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.     

Partial purification and property of pyridoxine (pyridoxamine)-5′-phosphate oxidase isozymes from wheat seedlings

Isozymes of pyridoxine (pyridoxamine)-5′-phosphate oxidase (EC 1.4.3.5) were isolated from the extract of wheat seedlings by column chromatographies. From DEAE-Sephadex A-50, two fractions having pyridoxine-5′-phosphate oxidase activity were separated by eluting with ~0.075 and ~0.125 m phosphate buffers (pH 8.0). These fractions were further fractionated on a Blue-Sepharose CL-6B column, from which again two activities were eluted by 1.0 m KCl solution. One fraction, designated as E-I, used only pyridoxine 5′-phosphate as substrate, whereas the other, designated as E-II, oxidized not only pyridoxine 5′-phosphate but also pyridoxamine 5′-phosphate with approximately equal rates. The mobility on polyacrylamide disc gel electrophoresis and the substrate specificity of these two fractions were different. Therefore, they were concluded to be isozymes.     

Identification of a second pyridoxine (pyridoxamine) 5′-phosphate oxidase in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Pyridoxine (pyridoxamine) 5′-phosphate oxidase (PPOX) is involved in the biosynthetic pathway of vitamin B₆, converting pyridoxine 5′-phosphate (PNP) or pyridoxamine 5′-phosphate (PMP) into pyridoxal 5′-phosphate (PLP). PLP is a well-known cofactor of numerous enzymes including transamination and decarboxylation reactions. We have previously identified a PPOX (AtPPOX-1) protein encoded by At5g49970 in Arabidopsis thaliana. Here, we report a second PPOX in Arabidopsis, which was named as AtPPOX-2 encoded by At2g46580. The RT-PCR amplified cDNA of AtPPOX-2 was cloned into an Escherichia coli expression vector and a yeast shuttle vector. Both PPOX enzyme assay and complementation of the oxidative stress sensitivity phenotype of a yeast PDX3 deletion mutant demonstrated that At2g46580 encodes a PPOX protein (AtPPOX-2). The catalytic efficiency of AtPPOX-1 is approximately 300-fold higher than that of AtPPOX-2 for PNP. Based on bioinformatic analysis, AtPPOX-2 has a putative mitochondrial transit peptide at the N-terminus. The truncated AtPPOX-2 without 18 amino acids at the N-terminal end lost PPOX activity, suggesting that the N-terminal 18 amino acids are necessary for the enzyme activity of AtPPOX-2. Phylogenetic analysis of AtPPOX-2 homologs from all domains of life suggests that AtPPOX-2 homologs in plants are the product of lateral gene transfer from the cyanobacterial endosymbionts from which plastids are derived.     

Regional distribution of pyridoxal-5′-phosphate in developing and mature brains and its depletion in pyridoxine deficiency

A study was made of (a) the distribution of the coenzyme pyridoxal-5-phosphate (PLP) in four discrete regions of developing and mature rat brains and (b) the effect of dietary pyridoxine deficiency on the distribution. There was an increase in PLP concentration of all the regions from infancy to adulthood. Highest concentration of PLP was found in the medulla and pons in both infants and adults. Pyridoxine deficiency resulted in a more marked reduction of PLP in all regions of the neonatal brain as compared with those in the mature brain. This is consistent with the vulnerability of the developing brain to nutritional stresses.     

Regulation of pyridoxal-5′-phosphate level by biogenic amines in mouse brain

We investigated the relationship between the concentration of pyridoxal-5′-phosphate (PLP) and biogenic amine in mouse brain. The production of PLP from pyridoxal (PL) by pyridoxal kinase (PLK) was inhibited by the addition of dopamine (DA), norepinephrine (NE) and 5-hydroxytryptamine (5-HT), but not by that of epinephrine and N-acetyl-serotonin. DA and NE were combined with PLP by a non-enzymatic reaction, whereas 5-HT was bound only slightly with PLP. The conjugated product of PLP with DA was also detected by HPLC analysis when PLK activity was assayed using PL as a substrate in the presence of DA. In an in vivo investigation, the depletion of DA and 5-HT in mouse brain after an intraperitoneal injection of 5 mg/kg reserpine, led to slight elevation of the PLP level to 120% of the control level. By contrast, the increase in DA in the brain caused by intraperitoneal administration of 150 mg/kg L-DOPA caused the PLP concentration to decrease to 70% of the control level. However, no change in PLK activity in the brain was observed when the mice were treated with either reserpine or L-DOPA. These results suggested that the level of PLP in mouse brain was partly regulated by the concentration of biogenic amines, such as DA, NE and 5-HT, without apparent induction of PLK.     

