Effect of binding to hemoglobin and albumin on pyridoxal transport and metabolism

Scatchard plot analysis indicated that pyridoxal binds to hemoglobin more than twice as tightly as it does to serum albumin. Comparison of the formation constants for hemoglobin and albumin, using standard competitive binding equations, indicated that the distribution ratio for pyridoxal between erythrocytes and plasma should be 6.5:1. This distribution was approximately the same as that observed when pyridoxal was incubated with whole human blood, suggesting that these two proteins are the primary determinants of the pyridoxal distribution in whole blood. With in situ perfused rat liver the uptake of [3H] pyridoxal from the perfusate was reduced by the inclusion of erythrocytes in the perfusate. This was reflected in the decreased production of 4-pyridoxic acid by the perfused liver from 3.8% to 1.2% of the dose by the addition of erythrocytes to the perfusate. The major labeled metabolites found in the liver were pyridoxal phosphate, pyridoxamine phosphate, and 4-pyridoxic acid for both types of perfusion. In intact animals, reduction of the erythrocytes concentrations to hematocrits of 30-40% increased the recovery in the urine of 3H from administered [3H] pyridoxal from control values of 27-35% to 40-50% of the dose within 48 h. Half of the label in urinary metabolites was in 4-pyridoxic acid.     

Metabolism of tritium-labelled pyridoxine and pyridoxine 5'-phosphate in the central nervous system

Abstract— [³H]Pyridoxine and [³H]pyridoxine 5′-phosphate have been injected into rats and mice. The uptake in brain tissue has been studied by comparing the concentrations of labelled compounds in serum, cerebrospinal fluid and brain tissue. Labelled pyridoxine passes rapidly into brain tissue, whereas the uptake of pyridoxine 5′-phosphate occurs at a much slower rate. Perchloric acid extracts of brain have been fractionated by ion-exchange chromatography and the distribution of isotope between the different forms of the vitamin has been determined at different times after the administration. The time sequence of the metabolic transformation is: pyridoxine+→ pyridoxine 5′-phosphate → pyridoxal 5′-phosphate → pyridoxamine 5′-phosphate. After the initial transformation period about 40 per cent of the isotope is recovered in each of the pyridoxal 5′-phosphate and pyridoxamine 5′-phosphate fractions.     

Evidence for the regulation of pyridoxal 5′-phosphate formation in liver by pyridoxamine (pyridoxine) 5′-phosphate oxidase

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) purified from rabbit liver is competitively inhibited by the reaction product, pyridoxal 5′-phosphate. The K_i, 3 μM, is considerably lower than the K_m for either natural substrate (18 and 24 μM for pyridoxamine 5′-phosphate and 25 and 16 μM for pyridoxine 5′-phosphate in 0.2 M potassium phosphate at pH 8 and 7, respectively). The K_i determined using a 10% rabbit liver homogenate is the same as that for the pure enzyme; hence, product inhibition $\text{in}\text{vivo}$ is probably not diminished significantly by other cellular components. Similar determinations for a 10% rat liver homogenate also show strong inhibition by pyridoxal 5′-phosphate. Since the reported liver content of free or loosely bound pyridoxal 5′-phosphate is greater than K_i, the oxidase in liver is probably associated with pyridoxal 5′-phosphate. These results also suggest that product inhibition of pyridoxamine-P oxidase may regulate the $\text{in}\text{vivo}$ rate of pyridoxal 5′-phosphate formation.     

Transport and metabolism of vitamin B6 in Salmonella typhimurium LT2.

Salmonella typhimurium LT2 concentrates radioactivity intracellularly from [3H]pyridoxal or [3H]pyridoxine up to 25 times the external concentration. After 1 min of uptake intracellular radioactivity is found as phosphorylated vitamin B6. The process is sensitive to temperature and is maximally active at pH 8.1, but under the conditions tested it is insensitive to monovalent cations or metabolic inhibitors, and does not require an exogenous energy source. The Km values for uptake of pyridoxine and pyridoxal are 2.0 x 10(-7) M and 1.2 x 10(-7) M, respectively; [3H]pyridoxamine is not transported. Evidence is presented for an uptake mechanism involving facilitated diffusion followed by trapping by pyridoxal kinase. S. typhimurium also appears to lack a periplasmic binding protein for vitamin B6.     

