Thiamine diphosphate-decomposing activities in the homogenates of rat brain, dog kidney' and bovine kidney were studied. Those in the autolysate of dog kidney, bovine kidney and yeast were fractionated, some of which were partially purified, and the enzymological properties were also studied with the following results.
1. When the liberation of inorganic phosphate was measured for determining thiamine diphosphate-decomposing activities, a turbidity caused by the reaction between thiamine and phosphomolybdate appeared. However, under suitable conditions, and in a settled concentration range, the extinction could be used as an indicator of degradation. Furthermore, the method of using ammonium chloride to extinguish the turbidity was retested and proved to be excellent and useful. It was applied to the present experiments.
2. β-Glycerophosphate-dephosphorylating activity in rat brain homogenates showed, under conditions used, two maxima at pH 9 and 5. On the contrary, thiamine diphosphate-decomposing activity showed the highest value at approximately pH 9, and, in addition, an accessary peak between pH 6 and 7. The degree of the inhibition for thiamine diphospate-decomposing activity by fluoride was much less than that for β-glycerophosphate-dephosphorylating activity, especially at pH 6.9. The activity for thiamine diphosphate was influenced to some extent by the change in osmotic pressure. The enzyme was presumed to exist in both soluble and insoluble fractions.
3. Thiamine diphosphate-degrading activity in dog kidney homogenates showed a maximum between pH 8 and 9, but the activity was also observed below pH 6. The accessory activity peak was observed adjacent to pH 4.9.
An enzyme preparation was obtained from the supernatant of the kidney homogenate by fractionating at various pH's. It decomposed thiamine diphosphate at acidic pH range, but its activity was not so high. Another enzyme preparation was obtained from the extract prepared by autolyzing kidneys in the mixed solvent containing water, acetone, toluene and ethyl acetate. This preparation degraded thiamine diphosphate more markedly at pH 9.2.
4. Bovine kidney homogenates showed strong decomposing activities for inorganic pyrophosphate over a wide pH range. Besides these, they showed relatively high decomposing activities for thiamine diphosphate at pH 9 and 6, and also for β-glycerophosphate at pH 9 and 5 (so-called alkaline and acid phosphatases).
5. Using the autolysate of bovine kidneys as the starting material, attempts for purification of the enzyme were made. In the partially purified enzyme preparations, no inorganic pyrophosphatase activity at pH 9.2 was found. However, decomposing activities for both thiamine diphosphate and β-glycerophosphate were found, and it was difficult to separate them. These two activities showed the same behaviour when tested with ordinary zone electrophoresis and column chromatography. With ammonium sulfate fractionation, trypsin treatment, and acetone fractionation, especially with repeated acetone fractionations, the activity ratio (TDPase/GPase) somewhat shifted to the larger. But it was not clarified whether ther could be separated or not. It was presumed that the thiamine diphosphate-decomposing enzyme and the β-glycerophosphate-decomposing enzyme would be of a very similar nature.
6. Thiamine diphosphate-decomposing enzyme preparations obtained from bovine kidneys could also decompose ATP, adenosine-5′-phosphate, thiamine monophosphate, and β-glycerophosphate.
Presumptive mechanism for degradation is as follows.
TDP→TMP+iP, TMP→T+iP
The activities for both thiamine diphosphate and thiamine monophosphate was inhibited by the increase in substrate concentration, but that for β-glycerophosphate was not. Thiamine showed no inhibitory action

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1. B₂G synthesized from maltose and B₂ by E. coli, compared with those obtained by the enzyme of rat liver and of Asp. oryzae. The chemical properties of these B₂G are found to be quite identical.
2. For the formation of B₂G, the optimum temperature and pH were found to be 30° and 6.9, respectively, and maltose was chosen as most suitable glucosyl donor among various saccharides.
3. The formation of B₂G was inhibited by both inorganic and organic phosphates, and the latter is attributed to the competitive effect on glucosyl-donating substances. Powerful inhibition revealed by the mild oxidizing reagents and SH reagents is due to the direct action on the enzyme itself.
4. The isoalloxazine ring of B₂ was found to play an important role in this kind of transglucosidation, and the glucosidation was inhibited profoundly by lumiflavin. The redox potential of B₂ is suitable for the glucosidation. The authors have postulated the mechanism of this transglucosidation and discussed on its significant role.

