

Abnormal regulation of plasma pyridoxal 5'-phosphate in patients with liver disease.

UNLABELLED: Pyridoxal 5'-phosphate (PLP), the coenzyme form of vitamin B6, is essential for many biochemical reactions in the body. Studies in experimental animals have suggested that the liver is a primary site for the formation of PLP circulating in the plasma, and that it may also participate in its degradation. This study evaluates, for the first time, the effects of liver disease in man on the regulation of plasma PLP. The plasma PLP level was measured before and sequentially after the rapid intravenous administration of 50 mg of pyridoxine to patients with alcoholic cirrhosis, acute hepatitis, and extrahepatic obstruction, and to normal control subjects. The base line plasma PLP concentration was significantly lower in cirrhotic patients than in normal persons (P less than 0.025), and there was a tendency for it to be reduced in patients with extrahepatic obstruction. After administration of pyridoxine there was a significant increase in the plasma PLP level over a 2- to 12-hr period, after which the concentration returned gradually toward the initial value. The area under the concentration/time curve was from 2 to 8 times smaller (P less than 0.002) in the patients with liver disease. To assess possible mechanisms of this change, 5 mg of PLP were intravenously administered to the various patient groups and the pharmacokinetics of the disposition were assessed. The initial and steady state volumes of distribution of PLP were comparable in cirrhotics and controls (P greater than 0.05), but the clearance of plasma PLP in cirrhotics was much faster (63.0 +/- 7.4 versus 31.7 +/- 2.7 ml per min, P less than 0.004). Similar findings were obtained in the other liver disease subjects. The in vitro plasma binding of PLP at supracirculatory concentrations was comparable in cirrhotics and controls (99.4 versus 99.5%, P greater than 0.05). IN CONCLUSION: (1) plasma PLP regulation in patients with liver disease is abnormal, (2) a significant factor in the decrease in plasma PLP after intravenous pyridoxine administration in these patients appears to be an increase in the total plasma clearance of the coenzyme, and (3) it is postulated that this may be due to increased degradation of PLP by the diseased liver.

Acute Disease↗
PubMed1981

Inhibitory effect of pyridoxal 5'-phosphate on the DNA binding site of ATP-dependent deoxyribonuclease from Bacillus laterosporus.

Bacillus laterosporus ATP-dependent deoxyribonuclease has been found to be inhibited by pyridoxal 5'-phosphate. The inhibition is specific for pyridoxal 5'-phosphate and pyridoxal which are required in relatively high concentrations. Pyridoxamine 5'-phosphate, pyridoxamine, and pyridoxine are ineffective. The inhibition is reversed by dilution or dialysis but can be changed to an irreversible inactivation by reduction of the enzyme . pyridoxal 5'-phosphate complex with sodium borohydride. The compound is a competitive inhibitor with respect to DNA but not ATP. Moreover, the presence of DNA substrate protects the enzyme against this inactivation but the presence of ATP shows no effect. The reduced enzyme . pyridoxal 5'-phosphate complex displays a new absorption maximum at 325 nm and a fluorescence emission at 390-400 nm when excited at 325 nm which are characteristic for epsilon-N-(phosphopyridoxyl)lysine. Thus, B. laterosporus DNase appears to have an essential lysine residue at the DNA binding site of the enzyme, and the enzyme possess two different active sites, a DNA binding site and an ATP binding site.

Bacillus↗
PubMed1975

Pyridoxal 5'-phosphate and analogs as probes of coenzyme-protein interaction in Baccillus alvei tryptophanase.

Trytophanase from Bacillus alvei was resolved from its coenzyme, pyridoxal phosphate, by treatment with cysteine followed by column chromatography. Spectrophotometric titration of apoenzyme with pyridoxal-P showed 1 mol of pyridoxal-P bound per 52,000 g of enzyme. Kinetic analysis of coenzyme binding showed hyperbolic activation curves with a Km of 1.6 muM. Pyridoxal-P was used as a natural active site probe in spectrophotometric studies to distinguish differences in the active center of holotryptophanase and reconstituted enzyme that were not apparent by other techniques. The pKa for holotryptophanase is 7.9 while the pKa for reconstituted apoenzyme is 8.4. Apotryptophanase binds 2-nor, 2'-methyl, 2'-hydroxy, 6-methyl, and N-oxide pyridoxal-P to form analog enzymes distinguishable on the basis of absorption spectra and relative activity in catalyzing both the alpha, beta-elimination and beta-replacement reactions of tryptophanase. Apoenzyme also binds pyridoxal but pyridoxal analog enzyme is not active.

