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PubMed2000

Role of pyridoxal 5'-phosphate in the structural stabilization of O-acetylserine sulfhydrylase.

Proteins belonging to the superfamily of pyridoxal 5'-phosphate-dependent enzymes are currently classified into three functional groups and five distinct structural fold types. The variation within this enzyme group creates an ideal system to investigate the relationships among amino acid sequences, folding pathways, and enzymatic functions. The number of known three-dimensional structures of pyridoxal 5'-phosphate-dependent enzymes is rapidly increasing, but only for relatively few have the folding mechanisms been characterized in detail. The dimeric O-acetylserine sulfhydrylase from Salmonella typhimurium belongs to the beta-family and fold type II group. Here we report the guanidine hydrochloride-induced unfolding of the apo- and holoprotein, investigated using a variety of spectroscopic techniques. Data from absorption, fluorescence, circular dichroism, (31)P nuclear magnetic resonance, time-resolved fluorescence anisotropy, and photon correlation spectroscopy indicate that the O-acetylserine sulfhydrylase undergoes extensive disruption of native secondary and tertiary structure before monomerization. Also, we have observed that the holo-O-acetylserine sulfhydrylase exhibits a greater conformational stability than the apoenzyme form. The data are discussed in light of the fact that the role of the coenzyme in structural stabilization varies among the pyridoxal 5'-phosphate-dependent enzymes and does not seem to be linked to the particular enzyme fold type.

Cysteine Synthase↗
PubMed1990

Acute ingestion of glucose decreases plasma pyridoxal 5'-phosphate and total vitamin B-6 concentration.

The effect of the acute ingestion of glucose on the concentration of pyridoxal 5'-phosphate (PLP) and total vitamin B-6 (TB6) in plasma was studied. Five males and four females were given 1 g D-glucose/kg body wt. A blood sample was collected at 0-10 min and 0, 0.5, 1, 2, 3, 4, and 5 h and the plasma was analyzed for PLP and TB6. There was a significant decrease in both plasma PLP and TB6 (p less than 0.05) at 2 h. All nine subjects exhibited a decrease, and by 5 h the mean change from fasting was 8-10 nmol/L for PLP and 8-12 nmol/L for TB6 (an 18-21% decrease from that when fasting). Ingestion of 300 mL water in four subjects resulted in a mean decrease of 3.4 nmol PLP/L plasma and indicated that some but not all of the decrease may have been due to fluid ingestion per se. Ingestion of glucose is associated with a decrease in both circulating plasma PLP and TB6; therefore, carbohydrate ingestion should be considered when plasma PLP and TB6 concentrations are used to evaluate vitamin B-6 status. In addition, fasting blood samples should be used when evaluating vitamin B-6 status.

Administration, Oral↗
PubMed1990

Chemical modification of adenylosuccinate synthetase from Escherichia coli by pyridoxal 5'-phosphate. Identification of an active site lysyl residue.

Incubation of adenylosuccinate synthetase from Escherichia coli with low concentrations of pyridoxal 5'-phosphate (PLP) resulted in a rapid loss of activity (92%), concomitant with the formation of a Schiff base. The inactivation of the enzyme by PLP is apparently first order with respect to PLP. The pseudo-first order rate constant, Kapp, showed a hyperbolic dependence on the concentration of PLP, indicating that a kinetically significant PLP.enzyme intermediate is formed during the inactivation process. Stoichiometry and peptide isolation studies showed that 2 lysine residues were modified during reaction of the enzyme with PLP. The three substrates of adenylosuccinate synthetase (GTP, IMP, and aspartate) showed different effects in their ability to protect the enzyme against PLP inactivation. Complete protection of the enzyme against inactivation can be observed only in the presence of high concentrations of GTP. One lysine residue was protected under these conditions. In contrast to GTP, addition of the other two substrates either alone or together to reaction mixtures did not render protection. Peptide mapping by digesting the enzyme with trypsin revealed that the lysine shielded by GTP is Lys140. Replacing the Lys140 with Ile140 by site-directed mutagenesis resulted in total loss of the activity. These results suggest that Lys140 may play an important role in enzymatic activity.

Adenylosuccinate Synthase↗
PubMed1992

Effect of interaction between gentamicin and pyridoxal-5-phosphate on functional and metabolic parameters in kidneys of female Sprague-Dawley rats.

