Pyridoxine kinase, pyridoxine phosphate phosphatase and pyridoxine phosphate oxidase activities in control and B-6-deficient rat liver and brain. (33/43)

The levels of pyridoxal phosphate in plasma, liver and brain and the activities of pyridoxine kinase, pyridoxine phosphate phosphatase and pyridoxine phosphate oxidase in liver and brain were measured over a 6-week period in rats fed pyridoxine-sufficient and pyridoxine-deficient diets. Consistently significant differences in enzyme activities between the two groups of animals were found only in pyridoxine kinase indicating that this enzyme plays a key role during the development of vitamin B-6 deficiency. Relative to control animals, the decrease observed in liver pyridoxine kinase acivity in animals fed pyridoxine-deficient diets is much greater than the decrease in brain pyridoxine kinase activity (50% decrease versus a 14% decrease after 5 weeks). In light of the suggestion that phosphorylation and binding to proteins serve to prevent the diffusion of B-6 vitamers out of cells, the differential response of pyridoxine kinase activity in liver and brain may be important in the maintenance of the vitamin B-6 supply in the central nervous system. During the course of this study, a new method for the determination of cellular phosphatase activity on a phosphorylated form of vitamin B-6 was developed. 3H-C4'-Pyridoxine phosphate was used as substrate and was separated from 3H-C4'-pyridoxine by means of anion-exchange filter paper disks.  (+info)

Metabolism of vitamin B6 in rat liver mitochondria. (34/43)

The capacity of rat liver mitochondria to transport and metabolize B6 compounds has been studied. In B6-sufficient rats, 20% of the B6 content in liver is located in mitochondria. The ratio of pyridoxal-P to pyridoxamine-P in isolated mitochondria is increased with 2-oxoglutarate as substrate and decreased with glutamate. Isolated mitochondria contain pyridoxal-P(pyridoxamine-P) hydrolase activity in the intermembranous space. They exhibit no pyridoxal kinase activity and less than 5% of the pyridoxamine-P(pyridoxine-P) oxidase activity in cytosol and cannot synthesize pyridoxal-P or pyridoxamine-P from pyridoxine, pyridoxal, or pyridoxine-P. When isolated hepatocytes are incubated with [14C]pyridoxine, [14C]pyridoxal-P synthesized in the cytosol is taken up by mitochondria at a rate approximating linearity. Additionally, as reflected by the lower specific radioactivity of [14C]pyridoxal-P in mitochondria than that in cytosol, the extent of mixing of newly synthesized and endogenous pools of pyridoxal-P in these subcellular compartments is heterogeneous. When cytosol from hepatocytes incubated with [14C]pyridoxine is dialyzed to equilibrium, a dialyzable pool of [14C]pyridoxal-P with specific radioactivity similar to that of the added [14C]pyridoxine is identified. It appears that pyridoxal-P from this newly synthesized pool is preferentially transported into mitochondria or secreted and degraded by hepatocytes.  (+info)

Vitamin B-6 metabolism and its relation to ornithine decarboxylase activity in regenerating rat liver. (35/43)

The metabolism of vitamin B-6 regenerating rat liver and liver from sham-operated control animals fed either a pyridoxine-sufficient or pyridoxine-deficient was investigated. The pyridoxal phosphate levels in plasma, regenerating liver and control liver were determined as were the activities of three enzymes involved in the metabolism of the vitamin, namely, pyridoxine kinase, pyridoxine phosphate oxidase, and pyridoxine phosphate phosphatase. In addition, total and holo-ornithine decarboxylase activities in the livers were measured. The results indicate that the metabolism of vitamin B-6 regenerating rat liver is different from that observed in Morris hepatomas (Thanassi et al. (1981) J. Biol. Chem. 256, 3370-3375). Vitamin B-6 metabolism in Morris hepatomas is concluded to be characteristic of the tumors rather than a property common to rapidly proliferating hepatic tissue. Regenerating liver ornithine decarboxylase holoenzyme activity in pyridoxine deprived rats was maintained at the same level as that in regenerating liver of pyridoxine-sufficient animals. The mechanism behind this maintenance of holo-enzyme activity appears to involve a pronounced increase in the amount of apoornithine decarboxylase. The time-dependent peak of ornithine decarboxylase activity following partial hepatectomy was shifted from four hours to twelve hours by vitamin B-6 deficiency.  (+info)

Brain pyridoxine-5-phosphate oxidase. A dimeric enzyme containing one FMN site. (36/43)

Pyridoxine-5-phosphate oxidase has been purified 2250-fold from pig brain by affinity chromatography. The enzyme of 60000 molecular weight is made up of two identical size subunits and binds 1 mol of FMN/mol dimer. 1 mol of the fluorescent inhibitor phospho-pyridoxal oxime interacts with 1 mol of the dimeric protein to yield a dissociation constant KD = 0.2 microM. Resolution of the holoenzyme into apoprotein and free FMN does not induce dissociation of the dimeric structure. The oxidase catalyzes the oxidation of the natural substrates pyridoxine 5-phosphate and pyridoxamine 5-phosphate, but phospho-pyridoxyl derivatives of the aromatic carboxylic acids, p-aminobenzoate and m-aminobenzoate, are also excellent substrates of the enzyme. Introduction of electron-withdrawing substituents into the structure of benzene increases the kcat values. A comparison of the kcat values obtained with several synthetic substrates suggests that electron-withdrawing substituents tend to stabilize carbanionic intermediates formed in the earlier stages of catalysis.  (+info)