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1. Inorganic Fe⁵⁵ was injected to B₁₂-deficient rats, and the incorporation of inorganic Fe⁵⁵ to hemin-Fe⁵⁵ was confirmed.
2. The relation between vitamin B₁₂ and the synthesis of hemin-Fe⁵⁵ from inorganic Fe⁵⁵ in bone marrow homogenate was studied, and the contribution of vitamin B₁₂ to hemoglobin synthesis in vitro was established.

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1. The activity of carboxylase in the tissues of B₁₂-deficient rats was studied, and the decrease in the evolution of CO₂ from pyruvic acid was recognized.
2. The animals receiving 0.2γ B₁₂per os daily showed an increased carboxylase activity.
3. These findings lead to the conclusion that vitamin B₁₂ contributes to the formation of carboxylase in rats.

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52 known stock strains of yeast-like fungi were tested for thiaminase activity and the following results were obtained.
1. Of the 51 strains tested, 16 (30per cent) decomposed more than 50per cent, 9 (18per cent) 30-50per cent, while 27 (52per cent) less than 30per cent of the thiamine added at pH 6.5 and 45° in 2 hours in the presence of 3γ thiamine.
2. The thiaminase activity of representative strains, Trichosporon cutaneum 0174, Torula lactis 0398, Torulopsis xylinus 0454, Cryptococcus albidus 0378 and C₄ strain of Yonezawa and Aoki was studied and found that the optimum pH was 6.0 and the optimum temperature 40°. Torulopsis xylinus, however, differed in that the optimum pH was 6.5 and the optimum temperature 60°.
3. Determination of the thiaminase potency according to Fujita showed that C₄ strain had the highest value, 146. Cryptococcus albidus 0378 had the value, 44.5 and Trichosporon cutaneum 0174, 30.5, only one-fourth that of C₄.
4. It is concluded that thiaminase is widely distributed in yeast-like fungi.

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For microanalysis of fat-soluble vitamins a procedure using paper chromatography was deviced, in which freshly prepared filter paper soaked with inorganic complex compounds, Ca(H₂PO₄)₂-NH₄OH or MgSO₄-NH₄OH, is used. Some examples of animal experiments using this method were given.

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1. FHD was synthesized from FAD by deamination with nitrous acid and the product was first isolated as barium salt. The yield and purity were, however, unsatisfactory by this method. Therefore, the product was extracted with cresol, followed by transfer in aqueous phase, and by subjecting to column chromatography, FHD was obtained in high purity in satisfactory yield.
2. Various physical and chemical properties of FHD were examined and its components were confirmed by hydrolysis.
3. Physiological activity of FHD was tested using D-amino acid oxidase but no remarkable activity was observed.
4. FHD formation from FAD by the action of adenosine deaminase obtained from Takadiastase could be proved both spectrophotometrically and chromatographically, but this method seems to be unadequate for preparation because of partial decomposition of FHD.
5. The spectroscopic characteristic of FHD was determined and compared with that of a mixture of FMN and inosinic acid.

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The addition of choline to the diet could prevent the elevation of blood pressure induced by high salt diet.

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By the stimulation of sound a significant decrease of thiamine content occurred in the body of mice.

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1. OMP is found to have a liver-disturbing effect.
2. The toxicity of OMP is most effectively prevented by vitamin B₆, but liver-protecting agents, as methionine and N-carbobenzoxy glutamylcholine, also have some effect even when administered alone.
3. The minimum disturbing dose of OMP is between 0.04 and 0.004mg/g.
4. The pyridoxine plus pyridoxamine content and the pyridoxal content of the liver are reduced when OMP is administered.

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