Daily intraperitoneal (i.p.) injection of genatmicin at a dose of 70 mg/kg for 11 days produced nephrotoxicity in female Sprague-Dawley rats as evidenced by increased excretion of urinary protein and trypsin inhibitory activity as well as rise in renal individual class and total phospholipid. The observed proteinuria was associated with a significant twofold fall in creatinine clearance and histopathological changes, including the presence of hyaline casts and flattened epithelial cells within the lumen of proximal convoluted tubules. Although pyridoxal-5-phosphate (50 mg/kg) administered i.p. did not significantly alter creatinine clearance, histopathology, proteinuria, and urinary trypsin inhibitory activity, an increase in individual class and total phospholipid was noted in kidney. In rats simultaneously administered gentamicin and pyridoxal-5-phosphate, the observed fall in renal gentamicin content was associated with a return of individual class and total phospholipid to control values. However, the decline in creatinine clearance, enhanced proteinuria, and increase in urinary trypsin inhibitory activity in the simultaneous-treated group was similar or greater than that seen in the gentamicin-only injected rats. Morphological examination of simultaneous-treated rats revealed extensive alterations in proximal tubules including numerous mitotic figures, large vesicular nucleii, and prominent nucleoli in epithelial cells as well as hyaline casts within the lumen. Our data combined with results of previous studies suggest that sex and type of rat strain are important factors in aminoglycoside-induced nephrotoxicity. It is evident that a specific concentration of pyridoxal-5-phosphate may be necessary to provide protection against all manifestations of aminoglycoside-induced renal damage.

Animals↗
PubMed1981

Preincubation of serum aspartate aminotransferase with pyridoxal 5'-phosphate in the SMAC: comparison with revised DuPont aca method and recommended IFCC method.

The method for continuous-flow assay of aspartate aminotransferase with the Technicon SMAC was modified to include preincubation of the serum enzyme with pyridoxal 5'-phosphate, to be consistent with the recommendations of IFCC and the Standards Committee of AACC. Preliminary estimates of the imprecision of the modified method on SMAC gave day-to-day standard deviations of 5.3 U/L at mean of 48 U/L (n = 66) and 6.2 U/L at 155 U/L (n = 61). Added bilirubin, sodium pyruvate, ascorbic acid, and endogenous lipids did not interfere. Comparison of results for 50 samples by this method with those by the manual IFCC method gave y = 1.1113x - 0.3 U/L, Sy/x = 4.4 U/L, and r = 0.997. Similar data are presented for the revised AST method for the DuPont aca discrete analyzer. Clinical data show that AST activities increase by as much as 200% when the serum is preincubated with pyridoxal 5'-phosphate.

Animals↗
PubMedDate not supplied

Plasma pyridoxal 5'-phosphate is a significant indicator of immune responses in the mechanically ventilated critically ill.

OBJECTIVES: This study assessed the effect of vitamin B6 status on immune responses in mechanically ventilated, critically ill patients and compared the results with those of healthy controls. METHODS: This was designed as a cross-sectional observational study. Forty patients in the intensive care unit successfully completed this study. Vitamin B6 intake was recorded for 8 d. Severity of illness (Second Acute Physiology and Chronic Health Evaluation score) was recorded. Thirty-eighty healthy controls were recruited from the physical check unit of Taichung Veterans General Hospital (Taichung, Taiwan). All control subjects were given instruction on how to complete a 24-d diet recall. Vitamin B6 status was assessed by direct measures (plasma pyridoxal 5'-phosphate [PLP] and 4-pyridoxic acid) and indirect measures (erythrocyte alanine and aspartate aminotransferase activity coefficients). Levels of serum albumin, hemoglobin, hematocrit, high-sensitivity C-reactive protein, and immune responses (white blood cell, neutrophil, total lymphocytes, T lymphocytes [CD3], B lymphocytes [CD19], T-helper cells [CD4], and suppressor cells [CD8]) were determined. RESULTS: Critically ill patients had sufficient vitamin B6 intake but showed marginal PLP deficiency (20.9 +/- 1.5 nmol/L). In addition, critically ill patients had significantly lower and abnormal immune responses than did healthy controls. There was no significant correlation of vitamin B6 intake and erythrocyte alanine and aspartate aminotransaminase activity coefficients with immune indices. Plasma PLP concentration was strongly negatively correlated with high-sensitivity C-reactive protein level. However, plasma PLP was significantly associated with immune responses after adjustment for age, sex, high-sensitivity C-reactive protein, and the other four vitamin B6 indicators. CONCLUSIONS: Plasma PLP is a significant indicator of immune responses in human subjects. Further research is warranted to study whether vitamin B6 supplementation in critically ill patients improves their immune responses.