Pyridox(am)ine 5′-phosphate oxidase (PNPO) deficiency in zebrafish results in fatal seizures and metabolic aberrations

Pyridox(am)ine 5′-phosphate oxidase (PNPO) catalyzes oxidation of pyridoxine 5′-phosphate (PNP) and pyridoxamine 5′-phosphate (PMP) to pyridoxal 5′-phosphate (PLP), the active form of vitamin B₆. PNPO deficiency results in neonatal/infantile seizures and neurodevelopmental delay. To gain insight into this disorder we generated Pnpo deficient (pnpo^−/−) zebrafish (CRISPR/Cas9 gene editing). Locomotion analysis showed that pnpo^−/− zebrafish develop seizures resulting in only 38% of pnpo^−/− zebrafish surviving beyond 20 days post fertilization (dpf). The age of seizure onset varied and survival after the onset was brief. Biochemical profiling at 20 dpf revealed a reduction of PLP and pyridoxal (PL) and accumulation of PMP and pyridoxamine (PM). Amino acids involved in neurotransmission including glutamate, γ-aminobutyric acid (GABA) and glycine were decreased. Concentrations of several, mostly essential, amino acids were increased in pnpo^−/− zebrafish suggesting impaired activity of PLP-dependent transaminases involved in their degradation. PLP treatment increased survival at 20 dpf and led to complete normalization of PLP, PL, glutamate, GABA and glycine. However, amino acid profiles only partially normalized and accumulation of PMP and PM persisted. Taken together, our data indicate that not only decreased PLP but also accumulation of PMP may play a role in the clinical phenotype of PNPO deficiency.     

N-(ϱ-coumaryl)-tryptamine and N-ferulyl tryptamine in kernels of Zea mays

N-(?-coumaryl)tryptamine and N-ferulyltryptamine were isolated from aqueous acetone extracts of ground kernels of Zea mays by successive column chromatography on partially sulfonated styrenedivinylbenzene copolymer resin, lipophilic Sephadex and preparative TLC. Identification of these compounds was made by GCMS of their trimethylsilyl derivatives and the trimethylsilyl derivatives of their acid hydrolysis products.     

Synthesis of 2′-O-(N-methylcarbamoylethyl) 5-methyl-2-thiouridine and its application to splice-switching oligonucleotides

The effect of 2′-O-(N-methylcarbamoyl)ethyl (MCE) modification on splice-switching oligonucleotides (SSO) was systematically evaluated. The incorporation of five MCE nucleotides at the 5′-termini of SSOs effectively improved the splice switching effect. In addition, the incorporation of 2′-O-(N-methylcarbamoylethyl)-5-methyl-2-thiouridine (s²T_MCE), a duplex-stabilizing nucleotide with an MCE modification, into SSOs further improved splice switching. These SSOs may be useful for the treatment of genetic diseases associated with splicing errors.     

Synthesis and fluorescent properties of 5-(1-pyrenylethynyl)-2′-deoxyuridine-containing oligodeoxynucleotides

Novel reagents for the fluorescent labeling of oligo- and polynucleotides have been prepared: 5-(1-pyrenylethynyl)-2′-deoxyuridine 3′-phosphoramidite and a solid support carrying this nucleoside. Oligo-nucleotides containing one or several modified units have been synthesized, and the fluorescence of these probes has been shown to change upon hybridization with the complementary sequence. Fluorescent Nucleosides. III. The previous communications, see [1, 2]. Prefix “d” in the oligodeoxynucleotide designations is omitted.     

