Pyridoxal Phosphate Phosphatase Research Articles

Identification of Proteins Regulated by Ferulic Acid in a Middle Cerebral Artery Occlusion Animal Model-A Proteomics Approach

Ferulic acid plays a neuroprotective role in cerebral ischemia. The aim of this study was to identify the proteins that are differentially expressed following ferulic acid treatment during ischemic brain injury using a proteomics technique. Middle cerebral artery occlusion (MCAO) was performed to induce a focal cerebral ischemic injury in adult male rats, and ferulic acid (100 mg/kg) or vehicle was administered immediately after MCAO. Brain tissues were collected 24 hr after MCAO. The proteins in the cerebral cortex were separated using two-dimensional gel electrophoresis and were identified by mass spectrometry. We detected differentially expressed proteins between vehicle- and ferulic acid-treated animals. Adenosylhomocysteinase, isocitrate dehydrogenase [NAD(+)], mitogen-activated protein kinase kinase 1 and glyceraldehyde-3-phosphate dehydrogenase were decreased in the vehicle-treated group, and ferulic acid prevented the injury-induced decreases in these proteins. However, pyridoxal phosphate phosphatase and heat shock protein 60 were increased in the vehicle-treated group, while ferulic acid prevented the injury-induced increase in these proteins. It is accepted that these enzymes are involved in cellular metabolism and differentiation. Thus, these findings suggest evidence that ferulic acid plays a neuroprotective role against focal cerebral ischemia through the up- and down-modulation of specific enzymes.

Characterization of enzymes involved in the interconversions of different forms of vitamin B6 in tobacco leaves

There are six different vitamin B(6) (VB(6)) forms, pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 5'-phosphate (PLP), pyridoxamine 5'-phosphate (PMP) and pyridoxine 5'-phosphate (PNP). PLP is a coenzyme required by more than 100 cellular enzymes. In spite of the importance of this vitamin, the understanding of VB(6) metabolic conversion in plants is limited. In this study, we developed a sensitive and reliable method to assay VB(6)-metabolizing enzyme activities by monitoring their products visually using high-performance liquid chromatography. With this method, the reactions catalyzed by PL/PM/PN kinase, PMP/PNP oxidase, PM-pyruvate aminotransferase, PL reductase and PLP phosphatase were all nicely detected using crude protein extracts of tobacco leaves. Under optimal invitro conditions, specific activities of those enzymes were 0.15±0.03, 0.10±0.03, 0.08±0.02, 0.64±0.13 and 23.08±1.98nmol product/min/mg protein, respectively. This is the first report on the conversion between PM and PL catalyzed by PM-pyruvate aminotransferase in plants. Furthermore, the PL reductase activity was found to be heat inducible. Our study sheds light on the VB(6) metabolism taking place in plants.

Long-Term Prednisolone Treatments Increase Bioactive Vitamin B6Synthesis In Vivo

The etiology of vitamin B(6) depletion in inflammation remains unknown. Hepatic vitamin B(6) decreased in adrenalectomized rats, and such reductions were restored by an acute muscle injection of a very high dose of glucocorticoids. We tested the hypothesis that long-term prednisolone treatment for treating inflammation restores vitamin B(6) status by induction of tissue B6 metabolic enzymes. Two independent in vivo models were used. Lewis rats and C57BL/6J mice received prednisolone regimens that reflected clinical prednisolone uses in treating human inflammation. We found: 1) prednisolone increased circulating B6 vitamer pyridoxal 5'-phosphate (PLP; bioactive B6 vitamer), pyridoxal (PL), and 4-pyridoxic acid without altering vitamin B(6) excretion; 2) prednisolone simultaneously induced the hepatic PLP-synthesizing enzyme pyridoxine kinase (PDXK) and pyridoxamine-5'-phosphate oxidase (PMPO) and suppressed PLP catabolic enzyme pyridoxal-5'-phosphate phosphatase (PDXP); and 3) elevations in circulating PL were caused by its release from the liver, not by PLP dephosphorylation (PDXP was suppressed and alkaline phosphatase was unaltered). We conclude that long-term prednisolone treatments promoted hepatic bioactive vitamin B(6) synthesis by inducing the synthesizing enzymes PDXK and PMPO and simultaneously suppressing the catabolic enzyme PDXP. Prednisolone increased circulating B6 vitamer without altering urinary B6 excretion. As the major form of vitamin B(6) across cell membrane, elevated circulating PL may facilitate the cellular uptake and utilization of B6. The elevated plasma PLP may increase vitamin B(6) supply to tissues with a higher B6 demand during inflammation. Results from two independent in vivo models suggested a potential advantage of clinical prednisolone use in treating inflammation with respect to vitamin B(6) status.