APACHE↗
PubMed1989

Determination of plasma pyridoxal 5'-phosphate by an enzymatic-high-performance liquid chromatographic procedure.

An enzymatic-HPLC procedure for the determination of plasma pyridoxal 5'-phosphate (PLP) has been established. The assay is based on the decarboxylation of L-3,4-dihydroxyphenylalanine using Streptococcus tyrosine decarboxylase apoenzyme, which requires PLP as cofactor. The product of the enzyme reaction, dopamine, is measured by Coulochem electrochemical detection with a series of oxidizing and then reducing electrodes. Trace amounts of PLP in the apoenzyme preparation were removed with the aid of cysteine-sulfinic acid and gel filtration. The detection limit for PLP by this method is 50 pM in plasma.

Chromatography, High Pressure Liquid↗
PubMed1983

Low muscle levels of pyridoxine in McArdle's syndrome.

Pyridoxal phosphate is a covalently bound cofactor of glycogen phosphorylase. Phosphorylase is a major muscle protein and therefore represents a significant pool of pyridoxal phosphate. Muscle pyridoxine content was measured in three patients with myophosphorylase deficiency (McArdle's syndrome) in whom there was a marked diminution or absence of phosphorylase protein as determined by acrylamide gel electrophoresis. Total muscle pyridoxine in the patients with McArdle's syndrome (0.55 +/- 0.08 microgram/g wet weight, mean +/- SD) was markedly reduced compared with 11 human control subjects who had normal levels of muscle phosphorylase (total muscle B6 = 2.49 +/- 0.47). Despite such drastically low levels of muscle pyridoxine, these patients had no evidence of pyridoxine "deficiency." These results suggest that low muscle B6 in McArdle's syndrome represents the specific loss of pyridoxal phosphate normally bound to phosphorylase apoenzyme and imply that phosphorylase pyridoxal phosphate accounts for 75 to 80 percent of the total pyridoxine in normal human muscle.

Adolescent↗
PubMed1987

Quantitative description of absorption spectra of a pyridoxal phosphate-dependent enzyme using lognormal distribution curves.

Ultraviolet-visible absorption spectra of cytosolic aspartate aminotransferase of pig hearts have been analyzed by resolution with lognormal distribution curves. These have been compared with spectra of reference Schiff bases of pyridoxal 5'-phosphate. Spectra of the free enzyme in two different states of protonation and of complexes with monoanions, dicarboxylates, the substrates L-glutamate, L-aspartate, and L-erythro-3-hydroxyaspartate, and the quasi-substrate 2-methylaspartate have been analyzed. Relative amounts of three tautomeric species have been estimated, as have amounts of various enzyme-substrate intermediates. Bandshape parameters which can be used as a guide to analysis of spectra of other pyridoxal phosphate-dependent enzymes are tabulated. Some formation constants and pKa values, which were evaluated at the same time as the spectra of the complexes, are also reported.

Animals↗
PubMed1985

Measurement of plasma pyridoxal 5'-phosphate by combination of an enzymatic assay with high-performance liquid chromatography/electrochemistry.

An assay method for the determination of pyridoxal 5'-phosphate (PLP) in plasma is presented. The procedure is rapid and requires only a small volume. The method includes PLP-dependent enzymatic decarboxylation of L-tyrosine to tyramine (enzyme used: L-tyrosine apodecarboxylase (EC 4.1.1.25) from Streptococcus faecalis) and measurement of tyramine by high-performance liquid chromatography with electrochemical detection. This technique confers specificity, rapidity, and low-cost measurement. Human plasma PLP from 30 normal healthy adults had a mean value of 65.10 +/- 4.70 nM. Sensitivity of the reaction was 1.3 nM and the coefficient of variation of the method (30 repeated assays of sample with a value of 60 nM) was 1.9%.