Inhibition of glycine cleavage system by pyridoxine 5′-phosphate causes synthetic lethality in glyA yggS and serA yggS in Escherichia coli

The YggS/Ybl036c/PLPBP family includes conserved pyridoxal 5′-phosphate (PLP)-binding proteins that play a critical role in the homeostasis of vitamin B₆ and amino acids. Disruption of members of this family causes pleiotropic effects in many organisms by unknown mechanisms. In Escherichia coli, conditional lethality of the yggS and glyA (encoding serine hydroxymethyltransferase) has been described, but the mechanism of lethality was not determined. Strains lacking yggS and serA (3-phosphoglycerate dehydrogenase) were conditionally lethality in the M9-glucose medium supplemented with Gly. Analyses of vitamin B₆ pools found the high-levels of pyridoxine 5′-phosphate (PNP) in the two yggS mutants. Growth defects of the double mutants could be eliminated by overexpressing PNP/PMP oxidase (PdxH) to decrease the PNP levels. Further, a serA pdxH strain, which accumulates PNP in the presence of yggS, exhibited similar phenotype to serA yggS mutant. Together these data suggested the inhibition of the glycine cleavage (GCV) system caused the synthetic lethality. Biochemical assays confirmed that PNP disrupts the GCV system by competing with PLP in GcvP protein. Our data are consistent with a model in which PNP-dependent inhibition of the GCV system causes the conditional lethality observed in the glyA yggS or serA yggS mutants.     

Comparison of mutagenicity and chemical properties of N-methyl-N′-alkyl-N-nitrosoureas

Thed mutagenic activities of 11 N-methyl-N′-alkyl-N-nitrosoureas were tested on Samonellatyphimurium TA1535 and compared with chemical properties (alkylating activity and decompostion rate). In their relative mutagenicities the N-nitrosoureas that had a cyclic N′-alkyl group showed far more mutagenic activity than those having a chain N′-alkyl group. M(1-A)NU and M(2-A)NU, which had the most bulky N′-alkyl group in this series, exhibited lethal effects at high concentrations. The mutagenicity showed a small positive correlation with decomposition rates but not with alkylating activities on 4-(p-nitrobenzyl_prridine. The highest mutagenicity in this series was observed in N-methyl-N′-cyclobutyl-N-nitrosourea.These results suggest that, in this series of N-methyl-M′-alkyl-N-nitrosoureas, structural differences in the N′-alkyl groups had great significance in mutagenicity.     

N-2′-Acetoxybenzoyl derivatives of chitosan, N-desulphated heparin and 2-amino-2-deoxy-d-glucose

N-2′-Acetoxybenzoyl (aspirin) derivatives (degree of substitution 0·35–1·00) of chitosan, N-desulphated heparin and 2-amino-2-deoxy-d-glucose were prepared by methods that gave yields in the range 65–86%. The salicylate of chitosan was isolated with a 98% yeild. Aspirin or salicylic acid was released much more slowly from N-(2′-acetoxybenzoyl)-chitosan than from the salicylate of chitosan, and much faster at 37°C in 0·1 m NaOH solution than in 2% aqueous acetic acid solution. Salicylic acid was isolated from the dialysate (0·1 m NaOH solution) of N-(2′-acetoxybenzoyl)-chitosan.     

Spectrophotometric determination of pyridoxal and pyridoxal 5′-phosphate with 3-methyl-2-benzothiazolone hydrazone hydrochloride, and their selective assay

A spectrophotometric method with 3-methyl-2-benzothiazolone hydrazone hydrochloride was developed for the determination of pyridoxal and pyridoxal 5'-phosphate, and for the selective determination of each in the presence of the other. Pyridoxal and pyridoxal 5'-phosphate react with the reagent to yield the azine derivatives, which give characteristic absorption spectra. The highest extinction values are obtained when pyridoxal and pyridoxal 5'-phosphate are incubated at pH values of about 3.4 and 8.0 respectively; their maxima are at 430nm. (in 2.74x10(4)) and 380nm. (in 2.24x10(4)) respectively. The azine of pyridoxal is only slightly soluble under the neutral and alkaline conditions, whereas that of pyridoxal 5'-phosphate is substantially insoluble in the acid pH range. This difference in solubility of the azines made possible the selective determination of pyridoxal and pyridoxal 5'-phosphate. alpha-Oxoglutarate and pyruvate are among the substances shown not to interfere with the assay of pyridoxal; their derivatives absorb appreciably only at wavelengths below 420nm. For the assay of pyridoxal 5'-phosphate in the presence of these compounds measurement at 390nm. is necessary.     

