¹⁴C-Labeled pyridoxal was synthesized from 2-¹⁴C-glycerol and 1, 3-¹⁴C-glycerol. Radioactivity of 2-¹⁴C-glycerol was incorporated in the pyridine moiety of pyridoxal, whereas that of 1, 3-¹⁴C-glycerol was incorporated in the pyridine moiety of pyridoxal and CO₂. From these results we suggested that 5, 6-positions of the carbon atoms of pyridoxal and that in hydroxymethyl attached to 5-C of pyridoxal came from glycerol and other carbons from leucine or its related compounds.

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In order to examine whether Pyridoxal phosphate could or could not be transferred into red blood cells in the form of a phosphate ester, the red blood cells were incubated with ¹⁴C-³²P-pyridoxal phosphate and the following results were obtained.
1. The radioactivity of the extracellular medium decreased with time, and the same amount of radioactivity was found in the cell fraction. The ratio of ¹⁴C:³²P in the medium or in the cells did not change throughout the reaction. 2. Most of the radioactivities present in the cells were recovered in the form of pyridoxal phosphate. The ratio of ¹⁴C:³²P of pyridoxal phosphate in the cells was the same as that in the medium. A slight amount of radioactivity of ¹⁴C of pyridoxal and ³²P of the inorganic phosphate were observed in the cells. 3. NaF and CH₂ICOOH inhibited the transfer of pyridoxal phosphate to the extent of 50%. At low temperature, the transfer of the radioactivity into the cell was remarkably depressed. 4. After hemolysis, the radioactivity in the red blood cells was liberated in the supernatant. By starch-gel electrophoresis and Sephadex-gel filtration, pyridoxal phosphate was found to be combined with hemoglobin. 5. The red blood cells containing pyridoxal phosphate was incubated with hemoglobin solution, but pyridoxal phosphate was not liberated from the cells. The pyridoxal phosphate was not hydrolyzed by alkaline phosphatase. These results suggested that pyridoxal phosphate was not merely adsorbed on the surface of cell membrane, but was transferred through the cell membrane. These observations lead to the conclusion that pyridoxal phosphate was transferred into the red blood cells without being hydrolyzed to pyridoxal and inorganic phosphate.

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The central problem inquired in this investigation is an attempt to elucidate how synthesis of the consecutive enzymes comprising the biosynthetic pathway directing to riboflavin is correlated with the life cycle of a flavinogenic fungus, Eremothecium ashbyii. The inhibition tests with three types of well known inhibitors to affect the fungal growth and riboflavinogenesis are employed for this purpose. The evidences presented as enumerated in the followings offers a powerful support for a suggestion that synthesis of the flavinogenic enzymes might be strictly controlled to progress within a confined period in the life cycle, which locates in an earlier stage of the fungal rapid growing phase. The life cycle can be roughly divided by three phases of the fungal growth, growth cessation and autolysis. The most striking attribute is an evidence that riboflavinogenesis can not be coupled with the growth: accumulation of the vitamin initiates just in coincidence with the growth cessation, and continues uninterrupted even at onset of the autolysis.
1. Riboflavin production is inhibited abruptly by 8-azaguanine within the narrow range of its low concentration. The flavinogenic inhibition can be paralleled with the growth injury of the fungi. The mycelial colonies under this situation are abnormal to develop just like lumps with hard paddings. Such a tendency is more remarkable in an agar slant culture containing the poison. This phenomenon is not a mutation, since transplantation of this fungi into a fresh slant devoid of the inhibitor can reverse both the growth and flavinogenic capacities identical with the normal strain. Effects of 8-azaguanine are tightly linked with the fungal life cycle. The agent is most effective as a potent inhibitor for riboflavinogenesis, when exists in an earlier stage of the growth phase. Addition of the drug after this stage causes only a slight effect on the vitamin formation. In order to explain these findings, a model is more reasonable to be proposed based on the accepted connotation, that synthesis of the enzymes organizing riboflavinogenic pathway is prevented by incorporating the abnormal base into the corresponding metabolic unstable ribonucleic acid strands such as messenger or transfer ribonucleic acids. This implies that the information from deoxyribonucleic acid molecules to the specific enzymes can be disturbed by the incorporation. The present evidences reveal that these genic processes of transcription and translation can proceed during the earlier period of the growth phase.
2. The model suggested by the previous experiments is ascertained by using an inhibitor specific to protein synthesis, chloramphenicol. A reciplocity is present between both inhibitions caused by the antibiotic of the growth and riboflavinogenesis. The inhibition of the vitamin production is intimately associated with the life cycle of the fungus. Induction of a strong blockage requires an essential existence of the inhibitor in the earlier stage of the growth phase. These two evidences can provide a rational suggestion that the enzymes implicated in riboflavinogenesis are synthesized during this period. This idea does not conflict with the data from 8-azaguanine functions.
3. 3-Amino-1, 2, 4-triazole prevents riboflavin yield without influencing the growth. The inhibitory power of the agent fails to be connected with the life cycle of the fungus, into some phases of which supplement with the antagonist raises a prompt inhibition of the vitamin production without a lag. These facts indicates that the triazole is restricted in its action: it can operate immediately on the intermediary step (s), but not on the synthesis of the enzymes, constructing riboflavinogenic pathway.

