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PubMed1972

Coenzymatic activity of pyridoxal 5'-sulfate and related analogues of pyridoxal 5'-phosphate.

The effect of changes in the substituent at the 5'-position of pyridoxal 5'-phosphate on the coenzymatic activity of six analogues of this coenzyme was determined for three bacterial enzymes. Pyridoxal 5'-sulfate showed substantial coenzymatic activity for arginine decarboxylase and tryptophanase, but not for D-serine dehydratase; alpha(5)-pyridoxalmethylphosphonate showed substantial activity for D-serine dehydratase, but not for the other two enzymes. These results demonstrate that neither the dianionic phosphate group nor the ester oxygen of pyridoxal 5'-phosphate is a general requirement for coenzymatic activity. Minor variations in orientation and charge of the group at position 5 exert major effects on the capacity for complex formation between analogue and apoenzyme, and on the catalytic efficiency of the resulting complex, but have comparatively little effect on the substrate affinity of the analogue-apoenzyme complexes. These effects show no general pattern from enzyme to enzyme, and do not correlate with the affinity of analogue for apoenzyme under the conditions tested.

Apoproteins↗

PubMed1995

Differentiation by pyridoxal 5-phosphate, PPADS and IsoPPADS between responses mediated by UTP and those evoked by alpha, beta-methylene-ATP on rat sympathetic ganglia.

1. The effect of pyridoxal 5-phosphate, and the 2',4' and 2',5'-disulphonic acid isomers of 6-azophenylpyridoxal 5-phosphate (PPADS and IsoPPADS respectively) on depolarization of the rat superior cervical ganglion evoked by alpha, beta-methylene-adenosine 5'-triphosphate (alpha, beta-Me-ATP) and uridine 5'-triphosphate (UTP) were determined by a grease-gap recording technique. 2. Pyridoxal 5-phosphate (10-100 microM) and PPADS (10-100 microM) enhanced UTP- and depressed alpha, beta-Me-ATP-evoked depolarizations but did not significantly alter depolarizations evoked by potassium or hyperpolarizations evoked by adenosine. IsoPPADS (10 microM) depressed alpha, beta-Me-ATP-evoked depolarizations but did not alter depolarizations evoked by UTP. Depolarizations evoked by muscarine were depressed by IsoPPADS but not by pyridoxal 5-phosphate. 3. It is concluded that pyridoxal 5-phosphate, PPADS and IsoPPADS are antagonists at P2x-purinoceptors but not at the receptors that mediate UTP-evoked depolarization of the rat superior cervical ganglion. These observations substantiate the recent proposal that the rat superior cervical ganglia possess distinct receptors for purine and pyrimidine 5'-nucleotides, i.c. P2x-purinoceptors and pyrimidinoceptors respectively.

Adenosine Triphosphate↗

PubMed1994

Stimulation of mouse brain gamma-hydroxybutyric acid transport by pyridoxal 5'-phosphate.

A synaptosomal preparation from mouse brain transported gamma-hydroxybutyric acid in a manner displaying saturation kinetics. A Kt of 48 +/- 11 microM was calculated. In the presence of pyridoxal 5'-phosphate, uptake was markedly enhanced. Stimulation of uptake was observed with increasing concentrations of pyridoxal 5'-phosphate, up to a maximum of about 3.5 mM. Increasing concentrations above this value led to a steady decrease in the stimulation of uptake, till at 7 mM pyridoxal 5'-phosphate and above there was a noticeable inhibition of uptake. The presence of increasing concentrations of gamma-hydroxybutyric acid gradually reduced the amount of stimulation, thus pyridoxal 5'-phosphate appeared to be producing its effect by reducing the Kt of the transport system rather than increasing its Vmax.

Animals↗

PubMed1984

Modification of hydroxymethylbilane synthase (porphobilinogen deaminase) by pyridoxal 5'-phosphate. Demonstration of an essential lysine residue.