Metabolism of pyridoxine in the liver of vitamin B-6-deficient rats

The metabolism of [6-³H]pyridoxine · HCl was investigated in the liver of vitamin B-6-deficient rats. Rats were made vitamin B-6 deficient by feeding adlititum for 42 days a diet lacking pyridoxine but otherwise optimal. Animals were each injected intraperitoneally with 33 μCi of [6-³H]pyridoxine · HCl and killed at different time intervals afterwards up to 7 days. Radioactively labeled hepatic B-6 compounds were extracted with acid and chromatographically separated on Dowex-X8 (H⁺) columns and the percent radioactivity for each vitamin compound was then calculated. Maximal uptake in control and deficient animals was observed 30 and 60 min, respectively, after administration of label. Radioactivity was not retained by the control animals but decreased steadily in a linear fashion after 30 min, reaching a low level after 3 h. On the other hand, vitamin deficient animals accumulated almost twice as much radioactivity in their liver as the controls and retained it through 7 days.In vitamin B-6-deficient animals 93% of the injected radioactivity was metabolized within 2 min at which time pyridoxine 5′-P and pyridoxal 5′-P reached 36 and 44% levels, respectively. Pyridoxine 5′-P dropped to minimal values (3%) within 15 min and remained unchanged for 7 days while pyridoxal 5′-P reached a peak (79%) level at 15 min and then began to drop linearly reaching a plateau (29%) at 5 days. Further, as the level of pyridoxal 5′-P was falling, pyridoxamine 5′-P was linearly synthesized reaching a plateau level (62%) in 5 days which also remained unchaged through 7 days. Some pyridoxal was also formed (7% at 1 h) which by 12 h had dropped to a plateau low level (3%). The specific activity level of pyridoxal kinase decreased 3.2 times and that of pyridoxine 5′-phosphate oxidase increased 1.5 times in the state of deficiency. The results presented show that metabolism of [³H]pyridoxine in deficiency is characterized by (a) a delayed, two-fold increase in label uptake as well as an extended label retention period, (b) a rapid pyridoxal 5′-P synthesis, and (c) a continuouus synthesis (and accumulation) of pyridoxamine 5′-P which is not utilized or further metabolized.     

Pyridoxamine-5'-phosphate oxidase exhibits no specificity in prochiral hydrogen abstraction from substrate

The stereochemistry for hydrogen removal from pyridoxamine 5'-phosphate with liver pyridoxine (pyridoxamine)-5'-phosphate oxidase was examined to determine whether or not there are significant steric constraints at the substrate region of the active site of the oxidase. For this, pyridoxal 5'-phosphate was reduced with tritium-labeled sodium borohydride in ammoniacal solution to yield racemically labeled [4',4'-3H]pyridoxamine 5'-phosphate which was then chemically or enzymatically oxidized to [4'-3H]pyridoxal 5'-phosphate. This latter was used as coenzyme with either L-aspartate (L-glutamate) aminotransferase and L-glutamate or L-glutamate decarboxylase and alpha-methyl-DL-glutamate to generate [4'-3H]pyridoxamine 5'-phosphate known to be labeled in the R-position. Reaction of the oxidase with the pro-R as well as the pro-R,S-labeled substrates followed by isolation of [4'-3H]pyridoxal 5'-phosphate and 3H2O revealed only half the radioactivity was abstracted from the original substrate in either case. Hence, the oxidase is not stereospecific and equally well catalyzes removal of either pro-R or pro-S hydrogen from the 4-methylene of pyridoxamine 5'-phosphate.     

Preparation and characterization of 3H-labeled α-bungarotoxin

α-Bungarotoxin has been labeled with [³H]pyridoxamine phosphate, by reaction with pyridoxal phosphate followed by reduction with sodium boro[³H]hydride. Specific activities of up to 27 Ci/mmol have been obtained. Mono- and dilabeled toxins bind irreversibly to the acetylcholine receptor from Torpedo electroplax, despite a change in isoelectric point from 9.2 for native toxin to 6.2 for dilabeled toxin. The ³H-labeled α-bungarotoxin is usable for over a year.     

The conversion of pyridoxine phosphate into pyridoxal phosphate in Escherichia coli

1. Evidence is presented for the presence of pyridoxine phosphate oxidase in aqueous extracts of Escherichia coli. Some comparison is made with pyridoxamine phosphate oxidase. 2. Isoniazid and iproniazid were found to combine with pyridoxal phosphate, but isoniazid did not combine with either pyridoxamine phosphate or pyridoxine phosphate. Both oxidase activities were somewhat inhibited by benzylamine and putrescine, but not by phenethylamine or cadaverine. 3. The significance of pyridoxine phosphate oxidase in cell metabolism is discussed.     