Apoenzymes↗
PubMed1977

Induction of pyridoxal phosphate-dependent enzymes in vitamin B-6 deficient rats.

The effectiveness of dietary and hormonal treatments in inducing several pyridoxal phosphate-(PLP)-dependent enzymes has been examined in vitamin B-6 deficient rats. Holo- and apoenzymes of serine dehydratase and ornithine aminotransferase were inducible in both control and deficient rats by feeding them 80% casein diets or by injecting them with glucagon. Holo- and apotyrosine aminotransferase were induced in both control and deficient rats by injecting them with glucagon or with dexamethasone phosphate. Phosphoenolpyruvate carboxykinase, a non-PLP-dependent enzyme, was inducible in both control and deficient rats by glucagon treatment if the rats were fed, but not if they had been starved. The degree of induction of certain enzymes depended upon whether rats were fed ad libitum, starved overnight, or fed a protein-free diet prior to the induction period. Phosphoenolpyruvate carboxykinase activities were about the same in both control and deficient rats. In vitamin B-6 deficient rats, both uninduced and induced activities of serine dehydratase, ornithine aminotransferase, and tyrosine aminotransferase assayed in the prsence of PLP, but not in its absence, either equaled or exceeded control values under most experimental conditions. Synthesis of excess of apoenzyme of PLP-dependent enzymes generally accounted for the high total enzyme activity in deficient rats. Differences between values for control and deficient rats could not be accounted for by differences in liver cyclic AMP concentrations nor were they apparently related to reduced food intake of the deficient rats. High apoenzyme concentration during depletion of coenzyme would tend to prevent depletion of active enzyme.

Animals↗
PubMed1981

Amino acid sequence around the pyridoxal 5'-phosphate binding sites of 6-phosphogluconate dehydrogenase.

6-Phosphogluconate dehydrogenase (6-phospho-D-gluconate: NADP+ 2-oxidoreductase(decarboxylating), EC 1.1.1.44) from Candida utilis is inhibited by reaction with pyridoxal 5'-phosphate. The aldehydic group of this compound forms a Schiff base with the epsilon-amino group of a lysine residue: reduction of this enamine with tritiated borohydride can label this amino acid. Two tryptic peptides, TS2 and TS3, have been isolated from the labelled protein and found to have the following amino acid sequences: TS2: Ile-Leu-Asx-Glx-Ala-Gly-Gly-Lys(P-Pxy)-Gly-Glx-Thr-Lys TS3: Thr-Val-Ser-Lys(P-Pxy)-Val-Asp-His-Phe-Ile-(Glx,Asx,Glx)-Ala-Lys where Lys(P-Pxy) indicates the modified lysine residue. The similarities between the amino acid sequences around the pyridoxal phosphate binding lysines of 38 peptides, obtained from enzymes which have pyridoxal phosphate as cofactor or inhibitor, are discussed and a prediction is made on the presence of reverse turns in these peptides.

Amino Acid Sequence↗
PubMedDate not supplied

Competition of pyridoxal 5'-phosphate with ribulose 1,5-bisphosphate and effector sugar phosphates at the reaction centers of the spinach ribulose 1,5-bisphosphate carboxylase/oxygenase.

