The effects of pyridoxal phosphate on rabbit liver and kidney fructose 1,6-diphosphatases.
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Adenine Nucleotides↗
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Pyridoxal phosphate catalyzed alpha-beta elimination of S-gamma-aminopropylcysteine.
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Chemical Phenomena↗
Structure-function relationships in Escherichia coli translational elongation factor G: modification of lysine residues by the site-specific reagent pyridoxal phosphate.
Translational elongation factor G (EF-G) of Escherichia coli was modified with the selective, site-specific lysine reagent pyridoxal phosphate (PLP). The reaction results in the modification of a maximum of 12 lysine residues, one of which is essential for guanosine 5'-triphosphate (GTP) binding and whose modification is inhibited by the presence of GTP. Formation of a reversible adduct between 2,3-butanedione and an essential arginine similarly located in the GTP binding site [Rohrbach, M.S., & Bodley, J. W. (1977) Biochemistry 16, 1360-1363] also protects EF-G from PLP inactivation, suggesting that these two residues are spatially close to each other in the native factor. The essential lysine residue was found in the trypsin-resistant fragment T4 (Mr 41 000). In addition to the lysine essential for GTP binding, at least one further lysine was found to be important for EF-G function, since GTP-protected, PLP-modified EF-G molecules fully competent in binding to 50S ribosomal subunits showed decreased activity in 50S- and 70S-dependent GTP hydrolysis. It is likely that a PLP-modified lysine impairs the interaction of the factor with 30S ribosomal subunits and/or a conformational change of the factor required for the hydrolysis of GTP.
Escherichia coli↗
Inactivation of pyridoxal phosphate dependent enzymes by mono- and polyhaloalanines.
beta,beta-Dichloro- and beta,beta,beta-trifluoroalanine irreversibly inactivate a number of pyridoxal phosphate dependent enzymes which catalyze beta- or gamma-elimination reactions. The inactivation is time dependent and the rate of inactivation is first order in enzyme concentration. This suggests that inactivation is due to covalent modification of the enzyme by a species generated at the active site from the polyhaloalanine (i.e., suicide inactivation). Monohaloalanines are substrates and do not inactivate. For gamma-cystathionase, covalent and stoichiometric attachment of [1-14C]beta,beta,beta-trifluoroalanine was shown. It is proposed that the mechanism of inactivation involves Schiff base formation between inactivator and enzyme-bound pyridoxal and subsequent elimination of HC1 from dichloroalanine or HF from trifluoroalanine. This results in the formation of a beta-halo-alpha,beta unsaturated imine, an activated Michael acceptor. Michael addition of a nucleophile at the active site leads to covalent labeling of the enzyme and inactivation. Alanine racemase is also inactivated by the two polyhaloalanines. Glutamate-pyruvate and gultamate-oxaloacetate transaminase are inactivated by monohaloalanines but not by polyhaloalanines.
Alanine↗
A micromethod for determination of plasma pyridoxal phosphate and its use in assessment of storage stability of the vitamer.
A widely used macromethod employing tyrosine apodecarboxylase for measurement of pyridoxal phosphate (PLP) concentration in 0.5-1.0-ml plasma was modified to a microscale utilizing 0.1-ml plasma. Mean PLP levels in 12 plasma samples were 160.6 +/- 32.8 pmol/ml (mean +/- SD) when analyzed by the macromethod, and were not significantly different compared to those obtained by the micromethod (158.4 +/- 28.2 pmol/ml). Results of the two methods were significantly correlated (r = +0.97, p less than 0.001). Plasma PLP concentrations of 11 samples determined by the micromethod (means = 151.8 +/- 30.0 pmol/ml) were similar and significantly correlated (r = +0.95, p less than 0.001) to levels measured in the same samples 1-2 years earlier (means = 145.1 +/- 26.2 pmol/ml). This suggests that plasma PLP content of the samples was stable for up to 2 years of storage when the micromethod was utilized for analysis. The strong significant correlation between macro- and micromethods attests that the micromethod is a reliable alternative to the macromethod. The micromethod is useful in instances where only small samples of plasma are available for measurement of PLP.
Blood Chemical Analysis↗
Reference values for alanine and aspartate aminotransferase (ALT and AST) optimized by addition of pyridoxal phosphate.