Synthesis and biophysical properties of 5′-thio-2′,4′-BNA/LNA oligonucleotide

Phosphorothioate modification of oligonucleotides is one of the most promising chemical modifications in nucleic acid therapeutics. Structurally similar 5′-thio or phosphorothiolate-modified nucleotides, in which the 5′-bridging oxygen atom is replaced with a sulfur atom, are attracting attention and gaining importance in oligonucleotide-based research. In our present study, we synthesized 5′-thio-2′,4′-BNA/LNA monomers bearing thymine or 5-methylcytosine nucleobase. The 5′-thio-2′,4′-BNA/LNA monomers were successfully incorporated into target oligonucleotides, and their nuclease stability and binding affinity with complementary strands were evaluated.     

Synthesis, characterization and properties of uridine 5′-(α-d-apio-d-furanosyl pyrophosphate)

1. A method was developed for synthesizing UDP-apiose [uridine 5'-(alpha-d-apio-d-furanosyl pyrophosphate)] from UDP-glucuronic acid [uridine 5'-(alpha-d-glucopyranosyluronic acid pyrophosphate)] in 62% yield with the enzyme UDP-glucuronic acid cyclase. 2. UDP-apiose had the same mobility as uridine 5'-(alpha-d-xylopyranosyl pyrophosphate) when chromatographed on paper and when subjected to paper electrophoresis at pH5.8. When [(3)H]UDP-[U-(14)C]glucuronic acid was used as the substrate for UDP-glucuronic acid cyclase, the (3)H/(14)C ratio in the reaction product was that expected if d-apiose remained attached to the uridine. In separate experiments doubly labelled reaction product was: (a) hydrolysed at pH2 and 100 degrees C for 15min; (b) degraded at pH8.0 and 100 degrees C for 3min; (c) used as a substrate in the enzymic synthesis of [(14)C]apiin. In each type of experiment the reaction products were isolated and identified and were found to be those expected if [(3)H]UDP-[U-(14)C]apiose was the starting compound. 3. Chemical characterization established that the product containing d-[U-(14)C]apiose and phosphate formed on alkaline degradation of UDP-[U-(14)C]apiose was alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate. 4. Chemical characterization also established that the product containing d-[U-(14)C]apiose and phosphate formed on acid hydrolysis of alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate was d-[U-(14)C]apiose 2-phosphate. 5. The half-life periods for the degradation of UDP-[U-(14)C]apiose to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP at pH8.0 and 80 degrees C, at pH8.0 and 25 degrees C and at pH8.0 and 4 degrees C were 31.6s, 97.2min and 16.5h respectively. The half-life period for the hydrolysis of UDP-[U-(14)C]-apiose to d-[U-(14)C]apiose and UDP at pH3.0 and 40 degrees C was 4.67min. After 20 days at pH6.2-6.6 and 4 degrees C, 17% of the starting UDP-[U-(14)C]apiose was degraded to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP and 23% was hydrolysed to d-[U-(14)C]apiose and UDP. After 120 days at pH6.4 and -20 degrees C 2% of the starting UDP-[U-(14)C]apiose was degraded and 4% was hydrolysed.     

Purification and Properties of Orotidine-5′-phosphate Pyrophosphorylase in Micrococcus glutamicus.

A coupled enzyme system of orotidine-5′-phosphate pyrophosphorylase and orotidine-5′-phosphate decarboxylase has been purified approximately 30-fold from cell-free extract of Micrococcus glutamicus 534 Co–147 by means of acid treatment and fractionations with ammonium sulfate and ethanol addition, and properties of the enzyme system have been studied.

Optima of pH, temperature and substrate concentrations for the activity of the purified enzyme system have been investigated, and compared with those of the same enzyme system from dried brewer’s yeast. Furthermore, effects of various inhibitors on the enzyme activity have been examined and it has become evident that the enzyme system is completely inactivated by addition of chelating agent such as EDTA, and regenerated by further addition of magnesium ion.     