The stimulation of the carboxylase reaction by effectors of ribulose 1,5-bisphosphate carboxylase/oxygenase displays higher sensitivity towards pyridoxal 5'-pyridoxal 5'-phosphate inhibition than the catalytical process itself. Pyridoxal 5'-phosphate binding to the enzyme is not affected by the modulators 6-phosphogluconate and fructose 1,6-bisphosphate at low concentrations at which these agents stimulate the carboxylation rate. At higher concentrations these sugar phosphates protect the enzyme against pyridoxal 5'-phosphate inhibition in a similar fashion like the substrate ribulose 1,5-bisphosphate. Such protection experiments in combination with spectrophotometrical studies of pyridoxal 5'-phosphate binding demonstrate two binding states of ribulose 1,5-bisphosphate at the reaction centers of the enzyme with different requirements for Mg2+. 6-Phosphogluconate functions as protector only in the presence of Mg2+. Our results imply a competition between pyridoxal 5'-phosphate and substrate or effector sugar phosphates at the reaction centers of the spinach carboxylase. It is proposed that the pyridoxal 5'-phosphate inhibition of the stimulatory activity of these effectors originates from a modification of the regulatory sites of the enzyme caused by pyridoxal 5'-phosphate binding to the catalytical sites.

Binding Sites↗
PubMed1986

Acetylcholinesterase is not inhibited by pyridoxal 5'-phosphate.

Acetylcholinesterase activity was assayed in the absence and presence of pyridoxal 5'-phosphate. If substrate hydrolysis was measured by the pH-stat method, its rate was not significantly affected by pyridoxal 5'-phosphate. In the spectrophotometric assay, however, this compound led to an apparent decrease in rate. The discrepancy between the two assays is explained by stray-light artefacts produced by pyridoxal 5'-phosphate at the wavelengths of the spectrophotometric assay.

Acetylcholinesterase↗
PubMed1982

A calorimetric study of the interaction of pyridoxal 5'-phosphate with aspartate apoaminotransferase and model compounds.

Schiff base formation between pyridoxal 5'-phosphate and model compounds of increasing complexity (i.e. L-valine and poly-L-lysine) and between pyridoxal 5'-phosphate and the apoenzyme of aspartate aminotransferase has been analyzed by microcalorimetric methods. The apparent pKa values and protonation enthalpy values for the relevant groups ionizing in the pH 4-9 range have been determined for the Schiff bases of L-valine and of poly-L-lysine. Upon Schiff base formation, the only noticeable change is the lowering of the ring nitrogen pK, accompanied by an increase of the relative delta H. In the poly-L-lysine Schiff base, however, the delta H relative to the protonation of the phosphate dianion becomes more negative. This behavior suggests a multiple interaction between the polymer and the ligand. The intrinsic heat of formation is small (congruent to -1 kcal/mol), of the same order of magnitude for both Schiff bases, and appears to be independent of the nature of the aminic reagent. The heat of reaction of pyridoxal 5'-phosphate with aspartate apoaminotransferase has been determined in the pH 6.2-8.8 range at 19 degrees C and at 25 degrees C. Each isotherm is characterized by a lack of proton evolution, a result that is unexpected on the basis of the known pK values of the reagents, and by a sharp pH dependence of the enthalpy change. Moreover, comparison of the two isotherms allows: (a) detection of a protonation-dependent effect (pK 7.5 at 25 degrees C), (b) exclusion of a preferential binding of the coenzyme to the apoenzyme in a particular ionization state; and (c) suggestion of a tightening of the protein molecule upon holoenzyme formation.

Animals↗
PubMed1976

Pyridoxal 5'-phosphate and the regulation of ornithine decarboxylase activity and stability.

There are two forms of ornithine decarboxylase with respect to pyridoxal 5'-phosphate (pyridoxal-P) affinity in exponentially-growing Swiss 3T3 mouse fibroblasts: form I (Km approximately 10 muM) accounts for 30% of the total activity, and form II (Km approximately 0.4 muM) the remainder. Each form of the enzyme is in rapid equilibrium with ornithine and pyridoxal-P; neither form recognizes the Schiff base between ornithine and pyridoxal-P as a substrate. Total pyridoxal-P concentrations indicate that both forms may normally be at least partially active in vivo. Upon stimulation of 3T3 cells by pituitary growth factors, form I becomes undetectable within 4 h. As total activity increases over 10-fold during this time and continues to increase thereafter, a possible conversion of form I to form II could account for this increase only if the Km change reflects other changes in preexisting enzyme. The rates of cofactor dissociation are apparently the same for each form and neither rate changes with the growth state. Since rapid equilibrium kinetics apply, the forms apparently differ in their rate of cofactor association. The half-lives of the two forms in vivo are the same in unstimulated cells when measured concurrently. Also, the half-life of total activity decreases markedly upon stimulation as form II becomes dominant. These and other observations are not consistent with pyridoxal-P serving a major protective function for the enzyme in vivo.