The increases of the activity of aspartate and alanine transaminases observed in sera of healthy male and female subjects by addition of pyridoxal phosphate have been determined. The reference values of the activated reactions, distinguished by sex and age classes, are presented.
Adult↗
Pyridoxal phosphate is better than pyridoxine for controlling idiopathic intractable epilepsy.
AIM: To study the difference between pyridoxine (PN) and its active form, pyridoxal phosphate, (PLP) in control of idiopathic intractable epilepsy in children. METHODS: Among 574 children with active epilepsy, 94 (aged 8 months to 15 years) were diagnosed with idiopathic intractable epilepsy for more than six months. All received intravenous PLP 10 mg/kg, then 10 mg/kg/day in four divided doses. If seizures recurred within 24 hours, another dose of 40 mg/kg was given, followed by 50 mg/kg/day in four divided doses. For those patients whose seizures were totally controlled, PLP was replaced by the same dose of oral PN. If the seizure recurred, intravenous PLP was infused followed by oral PLP 50 mg/kg/day. RESULTS: Fifty seven patients had generalised seizures (of whom 13 had infantile spasms) and 37 had focal seizure. Eleven had dramatic and sustained responses to PLP; of these, five also responded to PN. Within six months of treatment with PLP or PN, five of the 11 patients were seizure free and had their previous antiepileptic medicine tapered off gradually. Two were controlled with pyridoxine and the other three needed PLP to maintain seizure freedom. The remaining six responders needed PLP exclusively for seizure control. Six of the 11 responders to PLP had infantile spasms (46%); four of them needed PLP exclusively. The other five responders were in the remaining 81 patients with other seizure type. CONCLUSIONS: PLP could replace PN in the treatment of intractable childhood epilepsy, particularly in the treatment of infantile spasms.
Administration, Oral↗
[Reactivation and reconstitution of glutamate decarboxylase upon the interaction of its dimers with pyridoxal phosphate].
The relationship between the reactivation and reconstitution of the hexameric form of glutamate decarboxylase during the interaction of inactive apoenzyme dimers with pyridoxal phosphate (PLP) has been studied. It was shown that the restoration of enzymatic activity, appearance of spectral maximum at 340 nm, and reconstitution of the hexamer depend on the amount of PLP added; this reaction is completed when the PLP concentration reaches that of the initial enzyme. This native hexamer of the holo- and apoenzyme does not practically contain exposed sulfhydryl groups. Ten cysteine residues become available after DS-Na denaturation. The dimer of the apoenzyme contains 8 exposed and 2 buried cysteine residues. The hexamer formation from the dimers is accompanied by the burying of the cysteine residues. When half of the required PLP was added, 7 cysteine residues became buried in experiments with DTNB and six in experiments with 4.4'-DTDP. Further addition of PLP led to the disappearance of the exposed sulfhydryl groups.
Binding Sites↗
Effect of maternal vitamin B-6 restriction on pyridoxal phosphate concentrations in developing regions of the central nervous system in rats.
The effect of maternal vitamin B-6 deficiency on concentrations of pyridoxal phosphate (PLP) within four different regions of the central nervous system (CNS) of progeny, the corpus striatum (CS), hypothalamus (H), cerebellum (C), total brain and first cervical segment (C-1) of the spinal cord was determined at 7, 15, 21 and 50 days of age. PLP concentrations in each CNS region, as well as in total brain at 15, 21 and 50 days postnatally, paralleled the level of vitamin B-6 in the deficient (0.6, 0.8, 1.0 mg pyridoxine . HCl per kilogram diet) and control diets (7.0 mg pyridoxine . HCl per kilogram diet). By 21 days of age, PLP concentrations had essentially plateaued in CNS regions of controls, whereas in the vitamin B-6-restricted groups, a significant catch-up in concentration was observed between 21 and 50 days of age in all CNS regions except C-1 of the spinal cord. Throughout postnatal development, PLP concentrations in the C-1 region of spinal cord and in hypothalamus appeared least affected by vitamin B-6 restriction and levels in CS and C were most affected. These findings suggested that the mechanism for intracellular trapping of B-6 vitamers may develop in a caudal to rostral direction within the CNS.
Animal Nutritional Physiological Phenomena↗
Allosteric threonine synthase. Reorganization of the pyridoxal phosphate site upon asymmetric activation through S-adenosylmethionine binding to a novel site.