Metabolism of pyridoxine in the liver of vitamin B-6-deficient rats.

The metabolism of [6-3H]pyridoxine - HCl was investigated in the liver of vitamin B-6-deficient rats. Rats were made vitamin B-6 deficient by feeding ad libitum for 42 days a diet lacking pyridoxine but otherwise optimal. Animals were each injected intraperitoneally with 33 muCi of [6-3H] pyridoxine - HCl and killed at different time intervals afterwards up to 7 days. Radioactively labeled hepatic B-6 compounds were extracted with acid and chromatographically separated on Dowex-X8 (H+) columns and the percent radioactivity for each vitamin compound was then calculated. Maximal uptake in control and deficient animals was observed 30 and 60 min, respectively, after administration of label. Radioactivity was not retained by the control animals but decreased steadily in a linear fashion after 30 min, reaching a low level after 3 h. On the other hand, vitamin deficient animals accumulated almost twice as much radioactivity in their liver as the controls and retained it through 7 days. In vitamin B-6 deficient animals 93% of the injected radioactivity was metabolized within 2 min at which time pyridoxine 5'-P and pyridoxal 5'-P reached 36 and 44% levels, respectively. Pyridoxine 5'-P dropped to minimal values (3%) within 15 min and remained unchanged for 7 days while pyridoxal 5'-P reached a peak (79%) level at 15 min and then began to drop linearly reaching a plateau (29%) at 5 days. Further, as the level of pyridoxal 5-P was falling, pyridoxamine 5'-P was linearly synthesized reaching a platuau low level (3%). The specific activity level of pyridoxal kinase decreased 3.2 times and that of pyridoxine 5'-phosphate oxidase increased 1.5 times in the state of deficiency. The results presented show that metabolism of [3H]pyridoxine in deficiency is characterized by (a) a delayed, two-fold increase in label uptake as well as an extended label retention period, (b) a rapid pyridoxal 5'-P synthesis, and (c) a continuous synthesis (and accumulation) of pyridoxamine 5'-P which is not utilized or further metabolized.     

THE STABILITY OF VITAMIN B6 ACCUMULATION AND PYRIDOXAL KINASE ACTIVITY IN RABBIT BRAIN AND CHOROID PLEXUS

The effects of changes in the concentrations of pyridoxal phosphate and blogenic amines in brain on: (I) pyridoxal kinase (EC 2.7.1.35) activity in brain and choroid plexus; and (2) vitamin B₆ accumulation by brain slices and isolated, intact choroid plexuses were studied. New Zealand white rabbits were treated parenterally with 200 mg/kg pyridoxine-HCl for 3 days or 120 mg/kg 4-deoxypyridoxine HCI or 5 mg/kg reserpine I day before death. After these treatments the mean concentration of pyridoxal phosphate in brain was elevated by 39% by pyridoxine and decreased by 57% by 4-deoxypyridoxine. Reserpine had no effect. However, the ability of brain slices and isolated, intact choroid plexuses from the treated rabbits to accumulate [³H] vitamin B₆ (with [³H]pyridoxine in the medium) was not different from untreated controls. Also, the specific activity of pyridoxal kinase in brain and choroid plexus of treated rabbits was not different from controls. These results show that vitamin B₆ accumulation and pyridoxal kinase activity in brain and choroid plexus are independent of both pyridoxal phosphate and reserpine-sensitive biogenic amine concentrations in brain. In vitro studies with pyridoxal kinase showed that. in both choroid plexus and brain. pyridoxal kinase was a single enzyme with a molecular weight of 43.000 and a K_m , for pyridoxine of 2.0 μM Crude and partially-purified pyridoxal kinase from brain was not inhibited by biogenic amines (1 mM) or pyridoxal phosphate (5 μM). These in vitro data are consistent with the lack of effect of changes in pyridoxal phosphate and biogenic amine concentrations (in brain) on pyridoxal kinase activity in brain in vivo.     