Adult↗
PubMed1981

Histidine decarboxylase. Purification from fetal rat liver, immunologic properties, and histochemical localization in brain and stomach.

Histidine decarboxylase from fetal rat liver was purified to near-homogeneity. The purified enzyme has a molecular weight of 210,000, and appears to contain two subunits with molecular weights of 145,000 and 66,000, respectively. The enzyme is inhibited by heavy metals such as Hg2+ and Zn2+ and sulfhydryl-reactive compounds such as 5,5'-dithiobis-2-nitrobenzoic acid. The enzyme is partially dependent on exogenous pyridoxal phosphate. Extensive dialysis results in 50% loss of enzyme activity which can be fully recovered by adding pyridoxal phosphate. Affinity of pyridoxal phosphate for the apoenzyme is 0.1 microM at pH 6.8. Antibody against purified histidine decarboxylase was raised in rabbits. The antibody has been employed in immunohistochemical studies to visualize histidine decarboxylase containing cells and neuronal processes in rat stomach and brain, respectively. Immunologic studies indicate that histidine decarboxylase from brain, gastric mucosa, and fetal rat liver share common antigenic properties.

Animals↗
PubMed1989

Effect of ethanol and vitamin B6 deficiency on pyridoxal 5-phosphate levels and fetal growth in rat.

The effects of chronic ethanol consumption and dietary vitamin B6 levels on tissue pyridoxal 5-phosphate (PLP) contents and rat fetal development were investigated. Pregnant Sprague-Dawley rats were given 35% ethanol-calorie liquid diet with either adequate B6 (1.7 mg/liter) or deficient B6 (0.17 mg/liter), ad libitum, from gestation days 7 to 21. Control groups (adequate control and deficient control) were pair-fed with isocaloric sucrose substituted for ethanol. Rats were killed on gestation day 21. Ethanol groups had smaller fetuses than control groups, regardless of their dietary B6 levels. However, in B6 deficiency, ethanol affected fetal weight more severely than in B6 adequate state. Tissue PLP levels were determined by radioenzymatic method. In B6 deficiency, ethanol feeding reduced maternal liver PLP by 22%. PLP in other tissues were not affected by ethanol. These results confirmed that chronic alcohol consumption affected fetal growth and also provided evidence that B6 deficiency exacerbated ethanol effect.

Animals↗
PubMed2000

Crystal structure of the pyridoxal 5'-phosphate dependent L-methionine gamma-lyase from Pseudomonas putida.

L-Methionine gamma-lyase (MGL) catalyzes the pyridoxal 5'-phosphate (PLP) dependent alpha,gamma-elimination of L-methionine. We have determined two crystal structures of MGL from Pseudomonas putida using MAD (multiwavelength anomalous diffraction) and molecular replacement methods. The structures have been refined to an R-factor of 21.1% at 2.0 and 1.7 A resolution using synchrotron radiation diffraction data. A homotetramer with 222 symmetry is built up by non-crystallographic symmetry. Two monomers associate to build the active dimer. The spatial fold of subunits, with three functionally distinct domains and their quarternary arrangement, is similar to those of L-cystathionine beta-lyase and L-cystathionine gamma-synthase from Escherichia coli.

Amino Acid Sequence↗
PubMed1986

Intestinal hydrolysis of pyridoxal 5'-phosphate in vitro and in vivo in the rat: effect of ethanol.

The effect of ethanol on intestinal hydrolysis of pyridoxal 5'-phosphate (PLP), the first step in PLP absorption, was evaluated in the rat. Models included PLP decay during in vitro incubation and PLP luminal disappearance during in vivo perfusion of jejunal segments. Ethanol inhibited PLP in vitro decay (pH = 7.4 and 3.0) and in vivo disappearance (pH = 7.4) in a concentration-dependent manner (1-4% w/v). At pH = 7.4, 4% w/v ethanol inhibited in vitro PLP decay by 48% (p less than 0.001) and in vivo PLP disappearance by 55% (p less than 0.001). This inhibition was reproduced in vitro with other alcohols but not with osmotic controls, paralleled in vitro changes in phosphatase activity in the presence of ethanol, and was completely reversed in vivo after ethanol was removed from the perfusing solution. The inhibition of intestinal PLP hydrolysis may possibly be one of the mechanisms by which a high percentage of alcoholics become biochemically vitamin B6 deficient.

