Vitamin B6 metabolism in Morris hepatomas

The enzymes involved in the metabolism of vitamin B6 were measured in Morris hepatomas and livers of female Buffalo rats fed pyridoxine-sufficient and deficient diets. Pyridoxal phosphate levels in plasmas hepatomas, and livers were also determined. Nontumor-bearing animals were maintained as controls. Regardless of the B6 nutritional status, the concentration of pyridoxal phosphate was lower in the hepatomas than in the livers of the host animals. The apoenzyme levels of ornithine decarboxylase, a pyridoxal phosphate-dependent enzyme, were higher in the hepatomas from animals fed the B6-deficient diet. Liver pyridoxine kinase activity was higher in B6-sufficient animals. In contrast, tumor pyridoxine kinase activity was influenced by B6 intake and was significantly lower than that in host liver. Liver pyridoxine phosphate oxidase activity was not significantly affected by B6 intake or by the presence of tumor. In contrast, hepatomas had little or no pyridoxine phosphate oxidase activity. Pyridoxine phosphate phosphatase activity was elevated in tumors relative to livers. These data indicate that the metabolism of vitamin B6 is markedly different in the hepatomas than in host or control livers and suggest that the tumor is apparently incapable of the complete synthesis of co-enzymatically active pyridoxal phosphate from inactive precursor forms such as pyridoxine.     

Vitamin B6 fluorescence in cells]

The band of cell fluorescence with the maximum of 395-400 nm is registered. This band is exposed on the phone of the tryptophane by wavelength excitation lambdaex=250-260 nm, and in pure scape by lambdaex=310-326 nm. Pyridoxin - substrate of vitamin B6 has identical parameters of spectra excitation and emission of neutral (pH 7) and acid (pH 2) solutions. After temperature damage of cells the intensity of this band increases.     

A Synthesis of Vitamin B6

In the presence of basic catalyst condensation of dehydrolevulinate and cyanoacetamide afforded ethyl 2-methyl-5-cyano-6-pyridone-4-carboxylate [IV]. Starting from [IV], a total synthesis of vitamin B₆ was described.     

Studies on Vitamin B6 Metabolism in Microorganisms

A large number of bacteria were searched for the activity of the synthesis of pyridoxine 5′-phosphate by the transphosphorylation between pyridoxine and p-nitrophenyl phosphate. Several properties of the transphosphorylation by the partially purified enzyme prepared from one of the isolated bacteria, Escherichia freundii K–1, were investigated accompanying with phosphatase activity. The behavior of the phosphotransferase and phosphatase activities in various reaction conditions were almost parallel. It was pointed out that the transphosphorylation might be catalyzed by the function of acid phosphatase. The phosphoryl donor specificity for the enzyme system was found to be broad.

The enzyme which catalyzed the transphosphorylation of pyridoxine accompanying with the hydrolyzation of phosphoryl donor substrates was purified and crystallized from the cell free extract of Escherichia freundii K–1. The purification procedures involved heat treatment, ammonium sulfate fractionation and DEAE-cellulose, hydroxylapatite, and CM-sephadex column chromatographies. The crystalline enzyme showed the sedimentation coefficient of 7.5 S and the diffusion coefficient of 6.15 × 10^?7 cm²/sec. The molecular weight was calculated to be 120,000. Several properties of the purified enzyme were also investigated. It was recognized that the transphosphorylation of pyridoxine might be catalyzed by the action of acid phosphatase.     

Studies on Vitamin B6 Metabolism in Microorganisms

Pyridoxine-P and pyridoxamine-P oxidase in the extract of Alcaligenes faecalis was purified and some properties of the enzyme were investigated. Several lines of evidence indicated that both pyridoxine-P oxidation and pyridoxamine-P oxidative deamination were catalyzed with a single enzyme. The enzyme is a flavoprotein, and the treatment of the enzyme with acid ammonium sulfate resolved the enzyme into apo- and coenzyme. Flavin mononucleotide reactivated the apoenzyme for the oxidation of both substrates. Physiological role of the pyridoxine-P and pyridoxamine-P oxidase was suggested in relation to the transformation of vitamin B₆ in microorganisms.     