Purification, assay, and kinetic properties of sheep liver pyridoxal kinase

Pyridoxal kinase from sheep liver has been purified 1100-fold by ammonium sulfate precipitation and chromatography on DEAE-cellulose, Sephadex G-150, and ADP-Sepharose. Polyacrylamide gel electrophoresis indicated the enzyme to be nearly homogeneous. Initial velocity studies were consistent with a sequential mechanism. The Michaelis constants for pyridoxal and ZnATP²⁻ complex are 160 and 31 μ , respectively. A new assay was developed in which [³H]pyridoxine was used as substrate. The product, [³H]pyridoxine 5′-P, was separated from the substrate with DEAE-cellulose disks. Determination of the Michaelis constants for pyridoxine and the ZnATP²⁻ complex by this new method gave values of 110 and 32 μ , respectively.     

Effect of pyridoxine deficiency on nucleic acid metabolism in the rat

1. The nucleic acid metabolism in the pyridoxine-deficient rat has been investigated through studies on the incorporation of radioactivity from various isotopically labelled compounds into liver and spleen DNA and RNA. 2. In pyridoxine deficiency, the incorporation of radioactivity from sodium [¹⁴C]formate was apparently increased. The magnitude of this effect on incorporation into liver RNA and DNA and spleen RNA was approximately the same. The incorporation into spleen DNA was enhanced to a much greater degree. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [¹⁴C]formate. 3. In pyridoxine deficiency, the incorporation of radioactivity from dl-[3-¹⁴C]serine, [8-¹⁴C]adenine, [Me-³H]thymidine and [2-¹⁴C]deoxyuridine was decreased. The incorporation of radioactivity from l-[Me-¹⁴C]methionine was not affected. No noteworthy differences in the effect of pyridoxine deficiency on the incorporation of radioactivity from dl-[3-¹⁴C]serine into DNA and RNA were observed, whereas the effect of the deficiency on the incorporation of radioactivity from [8-¹⁴C]adenine into spleen DNA was somewhat greater than that into spleen RNA. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [3-¹⁴C]serine and [8-¹⁴C]adenine. 4. The adverse effects of pyridoxine deficiency on the biosynthesis of nucleic acids and cell multiplication are discussed in relation to the role of pyridoxal phosphate in the production of C₁ units via the serine-hydroxymethylase reaction.     

Pyridoxine-derived B6 vitamers and pyridoxal 5'-phosphate-binding proteins in cytosolic and nuclear fractions of HTC cells

The nuclear fraction of rat hepatoma-derived HTC cells contained approximately 8% of the total cellular pyridoxal 5'-phosphate. HTC cells were able to metabolize [3H]pyridoxine to coenzymatically active pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate. As HTC cells did not have any demonstrable pyridoxine-5'-phosphate oxidase activity, the conversion of pyridoxine to pyridoxal 5'-phosphate must have taken place by a nonconventional route. The ratio of pyridoxal 5'-phosphate to pyridoxamine 5'-phosphate in the nonnuclear fraction of HTC cells was approximately 1:1, whereas in the nuclear fraction it was approximately 17:1, indicating that there was selective acquisition of pyridoxal 5'-phosphate by the nucleus. With the aid of a monoclonal antibody specific for the 5'-phosphopyridoxyl group, it was shown that there was one major pyridoxal 5'-phosphate-binding protein in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-resolved nucleoplasmic extract of HTC cells. This finding was confirmed by radioautography of an SDS-PAGE-resolved nucleoplasmic extract obtained from cells grown in a medium containing [3H]pyridoxine. Isoelectric focusing followed by SDS-PAGE also indicated the presence of one major pyridoxal 5'-phosphate-binding protein in the nucleoplasmic extract of HTC cells having a relatively high isoelectric point (approximately 7). Data were obtained indicating that the protein might exist in a higher molecular weight form, probably a dimer. Currently, these findings constitute virtually all of the available information on vitamin B6 and the cell nucleus.     

4-Phospho-hydroxy-l-threonine is an obligatory intermediate in pyridoxal 5'-phosphate coenzyme biosynthesis in Escherichia coli K-12

Abstract We show that thrB -encoded homoserine kinase is required for growth of Escherichia coli K-12 pdxB mutants on minimal glucose medium supplemented with 4-hydroxy-l-threonine (synonym, 3-hydroxyhomoserine) or d-glycolaldehyde. This result is consistent with a model in which 4-phospho-hydroxy-l-threonine (synonym, 3-hydroxyhomoserine phosphate), rather than 4-hydroxy-l-threonine, is an obligatory intermediate in pyridoxal 5'-phosphate biosynthesis. Ring closure using 4-phospho-hydroxy-l-threonine as a substrate would lead to the formation of pyridoxine 5'-phosphate, and not pyridioxine, as the first B₆-vitamer synthesized de novo. These considerations suggest that E. coli pyridoxal/pyridoxamine/pyridoxine kinase is not required for the main de novo pathway of pyridoxal 5'-phosphate biosynthesis, and instead plays a role only in the B₆-vitamer salvage pathway.     