When hydroxymethylbilane synthase (porphobilinogen deaminase) from Euglena gracilis is incubated with pyridoxal 5'-phosphate at pH 7.0 and 0 degree C, it rapidly loses part of its activity. The proportion of activity that remains decreases as the concentration of the modifier increases up to approx. 2mM, above which no further significant inactivation occurs. Dialysis of the partly inactivated enzyme restores its activity, whereas reduction with NaBH4 makes the inactivation permanent. The maximum inactivation achievable from one cycle of the treatment with pyridoxal 5'-phosphate, then with borohydride, is 53 +/- 5%; taking this modified enzyme through second and third cycles causes further loss of activity. The enzyme from Rhodopseudomonas spheroides behaves similarly, but there are quantitative differences. Spectroscopic evidence indicates that the inactivation procedure modifies lysine residues, and labelling studies show that epsilon-N-pyridoxyl-L-lysine is a product when permanently inactivated enzyme is completely hydrolysed. Several lysine residues per molecule of the E. gracilis enzyme are modified by the treatment with pyridoxal 5'-phosphate and borohydride, but only one appears to be essential for enzymic activity, since porphobilinogen protects the enzyme against inactivation and then one fewer lysine residue per molecule of enzyme is affected. It is suggested that, during the biosynthesis of hydroxymethylbilane, the first porphobilinogen unit is covalently bound to the enzyme through the epsilon-amino group of the essential lysine.

Ammonia-Lyases↗

PubMed1967

The inactivation of pea-seedling diamine oxidase by peroxidase and 1,5-diaminopentane.

1. The rate of oxidative deamination of 1,5-diaminopentane by pea-seedling extracts, which contain diamine oxidase [diamine-oxygen oxidoreductase (deaminating), EC 1.4.3.6], was increased by adding pyridoxal or pyridoxal phosphate. 2. Evidence was obtained that pyridoxal does not activate the apoenzyme of diamine oxidase, but prevents the inactivation of the enzyme. 3. This inactivation only occurred when 1,5-diaminopentane was the substrate and depended on a second thermolabile factor in the extract besides the diamine oxidase. 4. Purified diamine oxidase, when catalysing the oxidation of 1,5-diaminopentane, was rapidly inactivated in the presence of peroxidase. 5. The inactivation was prevented not only by pyridoxal and pyridoxal phosphate but also by several unrelated compounds including alpha-oxoglutarate, catechol and o-aminobenzaldehyde. 6. It is suggested that peroxidase catalyses the further oxidation of the product of the oxidative deamination of 1,5-diaminopentane to a compound that inactivates diamine oxidase. 7. The results diminish the relevance of previous evidence that plant diamine oxidase contains pyridoxal phosphate.

Aldehydes↗

PubMed1988

Intestinal absorption of pyridoxal 5'-phosphate at physiological levels in rats.

The intestinal absorption of pyridoxal 5'-phosphate (PLP) at physiological levels (10(-7) -10(-6) M) was studied in comparison with that of pyridoxal (PL) in rat, using in vitro everted sac and an intestinal preparation that permitted continuous in situ collection of mesenteric venous blood. After PLP administration (10(-6) -10(-3) M) in situ, larger amounts of PLP were found in the mesenteric venous plasma than after PL administration at the same dose. The amount of PLP found in the mesenteric venous plasma was dependent on its dose at lower concentrations up to 10(-4) M but became independent at higher concentrations. After PL administration at various doses, the amount of PL found in the mesenteric venous blood increased linearly with the dose. When various concentrations of PLP were added to the mucosal side, under the in vitro condition with protection from alkaline phosphate hydrolysis, PLP was detected in the serosal side and the extent of PLP transport was dependent on the initial concentration of PLP in the mucosal side. When various concentrations of PL were added to the mucosal side, the extent of PL transport was independent of the initial concentration of PL in the mucosal side. In rat pretreated with actinomycin D, PLP transport in vitro was inhibited but not that of PL. N2-induced anoxia and pyridoxamine 5'-phosphate and anion transport inhibitor (4,4'-diisothiocyanostilben-2,2'-disulfonic acid disodium salt) showed no effect on PLP transport. These results suggest that PLP can be absorbed in the phosphorylated form and imply the presence of a saturable process for direct absorption of PLP itself and a diffusive process for PL absorption. In addition, the result of the in vivo neonatal experiment suggests that the neonatal intestine also can transport PLP in phosphorylated form.

Aging↗

PubMed1975

Pyridoxal phosphate-dependent conformational states of glycogen phosphorylase as probed by interconverting enzymes.

The interaction between pyridoxal 5'-phosphate and the convertible serine of glycogen phosphorylase has been investigated by using: specific interconverting enzymes, phosphorylase kinase and phosphorylase phosphatase; effectors, glucose and glucose 6-phosphate; and a protein kinase and trypsin. Both phosphorylase kinase and phosphorylase phosphatase utilized the native protein while having little influence on the apoprotein. Removal of a peptide containing the critical serine residue gave phosphorylase b' from which the pyridoxal 5'-phosphate in phosphorylase has an important effect on enzymic interconversion.

Apoenzymes↗

PubMed1977

L-Lysine-alpha-ketoglutarate epsilon-aminotransferease. Properties of the bound pyridoxal 5'-phosphate.