Proteomic analysis of aging brain in SAMP10 mouse: A model of age-related cerebral degeneration

Senescence-accelerated mouse prone 10 (SAMP10) strain is a model of age-related neurodegeneration in the limbic forebrain. To investigate changes in protein expression profiles involved in neurodegeneration, we performed two-dimensional fluorescence difference gel electrophoresis and compared protein expression in the limbic and non-limbic forebrains of SAMP10 and control mice at various ages. Among protein spots in which patterns of aging in expression in the limbic forebrain differed between SAMP10 and control, we identified three proteins by mass spectrometry: pyridoxal phosphate phosphatase (PLPP), collapsin response mediator protein 2 (CRMP-2) and α-internexin. Expression of PLPP was increased in the limbic forebrain of 3-month-old SAMP10 mice. Levels of CRMP-2 and phosphorylated α-internexin were increased in the limbic forebrain of SAMP10 mice at age 8 months and remained high until 14 months. Western blot revealed elevation in the level of phosphorylated CRMP-2 and the ratio of phosphorylation of α-internexin. Immunohistochemistry revealed that α-internexin was chiefly distributed in axons. Aging in SAMP10 mice was associated with abnormality of PLPP, CRMP-2 and α-internexin, all of which are known to be involved in brain cytoskeleton formation and associated with acute and chronic neurodegenerative conditions. These proteins are promising targets for further investigation of the mechanisms underlying brain aging.

Human brain pyridoxal-5'-phosphate phosphatase (PLPP): protein transduction of PEP-1-PLPP into PC12 cells

Pyridoxal-5'-phosphate phosphatase (PLPP) catalyzes the dephosphorylation of pyridoxal-5'-phosphate (PLP). A human brain PLPP gene was fused with a PEP-1 peptide and produced a genetic in-frame PEP-1-PLPP fusion protein. The purified PEP-1-PLPP fusion protein was efficiently transduced into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced PEP-1-PLPP fusion protein was stable for 36 h. The concentration of PLP was markedly decreased by the addition of exogenous PEP-1-PLPP to media pretreated with the vitamin B(6) precursors; pyridoxine, pyridoxal kinase and pyridoxine-5'-phosphate oxidase into cells. The results suggest that the transduction of the PEP-1-PLPP fusion protein can be one mode of PLP level regulation, and to replenish this enzyme in the various neurological disorders related to vitamin B(6).

Time course of changes in pyridoxal 5′-phosphate (vitamin B 6 active form) and its neuroprotection in experimental ischemic damage

In the present study, we investigated ischemia-induced changes of pyridoxal 5′-phosphate synthesizing enzyme and degrading enzyme and neuroprotective effects and roles of pyridoxal 5′-phosphate against ischemic damage in the gerbil hippocampal CA1 region. Pyridoxal 5′-phosphate oxidase and pyridoxal phosphate phosphatase immunoreactivities were changed in neurons up to 2 days after ischemia, while 4 days after ischemia their immunoreactivities were expressed in astrocytes. Pyridoxal 5′-phosphate oxidase immunoreactivity and its protein level were highest 12 h after ischemia, while those in pyridoxal phosphate phosphatase were highest 2 days after ischemia. Total activities of these enzymes were changed after ischemia, but specific activities of the enzymes were not altered. Treatment with pyridoxal 5′-phosphate into brains (4 μg/5 μl, i.c.v.) at 30 min before transient ischemia protected about 80% of CA1 pyramidal cells 4 days after ischemia and induced elevation of glutamic acid decarboxylase 67 immunoreactivity in the CA1 region. However, pyridoxal 5′-phosphate treatment into ischemic brains decreased GABA transaminase immunoreactivity in the CA1 region after ischemia. These results indicate that pyridoxal 5′-phosphate may be associated with the inhibitory discharge of GABA in the hippocampal CA1 neurons, and the increased level of GABA may protect hippocampal CA1 pyramidal cells from ischemic damage.

