In order to get informartion on the relationship between the partition coefficient and the molecular weight on Sephadex LH-20 in chloroform, the partition coefficient KD and K_<av> for 28 kinds of fat-soluble vitamins and related compounds were determined. It was found that the linear correlation between the molecular weight and the logarithm of KD was present and the compounds containing carboxyl or hydroxyl groups were retarded relative to the corresponding compounds having approximately similar molecular weight. It was also observed that for complete separation of the two solute with the partition coefficient KD_1 and KD_2 respectively, the volume of the sample should be less than 0.5・(KD_1 - KD_2)・Vi.

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The spectrophotomeric determination of vitamin A in mixture of vitamin A and α-tocopherol using hydrogenation method was applied to fish liver oils and to long-stored partially oxidized vitamin A palmitate to which known amounts of α-tocopherol were added. Recovery of vitamin A was almost complete and absorbance curve against hydrogenated blank resembled that of vitamin A used in preparing vitamin A ・α-tocopherol mixtures. It was also found that related compounds of vitamin A such as acetate, palmitate, aldehyde, acid, anhydrovitamin A, retrovitamin A, epoxide, vitamin D_2 and vitamin D_3 were hydrogenated under identical conditions used hydrogenation of vitamin A.

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In the previous studies of this series, it was investigated the influence of water content on the change of vitamin A acetate and anhydrovitamin A in aqueous ethanolic solution. This report describes the results of a similar study on the stability of retrovitamin A acetate, an intermediate product of vitamin A acetate in aqueous ethanolic solution. Retrovitamin A acetate was determined by spectrometric method (367mμ in ethanol). The amount of changed products of retrovitamin A acetate was examined by alumina column chromatography and thin layer chromatography. The stability of retrovitamin A acetate in 60-100% ethanolic solution, at 35 and 50℃ decreased gradually as water increased. Three substances were recognized as changed products of retrovitamin A acetate. (a) Retrovitamin A alcohol (Rf 0.29) , (b) unknown substance which seems to be the same substance of changed anhydrovitamin A, having maximum absorbance at 283 mμ (Rf 0.16) , (c) unknown substance (Rf 0.11). Anhydrovitamin A and unknown substances I, II. which were reported previously as the changed products of vitamin A acetate were not recognized. The reversible reaction of retrovitamin A acetate to vitamin A acetate or its alcohol was not observed.

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2'-nor-thiamine and 2'-ethylthiamine, 2'-methyl group of pyrimidine moiety of thiamine is substituted by H, C_2H_5,respectively, showed the activity of 22,89% to that of thiamine in the growth promotion of Kl. apiculata IFO 0630,No significant difference was observed in their permeability to the cells of the yeast and in their phosphorylation to 2'-nor-thiamine or 2'-ethylthiamine pyrophosphate by thiamine pyrophosphokinase. The lower activities of these thiamine derivatives to Kl. apiculeta should therefore be explained by their weak coenzymic activity to thiamine requiring enzymes such as pyruvate decarboxylase (PDC) or transketolase. The activities of 2'-nor- and 2'-ethylthiamine pyrophosphate to PDC were 5-6% and 25-27%, respectively, to that of thiamine pyrophosphate. The discrepancy between the growth promoting activity and PDC activating effect of these derivatives is discussed.

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Accumulation of thiamine on cells of Saccharomyces cerevisiae was completely inhibited at the presence of phenylthiazinothiamine (PTT) but at the presence of pyrithiamine (Pyri-B_1), it was partly inhibited with its simultaneous accumulation on cells. Accumulated Pyri-B_1 was not eluted by repeated washing with saline, although the cells showed to accumulate thiamine completely after the washing with saline. Cells preincubated with PTT accumulated thiamine incompletely even after the seventh washing with saline. Thiamine accumulated on cells was washed away with PTT, not with Pyri-B_1. Yeast cells can accumulate Pyri-B_1 quickly by active transport but sparingly accumulate PTT by simple diffusion. Mode of inhibition of thiamine accumulation on yeast cells by the two inhibitors is different, although inhibitors in the medium are more important than those in cells.