The preparation and use of pyridoxal [32P] phosphate as a labeling reagent for proteins on the outer surface of membranes

Pyridoxal [³²P] phosphate was prepared using [γ-³²P]ATP, pyridoxal, and pyridoxine kinase purified from Escherichia coli B. The pyridoxal [³²P] phosphate obtained had a specific activity of at least 1 Ci/mmol. This reagent was used to label intact influenza virus, red blood cells, and both normal and transformed chick embryo fibroblasts. The cell or virus to be labeled was incubated with pyridoxal [³²P] phosphate. The Schiff base formed between pyridoxal [³²P] phosphate and protein amino groups was reduced with NaBH₄. The distribution of pyridoxal [³²P] phosphate in cell membrane or virus envelope proteins was visualized by autoradiography of the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis.The labeling of the proteins of both influenza and chick cells appeared to be limited exclusively to those on the external surface of the virus or plasma membrane. With intact red blood cells the major portion of the probe was bound by external proteins, but a small amount of label was found associated with the internal proteins spectrin and hemoglobin.     

Metabolism of [3H]leupeptin by rat liver

Tritium-labeled leupeptin was used to study how this tripeptide proteinase inhibitor interacts with the liver, including the mechanism of its transport into the cell, its subcellular distribution after uptake, and its metabolism once in the tissue. Experiments were done in situ and in a perfused liver. At low concentrations (1 to 10 μm) the uptake of radioactive inhibitor was competed by chemically reduced leupeptin. At high concentrations at least up to 400 μm the uptake was directly proportional to the external concentration of tripeptide. Entry into the tissue essentially stopped at low temperature (<21 °C). [³H]Leupeptin initially was located in the soluble fraction of the liver homogenate and by 15 to 30 min became concentrated in the lysosome-rich fraction. During 2 h of perfusion almost 50% of [³H]leupeptin that had entered the liver was secreted intact into the bile. In addition, a portion of the leupeptin that remained in the liver was degraded during this time period.     

In vitro inhibition of cholesterol uptake in human and animal arteries by 7-ketocholesterol

Human and pig coronary arteries and rabbit aortas were perfused with pulsatile pressure in a modified Lindbergh apparatus with blood plasma obtained from the same species. Uptake of cholesterol by the arterial wall was measured using [³H]-cholesterol as tracer. Percent distribution of synthesized lipid fractions was determined by thin-layer chromatography and liquid scintillation counting. Inhibition of cholesterol uptake by the arterial wall was studied by the addition of 7-ketocholesterol (concentrations of from 0.05 to 1 μmoles/ml in the perfusate). The addition of 7-ketocholesterol to the perfusate reduced cholesterol uptake by the vessel by an average of 90%. At concentrations of from 0.1 to 1 μmoles/ml of perfusate, 7-ketocholesterol inhibition remained unchanged. Inhibition was reduced at concentrations of ketocholesterol of 0.05 μmoles/ml. Inhibition was present in all species, and was not due to oxidation of cholesterol to 7-ketocholesterol in the perfusate. The results suggest inhibition of cholesterol uptake in the arterial wall by a competitive process.     

Chemical,genetic and structural assessment of pyridoxal kinase as a drug target in the African trypanosome

Pyridoxal‐5′‐phosphate (vitamin B₆) is an essential cofactor for many important enzymatic reactions such as transamination and decarboxylation. African trypanosomes are unable to synthesise vitamin B₆de novo and rely on uptake of B₆ vitamers such as pyridoxal and pyridoxamine from their hosts, which are subsequently phosphorylated by pyridoxal kinase (PdxK). A conditional null mutant of PdxK was generated in Trypanosoma brucei bloodstream forms showing that this enzyme is essential for growth of the parasite in vitro and for infectivity in mice. Activity of recombinant T. brucei PdxK was comparable to previously published work having a specific activity of 327 ± 13 mU mg⁻¹ and a K_m^app with respect to pyridoxal of 29.6 ± 3.9 µM. A coupled assay was developed demonstrating that the enzyme has equivalent catalytic efficiency with pyridoxal, pyridoxamine and pyridoxine, and that ginkgotoxin is an effective pseudo substrate. A high resolution structure of PdxK in complex with ATP revealed important structural differences with the human enzyme. These findings suggest that pyridoxal kinase is an essential and druggable target that could lead to much needed alternative treatments for this devastating disease.     

