Changes in specific activity of total phospholipid and phosphatidylcholine, phospha-tidylethanolamine fractions in the rat's liver impaired experimentally by intraperitoneal administration of CCl_4 or ethionine using ^<32>P and effects of riboflavin or butyrate adminstration in different method on the phospholipid turnover were investigated. Ratio in liver weight to body weight of rat increased by experimental liver impairment and it was inhibited by oral administration of riboflavin or its butyrate. The amounts and specific activity of total phospholipid in the liver were found to be decreased as compared with that of control and these tendencies were prevented by riboflavin or its butyrate regardless of the method of administration. Single administration of ethionine or CCl_4 decreased phosphatidylcholine and increased phosphatidylethanolamine fraction but these variations were reduced by riboflavin administration. Furthermore, effects of continued riboflavin administration before and after administration of CCl_4 or ethionine were found to be largest on the turnover of phospholipid in impaired rat liver. It is suggested that CCl_4 or ethionine administration diminishes markedly the synthesis of phospholipid in the liver especially phosphatidylcholine fraction characteristically and riboflavin administration promotes the turnover of phospholipid in the rat's treated with CCl_4 or ethionine.

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For the detection of vitamin D_3-binding protein in rat intestinal mucosa, 2.0ml of mucosal supernatant (105,000×g, 60 min, protein 10mg) was incubated with 15 I.U. (0.97mμmoles, 15×10^4 cpm) of vitamin D_3-1,2-^3H (356mci/mM) in 2℃ for 20 min (total volume 3.0ml, pH 7.0). In the gel filtration of Sephadex G-200 (1.4×55cm), four protein peaks (1〜4) were observed in mucosal cytoplasma, and peak 1 fraction was bound with vitamin D_3-^3H. This peak 1 fraction was disappeared with pretreatment of mucosal supernatant by pronase, and somewhat modified by the RNase and heat treatment (50℃, 5min). DNase did not affect to the vitamin D_3-binding. Peak 1 fraction provided with single band on the polyacrylamide gel disc electrophoresis, and its molecular weight was calculated as about 350,000 (ca. 13S) by the sucrose density gradient centrifugaton. Vitamin D_3-binding protein in an intestinal mucosal cytoplasma was different from that of plasma with behaviours observed by the disc electrophoresis and sucrose density gradient centrifugation. The vitamin D_3 binding was not affected with equal amount of cholesterol, 17β-estradiol and testosterone.

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NTS cell-line are of a pure colonal clone separated from mass culture cells of newborn rat cerebellum which were maintaining in continuous cultivation since 54 months. Biochemical analysis of NTS cell-line revealed some difference in the quantitative ratio among free amino acids. Immunological tests with the serum antibodies from the sensitized rabbit disclosed that NTS cells have the similar protein components as the rat brain tissue. This cell-line was cultured in such a way that this appeared to be dependent upon vitamin B_<12>. The growth curve of this cell-line was dropped in the methionine- and choline-defficient medium, but this growth was enhanced only when the vitamin B_<12> was added in such defficient medium. The terminal reaction in methionine biosynthesis involves the transfer of a methyl group from 5-methyltetrahy-drofolate to homocysteine. The levels of 5-methylterahydrofolate-homocysteine transmethylase activities were elevated some 10-30 fold on NTS cells in the presence of methyl-B_<12> and homocysteine substituted for methionine and choline in the medium. Elevated enzyme levels were also obseved in primary culture of rat cerebellum maintained neurons and neuroglia cells in above medium. It is assumed above results that the methionine biosynthesis in the nervous tissue cells is required directly the vitamin B_<12>.

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It was found that riboflavin-β-galactosiae was formed from lactose and riboflavin by crystalline β-galactosidase from Escherichia coli.