Atom transfer radical polymerization of N-(ω-alkylcarbazolyl)methacrylates via the use of novel heteroleptic Ru(II) polypyridyl initiator

cis-Dichloro-bis(2-(2-pyridyl)-4-carbonylmethylquinoline)ruthenium (II) complex was synthesized and its structure, electrochemical, electronic absorption and emission properties were determined. A derivative Ru(II) complex with radical initiating sites was employed in the atom transfer radical polymerization (ATRP) of functional N-(ω^′-alkylcarbazoly)methacrylates to provide linear metallopolymers with the metal chromophores at one termini of the polymer chain. These polymers were characterized by gel permeation chromatography in combination with low-angle laser light-scattering, UV-Vis and emission spectroscopy to verify the covalent attachment of the metal chromophores to the polymer chain. The polymers thermal transitions and thermal stabilities were also investigated by differential scanning calorimetry and thermogravimetric analysis.     

Synthesis and hybridizing properties of isoDNAs including 3′-O,4′-C-ethyleneoxy-bridged 5-methyluridine derivatives

3′,4′-Ethyleneoxy-bridged 5-methyluridine derivatives with methyl groups in the bridge, (R)-Me-3′,4′-EoNA-T and (S)-Me-3′,4′-EoNA-T, were synthesized, and these two analogs and unsubstituted 3′,4′-EoNA-T were successfully incorporated into a 2′,5′-linked oligonucleotide (isoDNA). Their duplex-forming ability with complementary DNA and complementary RNA, and triplex-forming ability with double-stranded DNA, were evaluated by UV-melting experiments. The results indicated that isoDNAs, including these 3′,4′-EoNA analogs, could hybridize exclusively with complementary RNA. In particular, 3′,4′-EoNA-T and (R)-Me-3′,4′-EoNA-T modifications within isoDNA could stabilize the duplexes with complementary RNA compared with unmodified or 3′,4′-BNA-modified isoDNAs.     

Deuteration and Tritiation of 2′-Deoxyribonucleosides in the 5′ and 4′ Positions

Abstract

The deuterations of 2′-deoxyguanosine in the 4′ and 5′ positions have been described elsewhere (1). The starting material is the 5′-aldehyde formed by mild oxidation with N,N-dicyclohexyl carbodiimide in dimethyl sulphoxide of the fully protected nucleoside with free 5′-alcoholic function. The 5′4euteration was achieved by reduction with deuterated sodium borohydride. Incorporation of deuterium in the 4′-position was achieved v i a an enhanced keto-enol tautomerim by heating the aldehyde in 50/50 D20/pyridine, with subsequent reduction of the aldehyde with NaBH4. The 6-furanoid form was isolated from the I-lyxo by-product by reverse phase HPLC. Applied to pyrimidine 2′-deoxyribonucleosides, this method was shown to give deuterated 2′-deoxycytidine and thymidine in good yield.     

The effect of hydrogen bonding in two crystal modifications of aquabis(N,N-dimethylglycinato-κN,κO)copper(II): Experimental and theoretical study

From the crystals of trans aquabis(N,N-dimethylglycinato-κN,κO)copper(II) dihydrate (compound 1, space group P2₁2₁2₁) novel crystal structure of trans aquabis(N,N-dimethylglycinato-κN,κO)copper(II) (compound 2, space group Pbca) was obtained and analysed by X-ray diffraction. In the crystal structure 1, the O-H?O hydrogen bonds form three-dimensional network. In the crystal structure 2, two-dimensional layers stacking to each other are formed, with non-polar N,N-dimethyl groups placed on the opposite sides of the layers, and with the polar part in the middle forming CO?O-H and C-H?O hydrogen bonds. Different hydrogen bonding patterns in 1 and 2 do not pronouncedly affect molecular geometry of the title compound. Molecular mechanics force field suited for studying the properties of bis(amino acidato)copper(II) complexes in the solid state can follow the differences between the experimental molecular structures in the two diverse crystalline surroundings. To make possible direct comparison between crystal lattices, the force field was applied to predict unit cell packing of supposed anhydrous bis(N,N-dimethylglycinato)copper(II) in space group Pbca. Relative intermolecular energies of hypothetic anhydrous crystal and simulated 1 and 2 crystals are discussed. On the basis of experimental and theoretical results we conclude that the main effect of two water molecules of crystallisation in 1 is to stabilise the crystal packing via hydrogen bonding, whilst similar pyramidal copper(II) coordination geometry in 1 and 2 is due to axially coordinated water molecule and its intermolecular interactions.