Alkaline Phosphatase↗
PubMed1999

A prospective study on folate, B12, and pyridoxal 5'-phosphate (B6) and breast cancer.

To investigate the incidence of breast cancer and prediagnostic serum levels of folate, B12, and pyridoxal 5'-phosphate (B6), we conducted a nested case-control study using resources from the Washington County (Maryland) serum bank. In 1974, 12,450 serum specimens were donated, and in 1989, 14,625 plasma specimens were donated by female residents of Washington County. One hundred ninety-five incident breast cancer cases and 195 controls were matched by age, race, menopausal status at donation, and cohort participation as well as by date of blood donation. In both cohorts and all menopausal subgroups, median B12 concentrations were lower among cases than controls. Differences reached statistical significance only among women who were postmenopausal at donation (1974 cohort, 413 versus 482 pg/ml, P = 0.03; 1989 cohort, 406 versus 452 pg/ml, P = 0.02). Among women postmenopausal at blood donation, observed associations of B12 suggested a threshold effect with increased risk of breast cancer in the lowest one-fifth compared to the higher four-fifths of the control distribution [lowest versus highest fifth: 1974 cohort, matched odds ratio = 4.00 (95% confidence interval = 1.05-15.20); 1989 cohort, matched odds ratio = 2.25 (95% confidence interval = 0.86-5.91)]. We found no evidence for an association between folate, B6, and homocysteine and breast cancer. Findings suggested a threshold effect for serum B12 with an increased risk of breast cancer among postmenopausal women in the lowest one-fifth compared to the higher four-fifths of the control distribution. These results should stimulate further investigations of potentially modifiable risk factors, such as these B-vitamins, for prevention of breast cancer.

Adolescent↗
PubMed1978

Vitamin B6 nutriture during pregnancy.

The vitamin B6 nutriture was determined during pregnancy beginning at month II until term by means of determination of pyridoxal phosphate in serum of 16 healthy pregnant women. During pregnancy, a biochemical vitamin B6 deficiency occurs gradually. The most significant decrease of pyridoxal phosphate parallels the time of most intensive growth and energy retention of the fetus. Vitamin B6 nutriture of mothers at the onset of pregnancy as well as the decrease of pyridoxal phosphate during pregnancy are in a positive correlation with birth weight of newborns. The most important mechanism leading to maternal vitamin B6 deficiency seems to be the active diaplacental transport of pyridoxal phosphate from mother to fetus. A vitamin B6 supplementation during pregnancy is necessary.

Birth Weight↗
PubMed1993

Comparative modulating effects of captopril, diltiazem, dietary calcium and pyridoxal-5'-phosphate on gentamicin-induced nephrotoxicity in the rat.

1. Nephrotoxicity was induced in rats by injecting gentamicin intramuscularly (i.m.) at a dose of 80 mg/kg/day for 6 days. Treated animals demonstrated a typical pattern of aminoglycoside nephrotoxicity characterized histopathologically by necrosis of proximal tubular epithelium, and biochemically by increased serum creatinine and urea concentrations. Reduced glutathione (GSH) concentration in renal cortex was significantly decreased by gentamicin. 2. Simultaneous treatment of rats with gentamicin and either captopril or diltiazem significantly potentiated the gentamicin-induced increases in serum creatinine and urea and did not significantly affect the gentamicin-induced decrease in cortical GSH concentration. 3. Concomitant treatment with gentamicin and either Ca2+ or pyridoxal-5'-phosphate decreased serum urea level, did not significantly affect serum creatinine concentration, and significantly increased cortical GSH concentration in comparison to the values of these parameters following gentamicin treatment. 4. Histopathologically, the severity of gentamicin-induced renal damage was exacerbated by captopril, and even more so by diltiazem. Simultaneous treatment with gentamicin and either Ca2+ or pyridoxal-5'-phosphate produced only mild focal atrophy of renal tubular epithelium. Control rats had apparently normal histology. 5. In conclusion, captopril and diltiazem, at the doses used, significantly potentiated gentamicin-induced nephrotoxicity to a broadly similar extent. Although Ca2+ and pyridoxal-5'-phosphate, at the doses used, reduced significantly the severity of some of the manifestations of nephrotoxicity, they were equally ineffective in completely preventing the development of nephrotoxicity.

Animals↗