Studies on Vitamin B6 Metabolism in Microorganisms

Escherichia freundii alkaline phosphatase was found in a membrane fraction and was purified by procedures involving spheroplast formation with lysozyme and EDTA, and DEAE-cellulose and Sephadex G-150 column chromatographies. Then this enzyme along with other phosphatases was investigated on the ability to transfer the phosphoryl group from p-nitrophenyl phosphate to pyridoxine. It was found that the ability of the transphosphorylation varied with these phosphatases. The transphosphorylation to hydroxy compounds such as alcohols, sugars and nucleosides was also compared. Escherichia freundii acid phosphatase showed the highest activity of transphosphorylation among phosphatases tested. The mechanism of transphosphorylation was discussed.

An enzyme, pyridoxamine 5′-phosphate transaminase, was purified from the cell-free extract of Clostridium kainantoi. The purification procedures involved ammonium sulfate fractionation, protamine sulfate treatment and, DEAE-cellulose, hydroxylapatite, DEAE-Sephadex and Sephadex G-200 column chromatographies. The purified enzyme, which had approximately 2700-fold higher specific activity over the original extract, showed a single schlieren pattern in the ultracentrifuge. From the spectral analysis, it seemed that pyridoxamine 5′-phosphate transaminase did not contain pyridoxal 5′-phosphate as a prosthetic group. It was recognized that the transamination was accelerated by the addition of amino acid and was inhibited by diisopropyl phosphofluoride. Glutamic acid formed in the reaction was identified to be a D-isomer. A study on the substrate specificity showed that the enzyme might be possible to be specific for pyridoxamine 5′-phosphate.

The extracellular formation of vitamin B₆ was searched in marine and terrestrial microorganisms. Two bacterial strains were selected and were identified as Vibrio and Flavobacterium, respectively. Marine microorganisms showed the considerable formation of vitamin B₆ and the presence of vitamin B₆ in sea water was also recognized. The cultural and reaction conditions for vitamin B₆ formation by these strains were investigated. Glycerol was commonly the most effective compound on vitamin B₆ formation among the compounds tested. It was suggested that both bacteria did not have the control system on vitamin B₆ biosynthesis by the amount of possible end products.     

Studies on Vitamin B6 Metabolism in Microorganisms

Oxidation of pyridoxine-P and pyridoxamine-P to pyridoxal-P, inhibition and reactivation of the oxidases were investigated, using the Alcaligenes faecalis oxidase and the Azotobacter agilis oxidase catalyzing. Zone electrophoretic experiments indicated that the oxidases obtained from Alcaligenes faecalis and Azotobacter agilis moved to cathode and anode, respectively, under the same conditions. The oxidation-reduction potential of the both oxidase was found to be about ?50 mV. The oxidation of both pyridoxine-P and pyridoxamine-P was strongly inhibited by pyridoxal-P, pyridoxal, pyridine-4-aldehyde and 4-pyridoxic acid phosphate. This inhibition was markedly decreased by Tris-HCl buffer, and other amino compounds that form Schiff’s base of pyridoxal-P.

An enzyme “pyridoxamine-P transaminase” which catalyzed the transamination between pyridoxamine-P and α-ketoglutaric acid was found in certain anaerobic bacteria, such as Clostridium acetobutylicum, Cl. kainantoi, Cl. kaneboi and Cl. butyricum. The pyridoxamine-P transaminase in the cell-free extract of Cl. kainantoi was purified and some properties were investigated. α-Ketoglutaric acid appeared to be the dominant amino acceptor. Pyridoxamine-P was found to be active as amino donor, but other amino compounds were inert. Since the results were inconclusive, the possibility of vitamin B₆-enzyme of pyridoxamine-P transaminase was not shown by the inhibitor studies. Physiological role of the pyridoxamine-P transaminase was discussed in the relation to vitamin B₆ metabolism in anaerobic bacteria.     