Structural and Functional Analysis of Hypothetical Proteins in Mouse Hippocampus from Two-Dimensional Gel Electrophoresis

Protein profiling in five individual mouse strains showed strain-specific expression of three hypothetical proteins (HPs). As functional and structural assignment of HPs were based on predictions and low identity to known structures, HPs were identified by MALDI-TOF/TOF, and their proposed tentative function was determined by enzyme assays. Three identified HPs were extracted from gels and renatured, and pyridoxal phosphate phosphatase, inorganic pyrophosphate phosphatase, and antioxidant activities were revealed, findings in agreement with functional predictions.

Human Brain Pyridoxal-5'-phosphate Phosphatase: Production and Characterization of Monoclonal Antibodies

We cloned and expressed human pyridoxal-5\'-phosphate (PLP) phosphatase, the coenzymatically active form of vitamin B6, in Escherichia coli using pET15b vector. Monoclonal antibodies (mAb) were generated against purified human brain PLP phosphatase in mice, and four antibodies recognizing different epitopes were obtained, one of which inhibited PLP phosphatase. The binding affinities of these four mAbs to PLP phosphatase, as determined using biosensor technology, showed that they had similar binding affinities. Using the anti-PLP phosphatase antibodies as probes, we investigated their cross-reactivities in various mammalian and human tissues and cell lines. The immunoreactive bands obtained on Western blots had molecular masses of ca. 33 kDa. Similarly fractionated extracts of several mammalian cell lines all produced a single band of molecular mass 33 kDa. We believe that these PLP phosphatase mAbs could be used as valuable immunodiagnostic reagents for the detection, identification, and characterization of various neurological diseases related to vitamin B6 abnormalities.

A phosphatase for cofilin to be HAD

Regulating actin dynamics through cofilin dephosphorylation is the role of a newly identified phosphatase, chronophin, a member of the haloacid dehalogenase (HAD) superfamily. Chronophin, the second cofilin phosphatase to be identified, has high phosphoprotein specificity towards cofilin, but curiously it was also discovered independently as a pyridoxal phosphate (vitamin B6) phosphatase.

Human Pyridoxal Phosphatase: MOLECULAR CLONING, FUNCTIONAL EXPRESSION, AND TISSUE DISTRIBUTION

Pyridoxal phosphatase catalyzes the dephosphorylation of pyridoxal 5'-phosphate (PLP) and pyridoxine 5'-phosphate. A human brain cDNA clone was identified to the PLP phosphatase on the basis of peptide sequences obtained previously. The cDNA predicts a 296-amino acid protein with a calculated Mr of 31698. The open reading frame is encoded by two exons located on human chromosome 22q12.3, and the exon-intron junction contains the GT/AG consensus splice site. In addition, a full-length mouse PLP phosphatase cDNA of 1978 bp was also isolated. Mouse enzyme encodes a protein of 292 amino acids with Mr of 31512, and it is localized on chromosome 15.E1. Human and mouse PLP phosphatase share 93% identity in protein sequence. A BLAST search revealed the existence of putative proteins in organism ranging from bacteria to mammals. Catalytically active human PLP phosphatase was expressed in Escherichia coli, and characteristics of the recombinant enzyme were similar to those of erythrocyte enzyme. The recombinant enzyme displayed Km and kcat values for pyridoxal of 2.5 microM and 1.52 s(-1), respectively. Human PLP phosphatase mRNA is differentially expressed in a tissue-specific manner. A single mRNA transcript of 2.1 kb was detected in all human tissues examined and was highly abundant in the brain. Obtaining the molecular properties for the human PLP phosphatase may provide new direction for investigating metabolic pathway involving vitamin B6.

