JTC-3 cells were grown on a synthetic medium containing thiamine and its various derivatives at different concentration and the activity for cell growth was studied. Thiamine was proved to be essential for the growth of the cells and the latter was able to synthesize carboxylase from thiamine or its derivatives. The optimal concentration for thiamine, TATD, other disulfide-type compounds, excluding TATD, and phosphate-type derivatives were, in terms of thiamine, 0.1,1,0.001 and 0.1μg/ml, respectively. The difference in optimal concentration is considered to be due to the different activity of these compounds, and the difference between TATD and the other disulfide-type thiamine desivatives is supposed to be due the lipoic acid moiety of the former. The potency for cell growth at the optimal concentration of various thiamine derivatives did not show a significant difference.
The effect of thiamine and its derivatives at high concentrations over optimal on the growth of JTC-3 cells in a synthetic medium was investigated. As the concentration of the thiamine derivatives rose to 10^2,10^3 and 10^4 times of the optimal, the growth of the cells was impaired proportionally. At 10^4 times concentration, all of the compounds showed the impairment, especially conspicuous in TPD and TATD. At the concentration of 10μg/ml, the growth impairment was observed in TPD, TOED and BTMP, especially in the former.
Paperchromatographic separation of L-ascorbic and D-araboascorbic acids were carried out using HPO_3 treated filter paper and water saturated methyl-ethyl ketone employing ascending method. The filter paper, pretreated with 3% HPO_3 solution and developing at 25℃ was the most suitable condition for separating them. The moisture in stationary phase is quite important for the separation, and polyhydric alcohol and their derivative, such as ethylene glycol, glycerol, their chlorohydrines and ethers, can be used as partition liquid by adding to the filter paper. By increasing the concentration of glycerol in 3% HPO_3 solution, locations of the both compounds were varied without change of separating ratio (0.1 as Rf value). This technique may be effective for the separation of these substances from natural product containing some impurities having the same Rf values with those acids.
In recent publications, several studies relating to the separation and estimation of L-ascorbic and D-araboascorbic acids using acetonitrile・acetone・acetic acid・water system and descending method were reported. Separating ratio of the both compounds was only 0.05 in Rf values. The authors has reported to separate them using water saturated methyl-ethyl ketone and HPO_3-treated filter paper employing ascending method. In present paper, with the application of the new method, the both compounds were separated and were estimated by 2,4-dinitrophenylhydrazine method. The experimental results were treated by analysis of linear regression and 95% fiducial range of regression lines were determined. 95% fiducial limits were ±3-4 μg for both compounds at a range between 10 to 50 μg, and ±2-3μg for 4 to 10μg.
Activity of D-amino acid oxidase in the liver and kidney of rat administered with riboflavin-2'.3', 4', 5'-tetrabutyrate and tetrapalmitate was estimated. The results showed that the activity of D-amino acid oxidase in these organs of rat in case of riboflavin tetrabutyrate is almost the same with that of the normal rat, whereas that in case of riboflavin tetrapalmitate is lower than that of the normal one and a little higher than that of rat adminstered with riboflavin deficient diets.
Vitamin A-palmitate is prepared from its acetate by ester exchange reaction. The former, therefore, might be contained more or less with the latter. A simple and convenient method for the determination of acetate has been developed using infrared spectrophotometric technique. The absorption bands at 8.15 and 9.8lμ due to acetyl ester group were found to be applicable as key bands. The quantitative analysis was carried out with an 10% solution in carbon tetrachloride when acetate was contained in considerable amount. In case its content was rather small, defferential method had to be employed, with the error within 0.5%. The minimum quantity detectable by this method was 0.2-0.5%.
The physiological significance of Co^<58>-labeled 5,6-dimethylbenzimidazolyl cobamide coenzyme has been studied in terms of its absorption, tissue distribution and binding with human gastric juice. Schilling's urinary excretion test of coenzyme B_<12> gave the results of 1.6-2.6% in mormal human subjects and 0.2-0.6%, which were enhanced by hog intrinsic factor preparation, in patients with pernicious anemia in remission who showed the abolishment of vitamin B_<12> absorption. Coenzyme B_<12> was taken up preferentially by the liver and kidneys in Wistar strain rats, following either oral or intravenous administration. Human gastric juice binds with coenzyme B_<12> and makes it nondialyzable. Coenzyme B_<12> binders in the paper-electrophoregram of gastric juice revealed the same pattern as vitamin B_<12> binders.
It was concluded that the growth stimulating effect on rats exerted by mycelium of Aspergillus oryzae is not due to protein or inorganic substance, since an addition of purified casein or sodium chloride to the vitamin added basal diet described. On the other hand, the presence of a similar effective substance (s) in commercial crude casein was indicated and a combined addition of sodium choride and the mycelium caused a slight inhibition of the growth of rats.
In a previous publication, results on the purification, nature and mode of action of glutamic・pyruvic transaminase (GPT) from pig heart muscle were reported. In the course of the experiment, it was found that GPT was separated into two fractions on calcium phosphate gel column and starch zone electrophoresis, and they belong to the one in soluble fraction and mitochondria respectively. For solubilization of enzyme from mitochondria, several techniques, i. e., sonication (10Kc, 10min.) treatment with deoxycholate, freezing and thawing, and grinding with glass powder, gave the enzymes which have 60% of activity compared with original mitochondrial suspension. This loss is assumed to be attributed to the disruption of essential structure of mitochondria, or to the accumulation of oxalacetic acid in intact mitochondria which is measured as pyruvate. Digitonin inhibited the enzyme activity completely. GPT in soluble and in mitochondrial fractions were eluted with 0.02M phosphate buffer at pH 6.8 and 0.06M at pH 6.8 respectively on calcium phosphate gel column chromatography by stepwise and gradient eluting systems. On zone electrophoresis, they moved to cathode and separated each other. GPT preparation catalyzed the elemental reaction (C^<14>-alanine+pyruvate ⇄C^<14>-pyruvate+alanine, C^<14>-glutamate+α-ketoglutarate⇄C^<14>-α-ketoglutarate+glutamate) more strongly than original reaction (glutamate+pyruvate⇄alanine+α-ketoglutarate) with the same Km. GPT in soluble fraction has optimum pH at 9.7-9.5 and that in mitochondrial fraction at pH 8.5.
In previous report one of the authors has reported that glutamic・pyruvic transaminase located at mitochondria has quite different natures from one in soluble fraction. We have isolated glutamic-oxalacetic transaminase from pig liver mitochondria (GOTM) about 20 times active more than original mitochondria suspension using the technique of ammonium sulfate fractionation, calcium phosphate gel absorption and zone electrophoresis. On calcium phosphate gel column, GOTM is eluted with 0.1M phosphate buffer at pH 8.0 and GOT in soluble fraction (GOTS) with 0.04M phosphate buffer at pH 7.0. On starch zone electrophoresis, they showed very contrary pattern, i. e., GOTM moved to cathode and GOTS to anode. GOTM was more labile than GOTS at 70℃ heating. In detecting optimum pH and temperature, was not found any remarkable difference. Thus the presence of two different type of GOT in different cell fraction was demonstrated. We are now undergoing the efforts to elucidate the significance of their localization from the view point of respiratory regulation and protein synthesis.