L-Lysine-alpha-ketoglutarate epsilon-aminotransferase of Flavobacterium lutescens (Achromobacter liquidum) IFO 3084 shows positive circular dichroic bands at 340 and 415 nm where absorption maxima are observed, and fluorescence maxima at 380 and 490 nm on excitation at 340 and 415 nm, respectively. The pyridoxal 5'-phosphate absorbing at 415 nm is bound through an aldimine linkage to an epsilon-amino group of the lysine residue of the protein. Upon aging, the 415 nm pyridoxal 5'-phosphate changes to a less active form (lambda max, 325 nm), which is distinguishable from the 340 nm pyridoxal 5'-phosphate. This 325 nm bound pyridoxal 5'-phosphate is reduced with sodium borohydride and shows no circular dichroism. When the semiapoenzyme is aged under the same conditions, no spectral change is observed. These findings suggest that the pyridoxal 5'-phosphate bound through an aldimine linkage may be converted into a carbinol amine or some other related form by aging.

Apoenzymes↗

PubMed1984

Effect on the equilibrium between the Na+-form and the K+-form of the (Na+ + K+)-ATPase of modification of the enzyme with pyridoxal 5-phosphate.

An increase in pH shifts the equilibrium between the K+-form and the Na+-form of the (Na+ + K+)-ATPase towards the Na+-form. pK for the proton effect on the equilibrium is decreased by modification of the enzyme with pyridoxal 5-phosphate. The reactivity of the enzyme towards pyridoxal 5-phosphate is increased by an increase in pH. Modification by pyridoxal 5-phosphate of epsilon-amino groups on lysine, which has a pK of about 8 with the enzyme in the K+-form and of about 7.4 in the Na+-form, shifts the equilibrium between E1Na+ and E2 towards E2, and the equilibrium between E2(K+occ) and E2 towards E2, but has no effect on the overall equilibrium between E1Na+ and E2(K+occ). An additional modification of epsilon-amino groups on lysine, which has a pK of 9.5-10 with the enzyme in the K+-form and of about 7.7 with the enzyme in the Na+-form, shifts the equilibrium between E2(K+occ) and E1Na+ towards E1Na+; this is due to a shift in the equilibrium between E2(K+occ) and E2 towards E2, but with no effect on the equilibrium between E1Na+ and E2. The results show that the transition from the K+-form to the Na+-form decreases the pK of lysine epsilon-amino groups on the enzyme, and that the protonation of these groups influences the equilibrium between the two conformations.

Animals↗

PubMed1995

Vitamin B6 modulates expression of albumin gene by inactivating tissue-specific DNA-binding protein in rat liver.

The level of albumin mRNA in the liver of vitamin B6-deficient rats was found to be 7-fold higher than that of control rats. Since the transcriptional activity of the albumin gene, as measured by a nuclear run-on assay, was increased 5-fold in vitamin B6 deficiency, the higher concentration of albumin mRNA in the liver of vitamin-deficient rats could be attributed to the enhanced rate of transcription. The promoter proximal sequences of the albumin gene interact with a number of tissue-specific transcription factors including HNF-1 and C/EBP. We determined the binding activities of liver nuclear extracts to the HNF-1- and C/EBP-binding sites by gel mobility-shift assay and found that the activities of the extract prepared from liver of vitamin B6-deficient rats were greater than those of controls. As the concentrations of C/EBP in nuclear extracts from control and vitamin-deficient rats, estimated by Western-blot analysis, were essentially the same, the lower binding activity of the extract from control liver is probably due to inactivation of tissue-specific factors by pyridoxal phosphate and/or its analogues. We therefore examined the effect of pyridoxal phosphate and its analogues on the binding activity of nuclear extract in vitro and found that only pyridoxal phosphate effectively inhibited the binding. These observations indicate that vitamin B6 modulates albumin gene expression through a novel mechanism that involves inactivation of tissue-specific transcription factors by direct interaction with pyridoxal phosphate.

Albumins↗

PubMed1982

Pyridoxal 5-phosphate protection of trypsin cleavage site on the glucocorticoid receptor protein.

Pyridoxal 5-phosphate (PLP) treated rat liver triamcinolone acetonide complex was trypsin-digested followed by hydroxylamine treatment. This complex displayed a considerable fraction of DNA binding capacity of the intact complex, which was lost if trypsin digestion had not been preceded by PLP treatment. At the same time trypsin reduced the size of the complex regardless PLP pretreatment. These findings reveal that DNA binding does not require a large complex.