Evidence for a Phosphoenzyme Intermediate Formed During Catalysis by Pyridoxal Phosphatase from Human Erythrocytes

Pyridoxal phosphatase purified from human erythrocytes catalyzes the dephosphorylation of pyridoxal phosphate (PLP) and pyridoxine phosphate. The enzyme had phosphotransferase activity and transferred 20-25% of the phosphoryl group from either substrate to ethanol. Incubation of the enzyme with [32P]PLP, followed by quenching in acid, resulted in trapping 0.14-0.24 mol of 32P per mol of subunit. The incorporation of 32P was not due to Schiff base formation. Phosphorylation of the enzyme by [32P]PLP required catalysis by the enzyme and did not occur in the presence of excess pyridoxine phosphate or with denatured enzyme. The phosphoenzyme intermediate was relatively acid stable and very labile at high pH or in the presence of hydroxylamine. Woodward′s reagent K, which specifically modifies acidic amino acid residues, inactivated the phosphatase in a concentration- and time-dependent manner which followed pseudo-first-order kinetics. Substrates or Pi protected the enzyme from inactivation. It is concluded that PLP phosphatase catalyzes the hydrolysis of PLP by forming a covalent phosphoenzyme intermediate and the intermediate may be an acylphosphate. The 32P-labeled phosphatase was digested with pepsin, and two radioactive peaks were isolated by reversed-phase chromatography. However, definitive sequences were not obtained.

Alkaline Phosphatase Activity and Pyridoxal Phosphate Concentrations in the Milk of Various Species ,

Because pyridoxal phosphate does not normally cross membranes, it was intriguing that the concentration of pyridoxal phosphate is much higher in goat milk than in human milk. We also noted that, although the total vitamin B-6 concentration of bovine milk was similar to that of caprine milk, the bovine milk had lower pyridoxal phosphate. Preliminary data from five Alpine goats, five Brown Swiss cows, five Holstein cows and three humans suggested that there was an inverse relationship between pyridoxal phosphate concentration and phosphatase activity in the goats and cows but not in the humans. This was confirmed with additional data from Nubian goats, Jersey and Guernsey cows, and crossbred sows. Combining the animal data yielded the following relationship between pyridoxal phosphate (PLP, μmol/L) and alkaline phosphatase (P’ase) activity (mmol/(min-L): PLP = 2.03e(-2.26 P’ase) + 0.03. The human milk samples were low in both pyridoxal phosphate and alkaline phosphatase. We conclude that in goats, cows and pigs a significant fraction of the vitamin B-6 appearing in the milk is secreted as pyridoxal phosphate, probably bound to protein, and varying amounts may then be hydrolyzed back to pyridoxal depending on the alkaline phosphatase activity. Human mammary tissue apparently secretes very little pyridoxal phosphate.

Increased activity of pyridoxal kinase in tongue in Down's syndrome

The concentrations of B6 vitamins, and the activities of pyridoxal kinase, pyridoxamine phosphate oxidase and pyridoxal phosphate phosphatase were measured in tongue. Pyridoxal kinase activity was significantly greater (P less than 0.01) in Down's syndrome subjects compared with controls.

Subcellular localization and properties of pyridoxal phosphate phosphatases of human polymorphonuclear leukocytes and their relationship to acid and alkaline phosphatase

Using a novel fluorimetric assay for pyridoxal phosphate phosphatase, human polymorphonuclear leucocytes were found to exhibit both acid and alkaline activities. The neutrophils were homogenised in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. The alkaline pyridoxal phosphate phosphatase showed a very similar distribution to alkaline phosphatase and was located solely to the phosphasome granules. Fractionation experiments on neutrophils treated with isotonic sucrose containing digitonin and inhibitor studies with diazotised sulphanilic acid and levamisole further confirmed that both enzyme activities had similar locations and properties. Acid pyridoxal phosphate phosphatase activity was located primarily to the tertiary granule with a partial azurophil distribution. Fractionation studies on neutrophils homogenised in isotonic sucrose containing digitonin and specific inhibitor studies showed that acid pyridoxal phosphate phosphatase and acid phosphatase were not the result of a single enzyme activity. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (munits/mg protein) of alkaline pyridoxal phosphate phosphatase and alkaline phosphatase varied widely in the three groups and the alterations occurred in a parallel manner. The specific activities of acid pyridoxal phosphate phosphatase and of acid phosphatase were similar in the three groups. These results, together with the fractionation experiments and inhibition studies strongly suggest that pyridoxal phosphate is a physiological substrate for neutrophil alkaline phosphatase.

