2-Amino-4-hydroxy-6-carboxy-7, 8-dihydropteridine inhibited growth of Escherichia coli B, while 2-amino-4-hydroxy-6-carboxypteridine did not affect the growth. The half inhibition was achieved with the inhibitor concentration of 2.2×10^-4M. The inhibition could be reversed by 2-amino-4-hydroxy-6-formyl-7, 8-dihydropteridine. However, when the carboxydihydropteridine was absent in the culture medium, the formyldihydropteridine blocked the microbial growth with an inhibitory power about equal to the carboxydihydropteridine.

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Cell-free extracts of Escherichia coli B catalyze the production of 2-amino-4-hydroxy-6-hydroxymethyl-7, 8-dihydropteridine from 2-amino-4-hydroxy-6-formyl-7, 8-dihydropteridine. NADPH is essential as a cofactor for this reaction. The product is isolated by column chromatographies on Sephadex G-25 and Pcellulose. Structure of the product is determined on the basis of ultraviolet spectra, behavior on paper chromatography, pH-fluorescence curve, and activity as a substrate during enzymatic conversion to dihydropteroic acid.

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The B₁₂-binding principles isolated from cellular components of Lacotobacillus leichmannii ATCC 7830 were studied for the activity to augment vitamin B₁₂ uptake by intestinal mucosa homogenates (IMH) of the guinea pig, hog, rat, hamster, and man. It was found that the uptake of B₁₂ by, the IMH of these species was markedly augmented by these principles. The degrees of augmentation were much greater than that effected by the homologous intrinsic factor (IF). The IF-augmented uptake of B₁₂ by guinea pig IMH was reduced when calcium ion was deleted from the incubation medium, while the deletion of calcium ion had no effect on the uptake augmented by the B₁₂-binding principle. Boiling of IMH for 10min completely abolished the IF-augmented uptake of B₁₂ but the effect of the B₁₂-binding principle was not affected at all. These results indicate that difference exists between IF and the B₁₂-binding principle in the mode of attachment to the adsorptive surface of the small intestine. The B₁₂ uptake by everted sacs prepared from the distal portion of guinea pig ileum was slightly augmented by guinea pig IF but not by the B₁₂-binding princinciple. However, the mucous materials liberated into the medium from the everted sacs during incubation took up large amounts of B₁₂ in the presence of the B₁₂-binding principle as well as IF.

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With a view to study the formation of cross-links in BAPN treated rats by the simultaneous administration of certain vitamins, seven groups of weauling albino rats were raised for three weeks on basal dietalone or vitamin deficient basal diets with BAPN. One of the vitamins namely calcium pantothenate, vitamin E and vitamin B₁₂ was supplemented along with BAPN. The analysis of skins showed that α₁-and α₂-chains of neutral salt-soluble collagen appreciably increased, β₁₁-and β₁₂-chains and aldehyde content of the neutral salt-soluble collagen significantly decreased, whereas the solubility of insoluble collagen in 2 M/KCNS greatly increased under the influence of BAPN in the vitamin deficient groups. Among the vitamins tested, calcium pantothenate gave the maximum beneficial effect against these lathyragenic changes and vitamin E was the next best.

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The reconstitution reaction of glucose oxidase from FAD and apoenzyme was studied:
(1) Unimolecular reaction takes part in the reaction of glucose apooxidase with FAD as a rate limiting step.
(2) At the very initial stage of the reaction, intensity of the fluorescence derived from FAD was observed to increase rapidly and to be quenched slowly after reaching the maximum state.
(3) When glucose apooxidase was incubated with several nucleosides, nucleotides, FMN and riboflavin, FAD incorporation into apoenzyme was strongly inhibited by adenosine, moderately by guanosine and adenine, and was not inhibited by FMN and riboflavin.
(4) An allosteric transition induced by FAD molecule itself probably occurred in the course of the reconstitution reaction, and the second FAD molecule seemed to be likely incorporated spontaneously soon after the first FAD was incorporated.
(5) The mechanism that FAD is first bound to the apoprotein via adenosine residue was confirmed judging from the considerable strength of adenosine and the relative weakness of FMN or riboflavin for the inhibitory effect of the reconstitution reaction.

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The rate of lipid peroxidation, estimated by thiobarbituric acid (TBA) method, was 4.5 fold higher in liver homogenates of the vitamin E dificient rat than in those of the normal rat. But the peroxidation even in normal homogenates was elevated, when linolenate was added to the incubation medium. The increase of peroxidation was promptly reflected the linolenate addition and the rate of oxidation was almost proportionally increased with the incubation time. The optimal pH was sharply shown at 5.9, ATP and GSH increased the rate of the peroxidation, and NADPH accelerated the reaction than NADH. Cell fractionation revealed higher rate of peroxidation in every subcellular fraction.
The mechanism of lipid peroxidation in liver homogenates of E-deficient rat seems to be different from that of the normal animal. Since in the E-deficient system the elevation of peroxidation did not appear promptly but an induction period was observed in an early stage of the incubation. The optimal pH was broad from 5.8 to 6.2, ATP and GSH inhibited the peroxidation, and the reaction was more accerelated with NADH than with NADPH. Cell fractionation procedure decreased the rate of peroxidation in every subcellular fraction, and recombination of these fractions recovered the high rate of the lipid peroxidation.

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The colors of serotonin-FMN and tryptophan-FMN complexes are not changed upon addition of dimethyl sulphoxide at the concentration of 10%, which is routinely used for cell preservation, at 20°. However, the addition to the extent of 50% and above does affect the colors of these complexes. The detailed measurement of the differences in the absorption spectra suggested that dimethyl sulphoxide weakens both the serotonin-FMN and tryptophan-FMN interactions at 20°. In contrast with the observation at 20°, the addition of DMSO under freezing conditions results in the disappearance of colors of the charge transfer complexes while raising the temperature to 20° promptly restores the original color. This disappearance and reappearance of colors, accompanied by freezing and thawing, could be repeated several times.

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A thiamine regulatory mutant of Escherichia coli overproduced a compound which served to satisfy the nutritional requirement of Escherichia coli strain 70-17 for the pyrimidine moiety of thiamine. This compound was separated, purified, and identified as hydroxymethylpyrimidine on the basis of chromatographic, bioautographic and spectral data. In this mutant the synthesis of hydroxymethylpyrimidine was not controlled by thiamine or thiazole, but significantly inhibited by purines and some purine analogues.
Histidine reversed the inhibition of hydroxymethylpyrimidine synthesis by adenine.

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As a simple procedure for the separation of riboflavin tetrabutyrate and its hydrolysates, thin layer chromatography is suitable. To measure quantitatively the separated flavins, scratching out of the area of silica gel adsorbing flavin and direct application of the lumiflavin fluorescence method to the silica gel were examined. The results showed nearly 100% recovery, permitting application of the procedure of thin layer chromatography to the separating determination of synthetic riboflavin tetrabutyrate and riboflavin tetranicotinate and their partial hydrolysates as well as naturally, occurring flavins, free riboflavin, FMN and FAD.

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To reveal further the attitude of riboflavin 2′, 3′, 4′, 5′-tetrabutyrate in animal body, the process of its enzymic hydrolysis to riboflavin and butyric acid was studied. Using pancreatic lipase, the ester was hydrolyzed and the partial hydrolysates were analyzed, and it was found that the hydrolysis is initiated at ester bonding of either side and proceeds via stepwise cleavage.

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