Substance P-related antagonists inhibit vasopressin and bombesin but not 5′-3-O-(thio) triphosphate-stimulated inositol phosphate production in swiss 3T3 cells

The substance P (SP) analogues [DArg¹, DPhe⁵, DTrp^7,9, Leu¹¹] SP (AntD) and [Arg⁶, DTrp^7,9, MePhe⁸] SP (6–11) (AntG) inhibit the action of many different neuropeptides including SP. These analogues might be useful in the treatment of small cell lung cancer but their mechanism of action is unclear. Here, we analyzed the effect of AntD and AntG on neuropeptide vs. guanosine 5′-3-O-(thio) triphosphate (GTPγS) stimulated inositol phosphate generation in permeabilized Swiss 3T3 cells. AntD inhibited vasopressin and bombesin stimulated inositol phosphate formation (IC₅₀ of 0.75 μM and 2 μM, respectively). Similarly, AntG inhibited vasopressin-stimulated inositol phosphate generation with an IC₅₀ of 1 μM. Strikingly, neither AntD up to 10 μM nor AntG up to 20 μM was able to inhibit GTPγS-stimulated inositol phosphate generation. Dose-response curves of neuropeptide-induced inositol phosphate generation were dramatically displaced to the right by either 10 μM AntD or 20 μM AntG. However, neither antagonist affected the dose response of GTPγS-stimulated inositol phosphate generation. Furthermore, 20 μM AntD had no effect on AIF^?₄-induced inositol phosphates in COS-1 cells transfected with G_αq. AntD inhibited [³H]vasopressin binding competitively in intact Swiss 3T3 cells and both AntD and AntG inhibited [³H]vasopressin binding in Swiss 3T3 and rat liver membranes. Scatchard analysis revealed that AntD inhibited vasopressin binding by reducing receptor affinity without affecting receptor number in both intact and membrane preparations of Swiss 3T3 cells. The results strongly suggest that SP analogues AntD and AntG block the action of the Ca²⁺ mobilizing neuropeptides at the receptor level, rather than inhibiting G protein-stimulated inositol phosphate production. © 1995 Wiley-Liss, Inc.     

Identification and characterization of a pyridoxal reductase involved in the vitamin B6 salvage pathway in Arabidopsis

Vitamin B6 (pyridoxal phosphate) is an essential cofactor in enzymatic reactions involved in numerous cellular processes and also plays a role in oxidative stress responses. In plants, the pathway for de novo synthesis of pyridoxal phosphate has been well characterized, however only two enzymes, pyridoxal (pyridoxine, pyridoxamine) kinase (SOS4) and pyridoxamine (pyridoxine) 5' phosphate oxidase (PDX3), have been identified in the salvage pathway that interconverts between the six vitamin B6 vitamers. A putative pyridoxal reductase (PLR1) was identified in Arabidopsis based on sequence homology with the protein in yeast. Cloning and expression of the AtPLR1 coding region in a yeast mutant deficient for pyridoxal reductase confirmed that the enzyme catalyzes the NADPH-mediated reduction of pyridoxal to pyridoxine. Two Arabidopsis T-DNA insertion mutant lines with insertions in the promoter sequences of AtPLR1 were established and characterized. Quantitative RT-PCR analysis of the plr1 mutants showed little change in expression of the vitamin B6 de novo pathway genes, but significant increases in expression of the known salvage pathway genes, PDX3 and SOS4. In addition, AtPLR1 was also upregulated in pdx3 and sos4 mutants. Analysis of vitamer levels by HPLC showed that both plr1 mutants had lower levels of total vitamin B6, with significantly decreased levels of pyridoxal, pyridoxal 5'-phosphate, pyridoxamine, and pyridoxamine 5'-phosphate. By contrast, there was no consistent significant change in pyridoxine and pyridoxine 5'-phosphate levels. The plr1 mutants had normal root growth, but were significantly smaller than wild type plants. When assayed for abiotic stress resistance, plr1 mutants did not differ from wild type in their response to chilling and high light, but showed greater inhibition when grown on NaCl or mannitol, suggesting a role in osmotic stress resistance. This is the first report of a pyridoxal reductase in the vitamin B6 salvage pathway in plants.