A highly sensitive fluorimetric assay for pyridoxal phosphate phosphatase

A highly sensitive fluorimetric assay for pyridoxal phosphate phosphatase is described. The assay involves separation of the substrate and product by ion-exchange chromatography followed by treatment of pyridoxal with potassium cyanide under slightly alkaline conditions to form 4-pyridoxolactone, a highly fluorescent compound. Certain kinetic properties of the enzyme activities in human neutrophils are described.

Human Neutrophil Pyridoxal Phosphate Phosphatase and Its Relationship to Alkaline Phosphatase

Human Neutrophil Pyridoxal Phosphate Phosphatase and Its Relationship to Alkaline Phosphatase

Metabolism of [2- 14C]pyridoxine in riboflavin deficiency

To understand the mechanism by which riboflavin-deficient humans and rats maintain normal tissue levels of pyridoxal phosphate despite marked reduction in pyridoxaminephosphate oxidase activity, metabolism of [2- 14C]pyridoxine was studied in riboflavin-deficient and control (ad libitum-fed and pair-fed) rats. The animals were killed 3 or 5 and 10 min after intraperitoneal injection of [2- 14C]pyridoxine. Pyridoxal phosphate formation was markedly impaired in the liver of riboflavin-deficient rats. Contrary to expectation the major metabolic block was in phosphorylation of pyridoxine and pyridoxal. This block could not be ascribed to a decrease in pyridoxal kinase activity. Food restriction in control rats also impaired phosphorylation though to a lesser extent than in riboflavin deficiency. This may be due to the observed lowering of pyridoxal kinase activity in these rats. Pyridoxal phosphate phosphatase activity was not affected by food restriction or riboflavin deficiency. In the muscle most of the radioactivity was in the pyridoxine fraction. The data do not support the possibility of pyridoxal phosphate synthesis by a flavin-independent pathway in riboflavin-deficient rats.

Microassay and properties of pyridoxal phosphate phosphatase in rat pineal gland

1. 1. A sensitive and specifie method was developed capable of determining pyridoxal phosphate phosphatase in a single pineal gland weighing less than l mg. 2. 2. This enzyme which is paniculate bound, has a pH optimum between 8.5–9.0, is inhibited by inorganic phosphate and Zn 2+. 3. 3. It has a specific activity of 569 ± 27mμmol of pyridoxal phosphate hydrolyzed/mg protein/hr which is considerably greater than that of pyridoxal kinase.,

Purification and properties of brain alkaline phosphatase.

Abstract— Alkaline phosphatase from sheep brain has been purified to homogeneity. The method includes butanol extraction, fractional ethanol precipitation, ion‐exchange chromatography on DEAE‐cellulose, and on DEAE‐Sephadex followed by Sephadex G‐200 filtration. By these steps, the enzyme is purified 22,920‐fold with 15% recovery. The homogeneous enzyme is shown to be a sialoglycoprotein in nature. Neuraminidase treatment reduces the electrophoretic mobility of the enzyme. The enzyme shows pyridoxal phosphate phosphatase activity along with p‐nitrophenylphosphate phosphatase activity. Both these compounds behave as mutual alternate competitive substrates. The general properties of the enzyme are described.

Vitamin B 6 enzymes in normal and pre-eclamptic human placentae

Placentae from normal and pre-eclamptic women were analyzed for their content of three enzymes: pyridoxal kinase, pyridoxal phosphate phosphatase, and pyridoxine phosphate oxidase. The range of values for the specific activities of the phosphatase in partially purified extracts was identical in both normal and preeclamptic placentae. The mean value was 6.4 × 10 −3 μmoles/min/mg protein for both tissues. The ranges of values for the specific activities of pyridoxal kinase and pyridoxine phosphate oxidase in the same extracts on the other hand were lower in preeclamptic placentae than in normal ones. The mean value in normal placentae was 1.52 × 10 −3 μmoles/min/mg protein for the kinase and 1.68 × 10 −5 μmoles/min/mg protein for the oxidase. In extracts of placentae from pre-eclamptic women these values were 1.23 × 10 −3 μmoles/min/mg protein and 1.29 × 10 −5 μmoles/min/mg protein respectively.