Binding Sites↗
PubMed1989

Intracellular predominance of the pyridoxal 5'-phosphate form of aspartate aminotransferase in Escherichia coli B and reversible transformation of this form by extracellular substances.

The intracellular proportion of the pyridoxal 5'-phosphate form of aspartate aminotransferase to the total enzyme in E. coli B cells was determined by a newly devised method, dependent on selective inactivation of the intracellular pyridoxal 5'-phosphate form of the enzyme by extracellularly added sodium borohydride. A large portion (80-99%) of the intracellular aspartate aminotransferase was in pyridoxal 5'-phosphate form in both natural and synthetic medium-grown bacterial cells. The intracellular predominancy of pyridoxal 5'-phosphate did not vary during the growth of bacteria and during incubation of bacterial cells in various kinds of buffers with different pH values. In contrast, the saturation levels generally used to describe in vivo the proportions of the apo and holo vitamin B6-dependent enzymes did not reflect the intracellular amount of the pyridoxal 5'-phosphate (holo) form of aspartate aminotransferase probably because the intracellular pyridoxal 5'-phosphate form was changed to an apo form by the disruption of bacterial cells for preparing crude extract. Various extracellularly-added vitamin B6 antagonists decreased the intracellular amount of pyridoxal 5'-phosphate without decrease in the total intracellular activity of the enzyme. The modified forms were stable in E. coli B cells and reversed into pyridoxal 5'-phosphate form by incubation of the antagonist-treated cells in the buffer containing pyridoxal. The present results showed that the sodium borohydride reduction method can be used for further analysis of the in vivo interaction of pyridoxal 5'-phosphate and apoaspartate aminotransferase. The fact that about 50% of the intracellular pyridoxal 5'-phosphate form was changed to a modified form without impairment of cell growth in the presence of 4-deoxypyridoxine, and that about 50% of intracellular modified aspartate aminotransferase was reversed to pyridoxal 5'-phosphate by the removal of antagonist followed by incubation suggested that there exists characteristically 2 different fractions of pyridoxal 5'-phosphate forms of aspartate aminotransferase in E. coli cells.

Aspartate Aminotransferases↗
PubMed1997

Enhancement of the response to purinergic agonists in P2Y1 transfected 1321N1 cells by antagonists suramin and PPADS.

1. We have previously shown that both suramin and pyridoxal-phosphate-6-azophenyl-2',4' disulphonic acid (PPADS) act as antagonists at transfected P2Y1 receptors. Here we show that under certain experimental conditions these two P2 antagonists can enhance the response to agonists acting at these receptors. 2. The expression of either P2Y1 or P2Y2 receptors in 1321N1 human astrocytoma cells results, on a change of medium, in an elevation of basal (no added agonist) accumulation of [3H]-inositol(poly)phosphates([3H]-InsPx) compared to cells not expressing these receptors. This elevation is much greater in P2Y1 transfectants than in P2Y, transfectants. 3. Both PPADS and suramin reduced this basal level of [3H]-InsPx accumulation in the P2Y1 expressing cells. 4. When a protocol was used which required changing the culture medium, antagonists were added at a concentration which reduced the basal accumulation by about 50%, there was a significant stimulation in response to increasing concentrations of 2-methylthioadenosine 5'-triphosphate (2MeSATP), in the absence of antagonists there was no significant effect of the agonist. 5. However, when 2MeSATP was added in the absence of a change of medium and with no antagonist present, there was a several fold increase in [3H]-InsPx accumulation. These results show that a release of endogenous agonist activity (possibly ATP/ADP) from the P2Y1 expressing cells can create conditions in which a response to an agonist such as 2MeSATP can only be seen in the presence of a competitive antagonist.

Adenosine Triphosphate↗
PubMed1993

Vitamin B6 metabolism and binding to proteins in the blood of alcoholic and nonalcoholic men.

