1. The active protein of transglucosidase relating to B₂ was isolated in partially purified form from the cell extract of E. coil, and the specificity of this preparation was investigated.
2. It was demonstrated that the enzyme utilized only maltose as a glucosyl donor since it has α-glucosyl linkage, which is indispensable to the transglucosidation reaction.
3. The transglucosidase reacted with several isoalloxazine derivatives and synthesized the corresponding glucosides, isomaltosides and various oligosaccharides. Together with the evidence obtained from the inhibition experiments with B₂ analogues, these results confirm the preliminary posturation of B₂ that the isoalloxazine group plays an important role to interact with the enzyme molecule.
4. Quinacrine, quinine and other related compounds inhibited the transglucosidation, since they have relative affinity to the protein of transferase so as to reveal competition with B₂ in the reaction system.

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1. Successive transgalactosidation from lactose to B₂ took place by the action of E. coli, forming galactosyl oligosaccharide compounds of B₂.
2. By preparative chromatography of the reaction products three B₂ compounds were separated and identified as B₂-galactoside, B₂-galactobioside and B₂-galactotrioside, respectively.
3. B₂-galactoside produced by E. coli was isolated in a crystalline form and compared with the preparation obtained by Asp. oryzae. Both were found to be quite identical in chemical properties.

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1. An enzyme catalyzing the successive transfer of β-galactosyl group from lactose to B₂ was purified up to about 40 fold from the extract of E. coli. The enzyme showed a maximal activity at pH 7.3 and at 30° in glycine buffer.
2. It was found that the enzyme utilized only lactose as an efficient donor of galactosyl group among many saccharides tested.
3. The isoalloxazine part of the galactosyl acceptors was supposed to be responsible for the transfer reaction in actively associating with transgalactosidase.
4. The enzyme activity was slightly activated by univalent ions, while heavy metallic ions acted as powerful inhibitors.
5. The significance of B₂ in glycosyl-transferring mechanism, through which formation of higher oligosaccharides proceeds effectively, was discussed.

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1. Male albino rats maintained for 8 days on a standard diet, with 100mg α TAc per rat added as daily dietary supplement, had significantly higher levels of liver phospholipids, total and ester cholesterol than their controls.
2. During the course of incubation, with glycerol as substrate, the test homogenates synthesized significantly greater amounts of these lipids than the control homogenates.
3. The significance of these findings is discussed.

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1. The total and free vitamin B₁₂ of the serum of 40 normal human subjects was measured. The results showed that the total vitamin level ranged from 160 to 812μμg per ml with a mean value of 415μμg and the free form level from 0 to 84μμg per ml.
2. The bound form of vitamin B₁₂ in human serum is bound to the proteins in the α-(chiefly α₂) globulin fraction and the free form seems to be bound loosely to the protein in the β-globulin fraction.
3. α- and β-globulins have the capacity to bind added vitamin B₁₂ in excess of the quantity normally present. The former takes the binding type of the bound form, whereas the latter the binding type of the free form. The binding capacity of the former is stronger and greater, the saturation level being 1.0-1.7mμg/ml, whereas that of the latter 0.7-1.1mμg/ml.
4. The physiological significance of the two types of vitamin B₁₂ bound with proteins present in the serum and the relationship to liver function is discussed.

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1. When human serum is heated at 100° at pH 7.4, 6.8 or 4.6, dissociation of the bound form of vitamin B₁₂ occurs most rapidly at pH 4.6. The addition of a minute quantity of cyanide promotes the dissociation. No difference was found among the three pH levels when heated for 30 minutes and the addition of cyanide in this case had no effect.
2. When heated at pH 6.8 for 30 minutes and the dissociation of the bound form at 100° is taken as 100per cent, dissociation is 0per cent at 50°, 5per cent at 60°, 20per cent at 70°, 50per cent at 80° and 85per cent at 90°.
3. The bound form is dissociated almost 100per cent when it is allowed to react with trypsin for 2 hours and about 75per cent with pepsin.
4. Change in the binding capacity of the vitamin B₁₂-binding protein in α-globulin fraction of serum by treatment with oxidizing agents and other chemicals was examined by two different methods. It was found that the binding capacity was almost completely destroyed by periodate. From this finding the vitamin-B₁₂-binding protein in α-globulin fraction was assumed to be a kind of glycoprotein.

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Synthesis of cocarboxylase from thiamine hydrochloride was studied and an appropriate method was reported. Pure TDP crystal could be obtained using ion exchange chromatography and TDP hydrochloride with the purity of 80-90per cent was obtained with a relatively simple procedure and in a high yield.

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Investigations were made on the determination of cyanocobalamin present in natural materials together with a variety of the related factors by a static culture method with a protozoan, Ochromonas malhamensis.
1. The determination of vitamin B₁₂ is possible by a static culture method using Ochromonas malhamensis, although the assay range is narrower as compared with a shaking culture.
2. Assay test tubes of small size with aluminium cups are more convenient for the assay by a static culture method.
3. The higher the inoculum amount, the shorter the length of the incubation time necessary for assay. It. might be most convenient to use 1:10 dilution of 5-day culture of the test organism, since the heavy inoculum causes a high blank, and gives vigorous growth during a shorter incubation.
4. The use of cyanide at the concentration of about 1μg per tube facilitates the extraction of vitamin B₁₂ from natural materials.
5. For the purpose of widening the assay range in the dose-response curve of static culture, the effects of various kinds of surface-active agents were tested on the growth of the test organism. Especially effective agents could not be found except for Myri 52 with which approximately linear dose-response curve was obtained within the concentration of 0 to 0.2mμg of vitamin B₁₂.
6. Satisfactory recoveries were obtained with the extracts of beef liver and methane fermentation products of night soil and distillers' wastes.

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1. In the rat organs the disulfide forms of thiamine and of CoC are not detectable by employing the thiochrome method and utilizing the reducing action of cysteine.
2. Organ homogenate prevents the oxidation of thiamine to its disulfide, which easily occurs in a weakly alkaline solution.
3. The existence of a certain oxidizing factor of thiamine is noticed in human saliva, but not in the cerebrospinal fluid.

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1. In dilute solutions at a weakly alkaline pH range a certain amount of CoC is auto-oxidized, i.e., “masked” to form its disulfide which can be reduced by cysteine.
2. Ovalbumin and casein prevent this oxidation, while the cupric ion and gaseous oxygen accelerate it remarkably.
3. The change in the amount of the thiol group of CoC in the reaction with alkali is demonstrated by the PCMB method.
4. Rat liver homogenate reduces disulfide-CoC into the thiol almost completely.

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1. After TPD or B₁SSB₁ is reduced to free thiamine by cysteine or GSH, the re-formation of the disulfide-thiamine occurs in an aerobic and weakly alkaline medium at some mole ratios and concentrations.
2. B₁SSB₁ reacts similarily to TPD.
3. The reducing action of GSH upon the disulfide-thiamine is observed to be much stronger than that of cysteine.

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