Adrenalectomy↗

PubMed1975

[The influence of pyridoxal 5'-phosphate on the temperature relationships of aspartate aminotransferase isoenzymes].

The relationship between temperature and the behaviour of aspartate aminotransferase was investigated in the presence of pyridoxal 5'-phosphate. The addition in vitro of pyridoxal 5'-phosphate caused an increase in the activity and altered the thermal behaviour of aspartate aminotransferase. In choosing the temperature for the determination of enzymic activity, the concentration of the coenzyme must therefore also be considered.

Aspartate Aminotransferases↗

PubMed1991

Plasma pyridoxal and pyridoxal 5'-phosphate concentrations in response to ingestion of water or glucose polymer during a 2-h run.

The effects of ingesting a glucose polymer solution (GP) or water (W) on plasma pyridoxal 5'-phosphate (PLP) and pyridoxal (PL) concentrations were compared in six men (age: 30 +/- 2 y; VO2max: 57.4 +/- 3.2 mL.kg-1.min-1) under running (R) and control (C) conditions. Subjects ran for 2 h at 60-65% of VO2max for R and remained standing for C. For both R and C, 200 mL W or GP was ingested before (0-time) and every 30 min while running (30, 60, and 90 min). Plasma PLP decreased to 95% and 87% of 0-time at 180 min for WC and GPC and increased to 126% and 119% at 90 min and to 124% and 119% at 120 min for WR and GPR. By 60 min postrun, plasma PLP was 98% (WR) and 101% (GPR) of 0-time. There were no significant differences between W and GP conditions. Changes in PLP were not related to plasma volume or blood glucose, free fatty acids, lactate, alkaline phosphatase, aspartate aminotransferase, or alanine aminotransferase. No significant changes in plasma PL were noted. Exercise induces an increase in plasma PLP, perhaps due to transfer of B-6 vitamers from liver to skeletal muscle.

Adult↗

PubMed1991

Pyridoxal 5'-phosphate dependent enzymes in the nematode Nippostrongylus brasiliensis.

Eight classes of pyridoxal 5'-phosphate dependent enzymes have been investigated in Nippostrongylus brasiliensis in parallel with rat tissues. The range of decarboxylases detected in N. brasiliensis was limited in comparison with rat tissues. N. brasiliensis possessed a highly active L-serine hydroxymethyltransferase, but in contrast with rat liver, 5-aminolevulinic acid synthetase was absent. Similar levels of L-serine and L-threonine dehydratase activities were detected in N. brasiliensis and rat liver, and both organisms lacked L-alanine racemase, L-tryptophan synthetase and L-methionine gamma-lyase. The demonstration of cystathionine beta-synthase and gamma-cystathionase in N. brasiliensis suggests the presence of a functional trans-sulphuration sequence. The substrate specificities of the nematode cystathionine beta-synthase and gamma-cystathionase varied significantly from those of the corresponding mammalian enzymes. Particularly striking was the ability of N. brasiliensis cystathionine beta-synthase to catalyse the non-mammalian 'activated L-serine sulphydrase' reaction (L-cysteine + R-SH----cysteine thioether + H2S). N. brasiliensis and rat liver exhibited comparable abilities to transaminate amino acids via the 2-oxoglutarate: glutamate system.

Animals↗

PubMed1979

Pyridoxal 5'-phosphate levels in brain after treatments which impair cerebral glucose metabolism.

Pyridoxal 5'-phosphate (PLP) concentrations were measured in brains of rats to determine whether a deficiency of this coenzyme was a common feature in hepatic coma, ethanol intoxication, and in animals treated with L-dopa or with 5-hydroxytryptophan (5-HTP) alone or with inhibitors of MAO or of L-aromatic amino acid decarboxylase. These treatments have been shown previously to be associated with reduced conversion of glucose to amino acids in brain. Cerebral PLP concentrations were reduced after some of these treatments, notably injection of ethanol, or L-dopa alone or with beta-phenylisopropylhydrazine, an inhibitor of MAO, or of 5-HTP together with N-[beta-(chlorophenoxy)ethyl]cyclopropylamine hydrochloride, Lilly 51641, another MAO inhibitor. However, in other circumstances where inhibition of conversion of glucose to amino acids has been shown (treatment with 5-HTP, or with Lilly 51641 or with [N-(D,L-seryl)-N'-2,3,4-trihydroxybenzyl]hydrazine, an inhibitor of L-aromatic amino acid decarboxylase, together with L-dopa or with 5-HTP), PLP levels in brain were unchanged, or were increased (in hepatectomized rats).