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By observing the effects of tocopherylacetate (Toc-ac) and tocopheryl glycolate (Toc-glyc) on the stored citrate-phosphate-dextrose (CPD) blood and the blood added with EDTA-K₃, the following results were obtained.
1. By adding 10, 20, and 50mg, respectively, of Toc-ac per 100ml or 20 and 50mg Toc-glyc per 100ml, a daily decline of osmotic resistance of erythrocytes in the CPD-stored blood could be suppressed to a certain extent.
The range of effects revealed that 50mg per 100ml was the greatest in effectiveness and that Toc-ac and Toc-glyc had almost the same effectiveness.
2. It was found that the preservative action of Toc-ac and Toc-glyc for the osmotic resistance of erythrocytes was not due to the action of the solvent in tocopherol.
3. With respect to the specimens added with 10 or 20mg per 100ml of Toc-ac, more erythrocytes with normal morphology were observed on the 7th day after the blood sampling, and in the former there was no difference from the one added with the solvent. That is, the residual rate of normal erythrocytes in both cases was somewhat higher, and the solvent itself seemed to have more or less preservative effectiveness in a comparatively short period. In the specimens added with 20 and 50mg of Toc-glyc per 100ml, the residual rate of erythrocytes with normal morphology showed the same high value on the 6th day after the blood sampling, when compared with the group without addition and the group added with the solvent.
4. Both in the CPD blood added with 10, 20 and 50mg per 100ml of Toc-ac, 20 and 50mg per 100ml, respectively, of Toc-glyc and in the EDTA blood added with 50mg per 100ml of Toc-ac, the decrease of the number of leucocytes after the blood sampling was suppressed to a certain extent. Moreover in the latter group, there was neither the relative decrease of granulocytes in the differential count of leucocytes nor the increase of lymphocytes observed for 6 hours after the blood sampling.
5. Tocopherol added group showed the tendency of a hourly decrease of thrombocytes depressed in the EDTA blood. Furthermore, the preservative effect on the morphology of thrombocytes in the same group was comparatively good.

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Calcium activated biosynthesis of phospholipids was studied with liver mitochondria, inner and outer membranes obtained from vitamin D deficient and vitamin D₃ fed rats. Calcium activated D, L-serine-3-¹⁴C incorporation into phospholipids was observed at 37°, but did not occur at 4°. This reached maximum during the first 30min, and the optimal calcium concentration was 12mM. The inhibitors, such as cyanide, 2, 4-dinitrophenol, arsenate and oligomycin had no effect on the serine incorporation. N-ethylmaleimide had inhibitory effect. ⁴⁵Ca more easily binds to mitochondrial membrane in any incubational temperature. Calcium activated D, L-serine-3-¹⁴C incorporation into phospholipids was further stimulated by administration of vitamin D₃ in rat liver mitochondria and outer membranes of mitochondria. The site of calcium activated biosynthesis of phospholipids was confined to outer membranes and the action of calcium was to expose its reaction center.
From these results, it was recognized that vitamin D₃ effect on calcium stimulated phospholipid synthesis was not on calcium uptake or binding, but more complexed reaction involved with phospholipid biosynthesis. It was also confirmed that vitamin D₃ consisted in mitochondrial membrane, and concerned with transferring information or phospholipid precursors in the mitochondrial phospholipid biosynthesis.

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Dihydropteroate synthetase was widely distributed in the higher plants tested, and a high enzyme activity was found in green leaves. In the early germination stage in pea seedlings, most of the enzyme activity existed in the cotyledon. With growth, a remarkable increase in enzyme activity in the root plus shoot was observed. The amount of Streptococcus faecalis R-active substances in root plus shoot rapidly increased during the germination of pea seedlings.
A large percentage of the enzyme activity was located in the mitochondrial fractions of both pea and soybean seedlings. Most of the S. faecalis R-active substances in pea seedlings existed in the soluble fraction from the cytoplasm.

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Dihydropteroate synthetase was purified from extracts of one day-old pea seedlings by chromatographic methods on DEAE-cellulose, DEAE-Sephadex A-50 and Sephadex G-200. The final enzyme preparation was homogeneous in chromatographic and ultracentrifugal analyses. The purified enzyme utilized 2-amino-4-hydroxy-6-hydroxy-methyldihydropteridine as substrate in the presence of both ATP and Mg²⁺, as did its pyrophosphate ester in the presence of Mg²⁺, to produce dihydropteroic acid by coupling with p-aminobenzoic acid. The Km value for pyrophosphorylmethyldihydropteridine was 1.14×10^-5M. The enzyme was specific for the amino group at the para-position in benzoic acid, since the reaction was not influenced by the addition of ortho- or meta-aminobenzoic acid. The Km value for p-aminobenzoic acid was 1.11×10^-6M.
Enzymatic formation of dihydropteroic acid was competitively inhibited by several sulfonamides. Inhibition levels of the various sulfonamides corresponded relatively with the growth inhibition levels by these compounds for Escherichia coli.