Vitamin B 6 biosynthesis in Bacillus subtilis]

1) A vitamin-B6-producing mutant, BA 1, was selected by treatment of Bacillus subtilis with N-methyl-N'-nitro-N-nitrosoguanidine. Using gradient plates supplemented with the vitamin B6 antagonist isonicotinohydrazide, three mutants of BA 1 were isolated, which excrete 2-5 mg of vitamin B6/l of growth medium. 2) Mutation of the three vitamin-B6-producing strains BA 1, BA 11 and L 71 led to the isolation of 49 vitamin-B6 deficient mutants. All mutants are able to grow with pyridoxine, pyridoxal, pyridoxamine, and even with 4'-deoxypyridoxine. Glycolaldehyde or nicotinic acid do not support growth of the mutants. Some of these vitamin-B6-deficient mutants can also grow in the absence of vitamin B6, providing isoleucine is present. Others show a growth stimulation, when isoleucine is added to a medium containing a vitamin B6 compound. Isoleucine can be replaced by 3-methyl-2-oxovalerate. Cross-feeding experiments indicated a division of the mutants into two groups. Using chromatographic methods, substances which support growth of the mutants were purified, but have not yet been identified. Following the addition of 4'-deoxypyridoxine, 4'-deoxypyridoxine 5'-phosphate was isolated from the growth medium of a vitamin B6-deficient mutant. 3) Threonine dehydratase, transaminase B and transaminase C from wild-type Bacillus subtilis were compared with the enzymes from vitamin-B6-producing strains and with the enzymes from vitamin-B6-deficient mutants. Both the vitamin-B6-producing and the vitamin B6-deficient mutants show higher specific activities than wild type. In the mutant strains no multivalent repression of the threonine dehydratase and transminase B by isoleucine, leucine and valine could be demonstrated. Leucine dehydrogenase, the first enzyme of the isoleucine catabolic pathway, is constitutively produced in the vitamin-B6-producing and in the vitamin-B6-deficient mutants. In the vitamin-B6-deficient mutants, there is a correlation between growth yield in the presence of isoleucine and the specific activity of leucine dehydrogenase. In the crude extract of Bacillus subtilis no pyridoxamine-phosphate oxidase activity could be demonstrated, whereas pyridoxal kinase was readily detectable.     

Studies on Vitamin B6 Metabolism in Microorganisms

A new phosphotransferring reaction which phosphorylated pyridoxine through the phosphoryl group transfer from p-nitrophenylphosphate was found, and the distribution of the reaction in several microorganisms was investigated. The transferring activity was widely distributed in various kinds of microorganisms, especially in fungi belonging to genus such as Aspergillus. The phosphorylated product was isolated from the reaction mixture with the dried cells of Aspergillus flavus and identified as pyridoxine 5′-phosphate.     

Prebiotic photosynthetic reactions

Historically, numerous attempts have been made to mimic — by means of inorganic model reactions — the photosynthetic fixation of CO₂ by green plants. The literature in this field is strewn with claims and counter-claims. Two factors have led us to reexamine this subject: firstly; doubts concerning the highly reducing model for the atmosphere of the primitive Earth and secondly; recent results which demonstrate that photoreductive fixation is feasable on a suitable catalytic surface, for both CO₂ and N₂. The latter observation is of particular interest due to the well-known susceptability of NH₃ to photolytic destruction. Our review of the literature leads us to suggest that similar processes would have been plausible for the primitive Earth and could have been prebiotic precursors to an early development of CO₂-fixing autotrophs.     

Effect of amphotericin B on membrane-associated photosynthetic reactions in maize chloroplasts.

Superoxide dismutase of anaerobic purple sulfur bacterium, Chromatium vinosum, was purified to a homogeneous state. The enzyme contains two atoms of iron per mole and has a molecular weight of 41,000. It is composed of two identical subunits. Amino acid composition, absorption spectra, and the reaction rate constant with O₂^? are also similar to those of the Fe-superoxide dismutases from aerobes. The enzyme is sensitive to hydrogen peroxide and methylene blue-sensitized photooxidation. The functional and evolutional aspects of superoxide dismutase in anaerobes are discussed.     

Studies on the Biosynthesis of Vitamin B6

The production of vitamin B₆ (B₆) compounds with a cell-suspension of Achromobacter cycloclastes A.M.S. 6201 under various conditions were examined. An obvious accumulation of B₆ compounds in the incubation medium with a cell-suspension of the organism harvested at the middle to later part of exponential phase of growth was observed. γ-Aminobutyric acid or β-alanine was found to stimulate the B₆ production markedly, when they were added to the incubation mixture together with glycerol. Some discussion on the implication of these compounds as precursors of B₆ was presented.