5-Hydroxytryptophan↗

PubMed1989

Pig gastric (H+ + K+)-ATPase. Lys-497 conserved in cation transporting ATPases is modified with pyridoxal 5'-phosphate.

Pig gastric (H+ + K+)-ATPase can be covalently modified with pyridoxal 5'-phosphate (PLP) (about 1 mol/mol enzyme), and this modification is not observed in the presence of ATP, suggesting that PLP binds to a specific Lys residue in the ATP binding site or the region in its vicinity (Maeda, M., Tagaya, M., and Futai, M. (1988) J. Biol. Chem. 263, 3652-3656). The peptides labeled with radioactive PLP could be released from the gastric membrane vesicles quantitatively by chymotrypsin treatment, and two peptides were purified by high performance liquid chromatographies. These peptides were not obtained from vesicles incubated with PLP in the presence of ATP. The sequences of the two peptides were NH2-Asn-Ser-Thr-Asn-Lys-Phe-COOH and NH2-Ser-Thr-Asn-Lys-Phe-COOH, exactly corresponding to residues 493-498 and 494-498, respectively, of pig gastric (H+ + K+)-ATPase sequenced recently (Maeda, M., Ishizaki, J., and Futai, M. (1988) Biochem. Biophys. Res. Commun. 157, 203-209). Lys-497 was concluded to be the binding site of PLP, as pyridoxyl-Lys was identified at the corresponding position. This Lys residue is conserved in (Na+ + K+)- and Ca2+-ATPases. The possible amino acid residues in the catalytic site of gastric (H+ + K+)-ATPase are discussed.

Adenosine Triphosphatases↗

PubMed1982

Reexamination of the binding site for pyridoxal 5'-phosphate in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

The high specificity of pyridoxal 5'-phosphate (PLP) for an essential lysyl residue of ribulosebisphosphate carboxylase/oxygenase was confirmed, but half-of-sites reactivity was not observed in contrast to an earlier report [Robison, P. D., Whitman, W. B., Waddill, F., Riggs, A. F., & Tabita, F. R. (1980) Biochemistry 19, 4848-4853]. Subsequent to reduction with [3H]borohydride and tryptic digestion of the enzyme inactivated by PLP, the sole labeled peptide was purified by successive chromatography on DEAE-cellulose, SP-Sephadex, and Sephadex G-25. The peptide, recovered in good yield, appeared essentially homogeneous by amino acid analysis, peptide mapping, and sequencing. Automated Edman degradation established the peptide's sequence as Val-Leu-Gly-Arg-Pro-Glu-Val-Asp-Gly-Gly-Leu-Val-Val-Gly-Thr-Ile-Ile-(PLP)Lys -Pro-Lys instead of Ala-Leu-Gly-Arg-Pro-Glu-Val-Asp-(PLP)Lys-Gly-Thr-Leu-Val-Ile-Lys as reported by Robison et al. (1980) [Robison, P. D., Whitman, W. B., Waddill, F., Riggs, A. F., & Tabita, F. R. (1980) Biochemistry 19, 4848-4853]. The sequence -Ile-Lys-Pro-Lys- in the former is identical with that encompassing Lys-175 in the carboxylase/oxygenase from spinach, which reacts preferentially with PLP and two other affinity labels. This finding of homology greatly strengthens the supposition that Lys-175 in the spinach enzyme and the corresponding lysyl residue in the Rhodospirillum rubrum enzyme are active-site residues and furthermore increases the likelihood of their functionality in catalysis.

Amino Acid Sequence↗

PubMed1983

Serine hydroxymethyltransferase. 31P nuclear magnetic resonance study of the enzyme-bound pyridoxal 5'-phosphate.

The environment of the phosphate group of pyridoxal-P bound at the active site of cytosolic serine hydroxymethyltransferase has been investigated by 31P NMR spectroscopy. In the holoenzyme, the pyridoxal-P chemical shift is pH-dependent with a pKa of 6.45. The chemical shift of the bound pyridoxal-P is shifted upfield about 0.3 ppm from the signal for free pyridoxal-P. Saturation of the active site with the substrates L-serine, glycine, and tetrahydrofolate does not alter the chemical shift or the pKa of the phosphate group. The addition of these substrates does, however, alter the absorption and circular dichroism spectra of the bound coenzyme, reflecting environmental changes of the pyridine ring-Schiff's base system. We conclude from these studies that the phosphate group of the bound coenzyme is exposed to the solvent. The reorientation and conformational changes of the pyridoxal-P ring which take place during the formation of enzyme-substrate complexes do not appear to change the environment of the phosphate moiety of the coenzyme.

Animals↗