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Dihydropteroate synthetase purified from pea seedlings catalyzed the formation of dihydropteroic acid from 2-amino-4-hydroxy-6-hydroxymethyldi-hydropteridine and p-aminobenzoic acid in the presence of ATP and Mg²⁺. The reaction was stimulated by preincubation of the enzyme with hydroxymethyldihydropteridine, ATP and Mg²⁺. AT³²P was incorporated into the enzyme frac tion by incubation with ATP-γ-³²P, hydroxymethyldihydropteridine and Mg²⁺. Dihydropteroic acid was formed and pyrophosphate was liberated by the incubation with p-aminobenzoic acid from the AT³²P-incorporated enzyme solution. AMP was detected as the other product in the reaction.
The proposed systematic name of dihydropteroate synthetase is discussed.

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It was ascertained that riboflavin interacted with several indoles to form 1:1 complex in acid solution, and the formation of the complex was due to charge transfer forces judging from the absorption spectra. Charge transfer forces seemed to be very weak for the stabilization of the complexes. It was found that in relation to the solvent effect on stable complex formation, the extent of polarity as well as the acidity of the solvent was closely involved.

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1. Crystalline riboflavin-tryptophan complex was obtained from the acid solution (n-ButOH:12N HCI=7:3) in the existence of a small amount of CCI₄.
2. The crystalline complex was ascertained by X-ray powder diffraction method.
3. The crystalline complex consists of strictly equimolar amounts of riboflavin and tryptophan by spectrophotometric and colorimetric methods.
4. Spectral difference between the complex and two components was observed by infrared spectra.
5. The solubility of the complex for water increased 5 times as great as riboflavin alone.
6. The riboflavin moiety dissolved in acid and alkaline solutions as a complex showed considerable stability against photolysis.

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Tocopheronolactone showed an anti-inflammatory activity as effective as hydrocortisone acetate against dextran- and carrageenin-induced paw edemas.

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The purification and properties of vitamin D dependent calcium binding proteins were studied in rat intestinal mucosa. Two different vitamin D dependent calcium binding proteins were fractionated by the DEAE-cellulose column chromatography. Their properties were analyzed with the polyacrylamide gel disc electrophoresis and the sucrose density gradient ultracentrifugation. One of them is having a molecular weight about 24, 000 representing rapid migration on acrylamide gel electrophoresis and seems similar to the calcium binding protein obtained from chick intestinal mucosa in respect of its size and electrophoretic mobility. The other protein is a larger molecule (mol. wt. 145, 000) which is eluted in first fraction from DEAE cellulose chromatography. The smaller molecule of binding protein associates with one atom of calcium per one molecule of protein and the larger molecule would associate with more than 2 atoms of calcium.

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Influence of high cholesterol diet on riboflavin and choline requirements have been studied from the tissue levels of riboflavin and choline of albino rats fed high cholesterol diet. Albino rats fed high cholesterol diet for 12 weeks showed appreciable depletion of riboflavin and choline in liver and blood. When riboflavin and choline are given in massive doses along with high cholesterol diet, the depletion of these vitamins from these tissues was checked.

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Lowering incubation temperature resulted in a marked decreased transport of pyridoxine and pyridoxamine and a slightly decreased one of pyridoxal. Potassium fluoride inhibited the transport of pyridoxine, pyridoxamine and pyridoxal phosphate into the cells by 20, 15 and 60%, respectively, whereas the transport of pyridoxal was not inhibited. The conversion of pyridoxal to pyridoxal phosphate in the cells was also inhibited by the addition of potassium fluoride.
In the presence of deoxypyridoxine, the transport of pyridoxine and pyridoxamine into the cells was inhibited, while that of pyridoxal and pyridoxal phosphate was not. Further, pyridoxal phosphate concentrations in the cells after incubation with free forms of B₆ were decreased in the presence of deoxypyridoxine. Deoxypyridoxine phosphate did not inhibit the transport of various forms of B₆.
These evidences lead to the conclusion that the transport process of various forms of B₆ and their phosphorylation in erythrocytes are associated with the energy produced by intracellular glycolysis.

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Three successive experiments were conducted to evaluate the activity of a 5-hydroxybenzimidazole analogue of vitamin B₁₂ (B₁₂-FIII) in comparison with cyanocobalamin by chick growth assay. In all experiments, Single Comb White Leghorn chicks were used, bred from hens given all vegetable diet for 3-4 months. Chicks were divided into 4-6 lots at the end of the second week after hatching, and fed for three weeks with all vegetable, high protein diet supplemented with graded levels of cyanocobalamin or 3μg of B₁₂-FIII per 100g of basal diet. From the comparison of body weight gain during the experimental period, it may be concluded that the activity of B₁₂-FIII for chick growth is about 10% of that of cyanocobalamin.

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