Threonine synthase (TS) is a fold-type II pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the ultimate step of threonine synthesis in plants and microorganisms. Unlike the enzyme from microorganisms, plant TS is activated by S-adenosylmethionine (AdoMet). The mechanism of activation has remained unknown up to now. We report here the crystallographic structures of Arabidopsis thaliana TS in complex with PLP (aTS) and with PLP and AdoMet (aTS-AdoMet), which show with atomic detail how AdoMet activates TS. The aTS structure reveals a PLP orientation never previously observed for a type II PLP-dependent enzyme and explains the low activity of plant TS in the absence of its allosteric activator. The aTS-AdoMet structure shows that activation of the enzyme upon AdoMet binding triggers a large reorganization of active site loops in one monomer of the structural dimer and allows the displacement of PLP to its active conformation. Comparison with other TS structures shows that activation of the second monomer may be triggered by substrate binding. This structure also discloses a novel fold for two AdoMet binding sites located at the dimer interface, each site containing two AdoMet effectors bound in tandem. Moreover, aTS-AdoMet is the first structure of an enzyme that uses AdoMet as an allosteric effector.
Allosteric Site↗
[In vitro supplementation of pyridoxal phosphate for the optimisation of the determination of the catalytic activity of alanine aminotransferase and aspartate aminotransferase in kidney transplant patients (author's transl)].
Pyridoxal phosphate (PLP) is the coenzyme of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Thus, in vitro supplementation with PLP is important for the optimisation of the determination of catalytic activity of both enzymes. It patients with kidney transplants, stimulation by PLP is very important for ALT activity, which could be affected by plasma PLP deficiency. Furthermore, in this population catalytic activities are more frequently found increased using methods with PLP supplementation than without PLP supplementation (56% and 71% of patients for AST and ALT, respectively). These differences are not related to HBs antigen.
Adolescent↗
Mechanistic studies on the pyridoxal phosphate enzyme 1-aminocyclopropane-1-carboxylate deaminase from Pseudomonas sp.
The enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACPC deaminase) from a pseudomonad is a pyridoxal phosphate (PLP) linked catalyst which fragments the cyclopropane substrate to alpha-ketobutyrate and ammonia [Honma, M., & Shimomura, T. (1978) Agric. Biol. Chem. 42, 1825]. Enzymatic incubations in D2O yield alpha-ketobutyrate with one deuterium at the C-4 methyl group and one deuterium at one of the C-3 prochiral methylene hydrogens. Stereochemical analysis of the location of the C-3 deuteron was accomplished by in situ enzymatic reduction to (2S)-2-hydroxybutyrate with L-lactate dehydrogenase and conversion to the phenacyl ester. The C-3 hydrogens of the (2S)-2-hydroxybutyryl moiety are fully resolved in a 250-MHz NMR spectrum. Absolute assignment of 3S and 3R loci was obtained with phenacyl (2S,3S)-2-hydroxy[3-2H]butyrate generated enzymatically by D-serine dehydratase action on D-threonine. ACPC deaminase shows a stereoselective outcome with a 3R:3S deuterated product ratio of 72:28. 2-Vinyl-ACPC is also a fragmentation substrate with exclusive regiospecific cleavage to yield the straight-chain keto acid product 2-keto-5-hexenoate. The D isomer of vinylglycine is processed to alpha-ketobutyrate and ammonia at 8% the Vmax of ACPC, while L-vinylglycine is not a substrate. It is likely that ACPC and D-vinylglycine yield a common intermediate--the vinylglycine-PLP-p-quinoid adduct--which is then protonated sequentially at C-4 and then C-3 to account for the observed deuterium incorporation. The D isomers of beta-substituted alanines (fluoroalanine, chloroalanine, and O-acetyl-D-serine) partition between catalytic elimination and enzyme inactivation. Each shows a different partition ratio, arguing against the common aminoacrylyl-PLP as the inactivating species.
Butyrates↗
[Use of pyridoxal phosphate and L-dopa in the treatment of cardiac insufficiency in patients with mitral defects and the active phase of rheumatism].
The effects of pyridoxal phosphate (PALP) and L-DOPA on the central hemodynamics and myocardial contractility were studied in 61 patients with associated heart insufficiency and active rheumatic fever. Introduction of PALP into a complex of treatment measures in a dose of 0.06 g a day for 21--24 days provides a more demonstrable effect than that of L-DOPA in a dose of 1.5 g a day for the same time. It is advisable to include PALP into a complex treatment of heart insufficiency in patients with associated mitral valve and active rheumatic fever.