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Isoelectric point of pyruvate decarbaxylase from baker's, yeast was determined on both crude extract and partially purified preparation by isoelectric fractionation with Ampholine, a carrier ampholyte. The enzymic activity was distributed mainly at pI 5.6 fraction and weakly at pI 5.9 in case of crude extract. The activity at pI 5.6 fraction, however, disappeared in course of purification. Two kinds of pyruvate decarboxylase were separated by DEAE-cellulose column chromatography ; one of them was lost most of its activity by isoelectric fractionation, leaving trace of activity at pI 6.17 and pI 6.36 fractions, whereas the other was concentrated at pI 5.83 fraction. Though pI 5.83 preparation looked pure, the enzymic activity was lost by repeating isoelectric fractionation, leaving a trace activity at pI 6.1〜6.4 fraction. pI 5.83 preparation did not need Mg ion for its enzymic reaction ; Mg ion exhibited an inhibitory effect. Pyruvate decarboxylase reaction catalyzed by the preparation was analyzed according to Ingraham・Makower equation, a rate equation for two-substrate enzyme reaction, regard ing cocarboxylase as a substrate as well as pyruvate. The kinetic data were explained as follows : Rate of reaction was limited by forming enzyme-cocarboxylase-pyruvate ternary complex. The reaction may proceed through either one of two binary complexes, enzyme-cocarboxylase and enzyme-pyruvate, but mainly through enzyme-cocarboxylase complex. Affinity of cocarboxylase for enzyme-pyruvate complex was much less than that for free enzyme.

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By giving pantothenic acid deficient diet or large amount of pantothenic acid, the distribution of pantothenic acid, CoA and their intermediates in rat liver was observed. The result of this study obtained from the composition technique of CoA to phosphopantetheine by phosphatase was quite similar to our previous result derived from the biosynthetic method of CoA from phosphopantetheine or dephospho-CoA by dephospho-CoA pyrophosphorylase and dephospho-CoA kinase. Phosphopantetheine and dephospho-CoA contents in rat liver decreased by giving pantothenic acid deficient diet and the amount of phosphopantothenic acid and phosphopantothenoylcysteine increased by giving large amount of pantothenic acid. Therefore, CoA level was usually kept constant under above-mentioned two conditions. The activity of phosphopantothenoylcysteine decarboxylase was compaired under these different situations, but no significant difference was found.

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CH_3-B_<12> fraction was confirmed in rats fed freely, and after the administration of 4mg of CH_3-B_<12> total cobalamin in liver and kidney apparently increased and the ratio of CH_3-B_<12> fraction in total cobalamin fraction increased at least in liver. Following the parenteral adminstration of 2μg of CH_3-B_<12>-^<57>Co to the male rats, the hepatic and renal uptakes of ^<57>Co and the forms of cobalamins in both organs were measured for two weeks. Three hours after the administration, apporoximately ninety percent of the total activity in both organs was found in both fractions of OH-B_<12> and DBCC, while the CH_3-B_<12> fraction was demonstrated only in ten percent. After that, the radioactivity of OH-B_<12> fraction of liver came down gradually, with DBCC fraction increasing simultaneously. The activity of CH_3-B_<12> fraction also decreased slowly. The urinary excretion of CH_3-B_<12>-^<57>Co was higher than that of OH-B_<12>-^<57>Co. The kidney : liver uptake ratio of CH_3-B_<12>, on the other hand, resembled to DBCC.

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Eleven derivatives of 2-ethyl-4-aminopyrimidine and two derivatives of 2-methyl-4-aminopyridine were tested for the antivitamin B_6 potency against Lactobacillus arabinosus, Lactobacillus casei, Streptococcus faecalis, and Leuconostoc mesenteroides. Although all samples were not inhibitory to L. arabinosus, 5-CH_2OH-pyrimidine and 5-CH_2Br-pyrimidine were inhibitory to other three organisms. 5-CH_2OH-pyridine, when tested only on Leuc. mesenteroides, was shown to be inhibitory. Recovery of inhibited growth was observed on addition of pyridoxal or pyridoxamine. These compounds were proved to be antivitamin B_6 compounds having mhibitron indices as shown in Table 4,although their potencies were weaker than that of hydroxymethylpyrimidine.