Blood obtained from nonalcoholic and alcoholic subjects was incubated with 100 nM [3H]pyridoxine to study its uptake and metabolism by erythrocytes and the binding of vitamin B6 metabolites to proteins in plasma and erythrocytes. Erythrocytes of the alcoholics accumulated tritium faster than those of the controls; however, they contained the same total amount of tritiated compounds by 15 min. After incubation for 30 min, the erythrocytes had converted most of the pyridoxine to pyridoxal phosphate and pyridoxal. Pyridoxal-P remained in the erythrocytes, and approximately 40% of the pyridoxal diffused into the plasma. [3H]Pyridoxal and [3H]pyridoxal-P levels in the erythrocytes and plasma of the alcoholics were similar to those in the controls. However, dialyzed hemolysates of the alcoholics had more [3H]pyridoxal and a lower percentage of [3H]pyridoxal-P than those of the controls. The total concentration of plasma pyridoxal-P was lower in the alcoholics than in the controls and did not change upon incubation of whole blood with pyridoxine or upon dialysis. The erythrocytes of the alcoholics and controls had similar concentrations of pyridoxal-P that increased 2.5-fold upon incubation of whole blood with pyridoxine for 30 min and returned to the initial concentrations upon dialysis. The amount of [3H]pyridoxal and [3H]pyridoxal-P bound to protein was assessed by treating hemolysate and plasma samples with borohydride before dialysis. More 3H was bound to protein in the erythrocytes than in the plasma. The amount of protein-bound 3H in the erythrocytes of the alcoholics was lower than that of the controls, whereas the amount of protein-bound 3H in plasma was similar in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1999

Adequacy of maternal pyridoxine supplementation during pregnancy in relation to the vitamin B6 status and growth of neonates at birth.

To evaluate the adequacy of maternal pyridoxine supplementation during pregnancy for both maternal and neonatal status at birth, vitamin B6 status, assessed by plasma pyridoxal phosphate (PLP), pyridoxal (PL) and total aldehyde vitamer (PLP + PL) concentrations, and the growth of neonates, including weight, length, head and chest circumferences, were examined for 209 neonates whose mothers were supplemented with 0, 1, 2 or 3 mg pyridoxine.HCl (PN.HCl)/d during pregnancy. Maternal PN.HCl supplementations were positively correlated to both maternal (r = 0.62) and cord (r = 0.78) plasma PLP concentrations (p < 0.0001). Mothers supplemented with 2 or 3 mg/d PN.HCl had significantly higher plasma concentrations of PLP and total B6 aldehyde vitamer in maternal and cord blood compared with those receiving 0 or 1 mg PN.HCl/d. A growth benefit for neonates whose mothers had maternal and cord plasma PLP concentrations > or = 40 nM was revealed by the maternal supplementation of 2 mg PN.HCl/d during pregnancy. Thus, in healthy pregnant women, according to our study, a daily supplement of 2 mg PN.HCl provides the adequacy of maternal and neonatal vitamin B6 status and the satisfactory growth of neonates at birth.

Dietary Supplements↗
PubMed1992

Androgen hydroxysteroid dehydrogenases under the influence of pyridoxine derivatives.

The metabolism of testosterone in the rat ventral prostate, anterior pituitary, basal hypothalamus and amygdala was studied in vitro under the influence of vitamin B6 compounds. The influence of these compounds on the activity of 5 alpha-reductase (5 alpha-R), 3 alpha- and 17 beta-hydroxysteroid dehydrogenase (3 alpha-HSD, 17 beta-HSD) was determined for all the examined tissues. Pyridoxine hydrochloride significantly increased the activity of 5 alpha-R, 3 alpha- and 17 beta-HSD, but pyridoxal hydrochloride had an inhibitory influence on 5 alpha-R and showed no effect on 3 alpha-HSD activity at the prostate level. Male rat anterior pituitary, basal hypothalamus or amygdala incubated with pyridoxal phosphate and pyridoxal hydrochloride showed modified enzymatic activities. Pyridoxal hydrochloride showed an inhibitory effect on 5 alpha-R in the rat pituitary and basal hypothalamus as well as in the rat prostate.

17-Hydroxysteroid Dehydrogenases↗