A new process has been described for the preparation of CoA from pantothenic acid (PaA), cysteine, and adenine, adenosine or AMP by yeasts and bacteria. In yeasts, about 70〜80% of added AMP was phosphorylated to ATP but only a small amount of CoA (20〜200μg/ml) was accumulated. PaA, cysteine and ATP, when added to the cell suspension of Brevibacterium ammoniagenes, or PaA, cysteine, and adenine, adenosine or AMP added to its cultures, gave CoA in high yields (2〜5mg/ml). The product was obtained by using Duolite S-30,charcoal, and Dowex 1×2. This method is simple and rapid, and requires no special equipment. The yield of CoA from culture broth was 34% and the purity of CoA was 97%. PaA kinase was purified from cell extracts of Br. ammoniagenes and some properties of the enzyme were investigated. PaA kinase was inhibited by CoA and it was suggested that the enzyme regulated CoA levels in the cells.

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Dephosphorylation of dihydroneopterin triphosphate (I) formed from GTP by GTP cyclohydrolase has remained as an unknown step in dihydrofolate (II) biosynthesis. The present author and Brown have found in Escherichia coli at least two hydrolytic enzymes : one, named as dihydroneopterin triphosphate pyrophosphohydrolase, catalyzes the removal of PPi specifically from (I) to give dihydroneopterin monophosphate (III) as the product, and the other catalyzes the hydrolysis of (III) to dihydroneopterin (IV). 2-Amino-4-hydroxy-6-carboxydihydropteridine (V), but not 2-amino-4-hydroxy-6-carboxypteridine (VI), inhibits enzymatic synthesis of (II) from GMP by specific attacking an enzyme reaction catalyzed by 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine (VII) pyrophosphokinase. Also, (V) inhibits growth of E. coli, while (VI) does not affect the growth, demonstrating that (V) blocks the growth by antagonizing utilization of (VII) for (II) synthesis. The growth inhibition is reversed by 2-amino-4-hydroxy-6-formyldihydropteridine (VIII). But, when (V) is absent, (VIII) blocks the growth with an inhibitory power about equal to (V). The mechanism whereby this contradictory effect of (VIII) on the growth happens, and the biological significance of this phenomenon has been explored by following metabolism of (VIII) and 2-amino-4-hydroxy-6-formylpteridine (IX). Three possible directions of (IX) metabolism in vivo are estimated by testing the inhibitory powers on xanthine oxidase of various pteridines related to (IX) in their structures : the 1st conversion is to (VI), the 2nd to 2-amino-4-hydroxy-6-hydroxymethylpteridine (X), and the 3rd to (VIII). The most potent inhibitory action of (IX) is extremely reduced by these conversion, which are found in E. coli. Dihydropteroate (XI), a precursor of (II), is enzymatically formed from (VIII) and (IX) via (VII), since these transformations are inhibited strongly by (V). The enzymatic conversion of (VIII) to (VII) is catalyzed by a specific reductase dependent on NADPH, and the conversion of (IX) to (VIII) requires FAD and NADH as the cofactors. The transformation of (IX) to (VII) via (X) is not plausible, since the enzyme system is not found to reduce the pyrazine ring of (X). (VI) is produced from (IX) by a NAD^+-specific aldhyde dehydrogenase, but not by xanthine oxidase. The synthetic pathway of (II) from (IX) via (VIII) is characterized as a "salvage route" for formation of (II), which could function dominantly under reduction of folate coenzymes levels by blockage of the main biosynthetic pathway from GTP, resulting in reversal by (VIII) of E. coli growth inhibition produced by (V). Mathis and Brown (1970) have found the strong inhibition by (VIII) of dihydroneopterin aldolase in the biosynthetic pathway of (II). If (VIII) is produced as the catabolic product from the folate coenzymes, this inhibition may have a nature of "feed-back inhibition", whereby the physiological levels of folate coenzymes could be maintained in the cells, and excess addition of (VIII) results in inhibition of E. coli growth.

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The biological activities of tocopherol related compounds which have different isoprene chain ; dl-α-tocopherol (1), 2,5,7,8-tetramethyl-2-(4', 8'-dimethylnonyl)-6-hydroxychroman (2), 2,5,7,8-tetramethyl-2-(4'-methylpenthyl)-6-hydroxychroman (3) and 2,2,5,7,8-pentamethyl-6-hydroxychroman (4), were studied by the hemolysis test using dialuric acid. In vivo test, the activity of (2) was found to be 3〜5% of (1), while (3) and (4) had no activities in 20 mg doses. On the other hands, the activities of (2), (3) and (4) were much higher than that of (1) in vitro test; those were 850,660 and 400% of that of (1) respectively. When the red blood cells treated with these compounds were washed, however, the activities of these compounds except (1) reduced less than half of that before wash. The decreasing ratio of activities of (1), (2), (3) and (4) by washing were 0,50,60 and 90%, respectively.

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The diazotization product from 5-chloroaniline 2,4-disulfonyl chloride was found to be suitable and satisfactorily applicable as a color-producing reagent to an automated colorimetric analysis of pyridoxine (PIN), pyridoxal (PAL) and pyridoxamine (PAM). Optimal conditions for coupling and color development determined and the characteristics of the reaction revealed were as follows : (1) Respective product from PIN, PAM and 4-deoxypyridoxine (DOP) after coupling with the diazotized reagent showed an absorption maximum at 467 nm, and that from PAL at 450 nm. (2) The coupling reaction proceeded fairly rapidly and the color developed was significantly stable when the reaction medium was adjusted to pH 5.4 to 6.0. (3) The coupling reaction with either one of PIN, PAM, PAL and DOP completed within 2 min after mixing when the reaction mixture was adjusted to pH 5.4 and kept at 55℃. (4) Absorbancy of the mixture at absorption maxima was proportional to the amount of vitamin B_6 complex within the range of up to 12.5μg per ml. (5) Coupling capacity of the diazotized reagent with the vitamins was found unchanged after storage for 24 h in a refrigerator. The reagent could be thus stored with perfect impunity for at least 24 h at a refrigerator temperature.

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