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The antivitamin B_6 potencies of eleven derivatives of 2-ethyl-4-aminopyrimidine and two derivatives of 2-methyl-4-aminopyridine on the growth of Saccharomyces carlsbergensis 4228 were investigated. Of all samples, ten compounds showed appreciable growth-inhibitory activity. The inhibition indices of these active samples were shown in the following order of their antagonistic potency. 5-C_2H_5-pyridine > 5-C_2H_5-pyrimidine > 5-C_3H_7-pyrimidine> hydroxymethylpyrimidine ≒ 5-CH_2OH-pyrimidine > 5-CH_2OH-pyridine > 5-CH_2OC_2H_5-pyrimidine > 5-CH_2NHCOC_2H_5-pyrimidine > 5-CH_2NH_2-pyrimidine> 5-CH_2Br-pyrimidine> 5-CH_3-pyrimidine. The first three compounds were proved to be three to eight times higher than the potency of hydroxymethylpyrimidine. The other three compounds of 6-substituted 5-C_2H_5 (or-C_3H_7) -pyrimidine showed no growth inhibition.

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Six 2-methyl-4-amino- or 2-ethyl-4-amino-pyrimidine compounds having -CH_2OH, -CH_2Br or -CH_3 at 5C were added to modified Atkin's media (thiamine- and B_6-free) in concentration of 10^<-9> - 10^<-2> M, and their effect on the growth of Saccharomyces carlsbergensis 4228 was investigated. 5-CH_3-compounds showed gradual growth-stimulation according to increasing concentration, while 5-CH_2OH- or 5-CH_2Br-compounds showed a tendency of growth-inhibition inversely proportional to the concentration over 10^<-8> or 10^<-7> M. In addition to any pyrimidine compound in concentration to stimulate the growth, thiamine was able to inhibit the growth, while pyridoxal and any pyrimidine compound in concentration to inhibit the growth showed antagonistic recovery of the growth.

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Growth of Sacch. carlsbergensis in varied age of preincubation was estimated on shaking culture of the broth containing thiamine or pyridoxine with heavy inoculum such as 19-22 mg% (dry weight of cells to the broth) . Growth response of the yeast to thiamine varied with age of inoculum : Growth of cells from exponential growth phase was stimulated by thiamine but it from apparent stationary phase was inhibited by thiamine. Maximum growth inhibition was shown at concentrations of thiamine above 500μg%. Growth of the yeast was always stimulated by pyridoxine regardless of age of the inoculum, and the fastest growth of the yeast was observed by addition of pyridoxine 2μg% and thiamine 500μg%.

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Relation between vitamin B_6 content of Sacch. carlsbergensis and its growth inhibition by thiamine on shaking culture was investigated. B_6 content of growing cells in the broth with thiamine was found to be always lower than that in the broth without thiamine. B_6 content of the inoculum and its increase rate according to the growth were important to regulate growth of the yeast in the broth with thiamine. Growth inhibition by thiamine was observed only when inoculum (low B_6 content) from the apparent stationary phase was used. Increase of B_6 content was started just before the beginning of growth and B_6 content in cells was the highest in exponential growth phase and the addition of thiamine reduced the increase of B_6 content. Similar decrease of B_6 content by thiamine was observed in thiamine-adapted Sacch. carlsbergensis and two strains of Sacch. cerevisiae of which the growth was not inhibited by thiamine. However, B_6 contents of inoculum and growing cells were the lowest in Sacch. carlsbergensis. Growth retardation of Sacch. carlsbergensis by thiamine is reasonably explained by the disturbance of biosynthesis of vitamin B_6 in the cells.

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