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Tryptophan oxygenase was become to be activated fully by low concentration of ascorbic acid after catalase was eliminated from tryptophan oxygenase fraction. Furthermore, tryptophan oxygenase free from catalase was inhibited by the addition of high concentration of ascorbic acid. The activation was dependent upon the increase of V_<max>. On the other hand, the inhibition of tryptophan oxygenase by the addition of high concentration of ascorbic acid showed competitive type. This mechanism would be dependent upon the coordination of ascorbic acid to heme-iron, because ascorbic acid could coordinate with iron ion and hematin and o-phenanthroline, a chelator as Fe^<2+>, could competitively inhibited tryptophan oxygenase. Interestingly, ascorbic acid and o-phenanthroline could not inhibit tryptophan oxygenase to which catalase was added. This fact suggests that tryptophan oxygenase forms a complex enzyme with catalase.

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A temperature-sensitive thiazoleless mutant, E. coli KG 801,was isolated from E. coli K12 after treatment with N-methyl-N'-nitro-N-nitrosoguanidine. The mutant grew in minimal medium with glucose or glycerol at 30℃, whereas it failed to grow with glucose at 37℃ and required thiazole for its growth. Among various amino acids tested, glycine was found to be able to replace thiazole for the growth of the mutant in the presence of glucose at 37℃. Glyoxylic acid and trimethyleneglycol were somwhat effective on the growth. On the other hand the growth of the mutant was inhibited when glucose, fructose or glyceraldehyde was added to a culture growing in the presence of glycerol at 37℃.

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Thiamine production by growing cells of a temperature-sensitive thiazoleless mutant derived from E. coli K12,KG 801,was about a half that of its parent. Glycine, which was found to be able to replace thiazole for the growth of this mutant, stimulated the production of thiamine in the presence of a excess of hydroxymethylpyrimidine by washed cell suspension of the mutant, which is presumed to depend on the synthesis of thiazole in the mutant. A possibility of glycine as precursor in the biosynthesis of thiazole in E. coli was discussed.

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The enzymatic reduction of disulfide type thiamine derivatives to thiamine was investigated. In the homogenates of rat tissues except blood, there exist an enzyme which catalyzes the thiol-disulfide exchange reaction between reduced glutathione and disulfide type thiamine derivatives and the enzyme was named GSH-thiamine derives transhydrogenase. The enzyme was partially purified from rat liver and its substrate specificity was studied. This enzyme appeared to be different from some reported enzymes which catalyzed thiol-disulfide exchange reaction and seemed to play an important role for the reduction of disulfide type thiamine derives to thiamine in the body.

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In the previous paper, it was reported that the enzyme, which catalyzes the reduction of disulfide type thiamine derivatives by glutathione, occurs in rat liver supernatant. In this paper, it was shown that the reaction of S-acyl type thiamine derivatives and glutathione was accelerated by the similar enzyme in rat liver. The specific activity of this enzyme for S-acyl type thiamine derivatives was 20-60 times greater than that of thioesterase and it seemed that the conversion to thiazole type thiamine of S-acyl type thiamine derivatives in the body may be performed by this enzyme rather than thioesterase. On a SE-Sephadex column chromatography, the activity for the disulfide type thiamine derivatives was separated into four fractions, the three of which were active for S-acylthiamine. It was assumed that the two activities in the three fractions were due to the same enzyme, respectively,

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General natures of GSH-thiamine derivatives transhydrogenase were found to agree approximately with that of GSH-S-aryltransferase, which concerned with the mercapturic acid formation, and the identity of these two enzymes was investigated. GSH-S-aryltransferase in rat liver was separated into three fractions on SE-Sephadex column chromatography, and each fraction showed GSH-thiamine derivatives transhydrogenase activity. In these fractions, the activity for thiamine derivatives was inhibited with the addition of 3,4-dichloronitrobenzene, the substrate for GSH-S-aryltransferase. From these finding, it was assumed that the three active enzymes of five distinct fractions of GSH-thiamine derivatives transhydrogenase in rat liver were the same as GSH-S-aryltransferase.

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In applying with the new method for the determination of OBT (O-benzoyl thiamine), reported by the author, the urinary excretion of OBT has been examined. As the result of the experiment, it has become clear that a pretty amount of OBT was excreted in the urine, when OBT or its related compound were given to rabbit or rat.

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