Kinetic properties of pyridoxamine (pyridoxine)-5'-phosphate oxidase from rabbit liver. (37/43)

The kinetic properties of pyridoxamine (pyridoxine)-5'-phosphate oxidase have been studied using the physiological substrates pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) at 25 degrees C and pH 8.0. Under steady-state conditions with different concentrations of PNP and O2, a series of parallel lines and competitive substrate inhibition with a KI of 50 microM are seen in double reciprocal plots. This is suggestive of a binary complex mechanism. Secondary plots yield a turnover number of 42 min-1 and Km values for both PNP (8.2 microM) and O2 (182 microM). A large deuterium isotope effect, VH/VD of 6.5, was observed with [4',4'-2H]PNP. In analogous studies using PMP, a turnover number of 6.2 min-1 and respective Km values for PMP and O2 of 3.6 and 85 microM were calculated. No significant substrate inhibition and a small deuterium isotope effect (VH/VD = 1.1) were observed with PMP. Anaerobic stopped flow data showed that the enzyme-bound flavin was reduced at a rate similar to catalytic turnover with PNP as a substrate, whereas with PMP, the rate of enzyme reduction is 500-fold faster than turnover. Stopped flow kinetic data also showed the reduced enzyme to react with O2 at rates at least 10(2)-10(3) faster than turnover. These results indicate that enzyme reduction is rate-limiting when the alcohol form (PNP) is the substrate, but in the case of the amine (PMP), the rate-limiting step in catalysis occurs subsequent to reduction. With PMP as substrate, release of product from the complex with reduced enzyme is 15-fold slower than turnover, and thus, it is suggested that oxygen reacts with the complex. The pH dependence of the deuterium isotope effect and the Km for PMP showed substantial change in the pH range between 6.0 and 7.5, whereas little or no pH dependence was observed for PNP. These data show that the kinetic mechanism of pyridoxamine (pyridoxine)-5'-phosphate oxidase can function via either a binary or ternary complex mechanism, depending upon the nature of the substrate.  (+info)

Effect of strain, sex and dietary riboflavin on pyridoxamine (pyridoxine) 5'-phosphate oxidase activity in rat tissues. (38/43)

Weanling male rats of four strains--Buffalo, Long-Evans, Sprague-Dawley (SD) and Wistar--were fed a control or a riboflavin-deficient purified diet for 6 weeks. Both strain and diet had significant effects on pyridoxamine (pyridoxine) 5'-phosphate (PMP) oxidase activity in liver and kidney. No single strain had extreme PMP oxidase values or was consistently more responsive than the others to riboflavin deficiency. Weanling male and female SD rats were fed purified diets containing 2.5, 1.0, 0.5 or 0 mg of riboflavin/kg diet for 9 weeks. PMP oxidase values generally were lower in the females than in the males. In both sexes, increases in dietary riboflavin level were reflectd in increases in PMP oxidase activity in liver and kidney. These results confirm that PMP oxidase activity is a sensitive indicator of riboflavin status in the rat.  (+info)

Genetic map position of the pdxH gene in Escherichia coli. (39/43)

The gene for pyridoxine phosphate oxidase, pdxH, is located 1.2 min beyond aroD, proximal to trp.  (+info)

Kinetic limitation and cellular amount of pyridoxine (pyridoxamine) 5'-phosphate oxidase of Escherichia coli K-12. (40/43)

We report the purification and enzymological characterization of Escherichia coli K-12 pyridoxine (pyridoxamine) 5'-phosphate (PNP/PMP) oxidase, which is a key committed enzyme in the biosynthesis of the essential coenzyme pyridoxal 5'-phosphate (PLP). The enzyme encoded by pdxH was overexpressed and purified to electrophoretic homogeneity by four steps of column chromatography. The purified PdxH enzyme is a thermally stable 51-kDa homodimer containing one molecule of flavin mononucleotide (FMN). In the presence of molecular oxygen, the PdxH enzyme uses PNP or PMP as a substrate (Km = 2 and 105 microM and kcat = 0.76 and 1.72 s-1 for PNP and PMP, respectively) and produces hydrogen peroxide. Thus, under aerobic conditions, the PdxH enzyme acts as a classical monofunctional flavoprotein oxidase with an extremely low kcat turnover number. Comparison of kcat/Km values suggests that PNP rather than PMP is the in vivo substrate of E. coli PdxH oxidase. In contrast, the eukaryotic enzyme has similar kcat/Km values for PNP and PMP and seems to act as a scavenger. E. coli PNP/PMP oxidase activities were competitively inhibited by the pathway end product, PLP, and by the analog, 4-deoxy-PNP, with Ki values of 8 and 105 microM, respectively. Immunoinhibition studies suggested that the catalytic domain of the enzyme may be composed of discontinuous residues on the polypeptide sequence. Two independent quantitation methods showed that PNP/PMP oxidase was present in about 700 to 1,200 dimer enzyme molecules per cell in E. coli growing exponentially in minimal medium plus glucose at 37 degrees C. Thus, E. coli PNP/PMP oxidase is an example of a relatively abundant, but catalytically sluggish, enzyme committed to PLP coenzyme biosynthesis.  (+info)