Adult↗
Restoration of the poor oxygen transport function of ACD-stored blood by pyridoxal 5'-phosphate.
Pyridoxal 5'-phosphate is easily incorporated into ACD-stored erythrocytes without decrease of ATP, and restores the poor oxygen transport function with a similar effect to 2,3'-diphosphoglycerate.
Adenosine Triphosphate↗
Conditions for the formation of cyclic compounds between pyridoxal phosphate and 5-hydroxytryptamine or 5-hydroxytryptophan.
5-Hydroxytryptamine (5HT) and 5-hydroxytryptophan (5HTP) form cyclic compounds (probably of the tetrahydro-beta-carboline type) with pyridoxal phosphate (PLP). In the first step of reaction a Schiff's base is formed; during incubation it is transformed into a cyclic compound with a maximum absorption spectrum at 330 nm. The degree of cyclization depends on pH and substrate concentrations. One mole of 5HT or 5HTP reacts with one mole of coenzyme. The velocity of cyclization increased with an excess of either 5HT (5HTP) or PLP, without any change in the mole to mole ratio. The formation of cyclic compounds was confirmed by the use of isotopes, separation from substrates being achieved by high-voltage electrophoresis.
5-Hydroxytryptophan↗
Effects of the P2-purinoceptor antagonists suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid on glutamatergic synaptic transmission in rat dorsal horn neurons of the spinal cord.
The effects of suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) on glutamatergic synaptic transmission were studied on dorsal horn lamina II neurons of rat spinal cord slice preparation and cultured dorsal horn neurons. Suramin at 100 microM significantly suppressed the amplitude of the evoked excitatory postsynaptic currents (EPSCs) by 33%, miniature EPSC (mEPSC) amplitude was decreased by 46% and the mEPSC frequency also decreased by 41%. PPADS at 50 microM had little effect on either the evoked EPSCs or mEPSCs. The lack of effect of PPADS on glutamatergic synaptic transmission suggests that the effect of suramin is less likely to be mediated by P2x receptors. When whole-cell (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) currents evoked by glutamate were examined, both suramin and PPADS showed no inhibition of peak amplitude. However, the onset of glutamate-evoked whole-cell currents became significantly slowed by suramin but not by PPADS. The suppression of synaptic transmission by suramin may be due, in part, to the slowed onset of glutamate-evoked AMPA currents. These results suggest that the analgesic effects of suramin shown in cancer patients and animal pain models may not be solely due to its antagonism to purinoceptors. PPADS is probably a more suitable antagonist for the study of synaptic P2x receptor function at excitatory synapses mediated by AMPA receptors.
Animals↗
Plasma pyridoxal phosphate in anxiety disorders.
One hundred and eighty-nine subjects with either generalized anxiety disorder, panic disorder, or obsessive-compulsive disorder were evaluated for plasma pyridoxal phosphate (PLP) levels and compared with normal controls. There was no difference in plasma PLP levels between the anxiety disorder groups and normal controls. Low levels of plasma PLP were found in 42% of the controls. Our results suggest that previous reports of low PLP levels in psychiatric patients are unlikely to be significant in the etiology of the psychiatric disorders.
Adult↗
Identification of the influenza virus transcriptase by affinity-labeling with pyridoxal 5'-phosphate.
Pyridoxal 5'-phosphate (PLP), a reversible inhibitor of in vitro transcription by fowl plaque virus, has been used to identify the transcriptase. Kinetic analyses showed that PLP competitively inhibits the addition of each nucleoside triphosphate in ApG-primed reactions, suggesting that both initiation and elongation are affected. The irreversible inhibition by PLP following reduction with borohydride was prevented by preincubation with the first substrate: GTP in unprimed reactions or CTP in the presence of ApG. On reaction of FPV proteins with PLP and [3H]borohydride the core protein PB1 was preferentially labeled and the labeling was selectively blocked by GTP or ApG + CTP. These data suggest that PB1 has the nucleotide-binding site of the transcriptase, is responsible for both initiation and elongation, and is apparently associated with the 3' ends of template RNAs in virions.
Affinity Labels↗