A significant influence of dietary biotin has been observed in enhancing the blood, liver and fecal levels of unconjugated reduced folate cofactors in folate deficient rats. Total folate levels were, however, not influenced by dietary biotin. The observed decrease of liver folylconjugate synthetase activity in the presence of dietary biotin may plausibly explain the increased tissue levels of unconjugated folates in biotin fed rats.

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Experiments for investigating thiamine metabolism in magnesium-deficient rats were carried out, and the following results were obtained.
1. Thiamine concentration in the sciatic nerve, liver, heart, and kidneys of magnesium-deficient rats was lower than that in the magnesium-sufficient rats. A decrease in thiamine was not detected in the central nervous system of magnesium-deficient rats.
2. In subcellular fractions of the liver of magnesium-deficient rats, thiamine content was most markedly decreased in the mitochondrial fraction.
3. When thiamine-¹⁴C was administered to magnesium-deficient rats, radioactivity in 24-hour urine decreased, and the radioactivity in the blood, liver, kidneys, heart, and sciatic nerve increased as compared to magnesium-sufficient rats.
4. With either oral or parenteral administration of thiamine, thiamine content in the heart, liver, and kidneys decreased at the same level in magnesium-deficient rats.
A plausible mechanism is suggested by these results.

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The determination of nicotinamide adenine nucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) levels in the liver and urinary excretion of tryptophan niacin metabolites were made in rats fed a tryptophan-deficient diet.
Under experimental conditions excretion of N¹-methyl-2-pyridone-5-carboxiamide (pyridone) decreased by approximately one-third in the tryptophan-deficient group when compared with the control group. A rapid decrease in body weight was shown in the tryptophan-deficient group when compared with the control group. The level of NAD⁺ and NADP⁺ and total PN⁺ in the tryptophan- and niacin-deficient group was decreased significantly when compared with other deficient group (niacin- or nicotinamide-added group) and the control group except for NADP⁺. No statistical difference was seen on the addition of nicotinic acid or nicotinamide. But a slightly lower level of urinary nicotinic acid was observed in the group with nicotinamide added when compared with group with nicotinic acid added. Simultaneously, a high level of NAD⁺ was observed in the group with nicotinamide added. Thus, a difference in availability between nicotinic acid and nicotinamide might be expected.

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Experiments were carried out in regard to the effect of tryptophan and vitamin B₆ deficiency on tryptophan and niacin metabolites. Tryptophan-deficient rats promptly lost weight, and the administration of tryptophan to the diet resulted in a rapid gain in weight. Nitrogen loss was found in tryptophan-deficient rats, and nitrogen retention occurred when tryptophan was supplemented to the diet. There was no apparent effect of vitamin B₆ supplement on the excretion of nitrogen. The increase of urinary xanthurenic acid by vitamin B₆-deficient rats was seen when tryptophan was supplemented.
Vitamin B₆ had no effect on the excretion of niacin and N¹-methylnicotinamide (MNA) and N¹-methyl-2-pyridone-5-carboxiamide (pyridone).

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A considerably high activity of quinolinate phosphoribosyltransferase, an intermediary enzyme in the de novo NAD biosynthetic pathway, was found in a soluble fraction of the “Shiitake” mushroom extract. Highly purified enzyme (approximately 1, 000-fold, chromatography-cally pure) was extracted for the first time from mushrooms and its general properties were investigated. An apparent molecular weight of 1.6×10⁵ was estimated by the gel filtration method. The optimum pH for the reaction was 6.5. The reaction required a divalent cation in addition to quinolinic acid and PRPP. Michaelis constants for quinolinic acid, PRPP and Mg⁺⁺ were 1.1×10^-5 M, 2.3×10^-5M and 2.0×10^-4M, respectively. The reaction product was identified as nicotinic acid mononucleotide by KCN addition reaction and by paper partition chromatography.

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A highly purified preparation of quinolinate phosphoribosyltransferase was obtained from the “Shiitake” mushroom and some characteristics of this enzyme were investigated. Maximum activity was observed at an incubation temperature of 50°C. Monovalent cations generally had no effect, but the divalent and trivalent cations were more or less inhibitory. Fe²⁺, Ni²⁺, Zn²⁺, Al³⁺ and Fe³⁺ strongly inhibited the reaction and Cl^- and NO^3- were also inhibitory. The PRPP saturation curve of this enzyme was sigmoidal. PRPP may be an allosteric effector to this enzyme. Quinolinic acid analogues (pyridine di- and mono-carboxylic acids) exerted no influence on the reaction. Some nucleotides were inhibitory, especially ATP, which was the most potent inhibitor. SH reagents and albocycline did not affect the activity. Possible regulations of the de novo biosynthesis of NAD in the mushroom are discussed.

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During purification of D-erythrulose ruductase, the existence of a protecting factor which prevents the enzyme from cold inactivation was confirmed. The factor was purified by ion exchange and paper chromatography and was identified as NADP⁺ by paper chromatography, paper electrophoresis, determination of the components, and spectroscopic properties.

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Riboflavin synthetase catalyzes a conversion of two molecules of 6, 7-dimethyl-8-ribityllumazine to one molecule each of riboflavin and 4-ribitylamino-5-aminouracil. A riboflavinogenic Eremothecium ashbyii produces this enzyme only within an earlier stage of the growth phase of its fungal life cycle prior to an initiation of vitamin overproduction, and the enzyme persists to function throughout the cycle. The specific enzyme activity of the cell-free extract is high, 72 to 165 mμmoles of riboflavin formed per hour per mg of protein at 37°C, approximately 100 times higher than those of the vitamin-producing microbes examined to date.
The enzyme exhibited a broad plateau on its activity curve between pH of 5.7 and 7.0 with a maximum point at pH 6.5. The optimal activation with temperature occurred at 47°C, but the enzyme was completely inactivated at 60°C. The apparent activation energy was 11, 000 calories per mole between 42° and 20°C. The initial reaction velocity showed a second order dependency on the lumazine concentration in the range 1.5×10^-4 to 6.3×10^-6M, i, e., a sigmoidal kinetic response instead of a hyperbolic Michaelis-Menten behavior, suggesting that affinities of the two substrate binding sites present on the enzyme have similar orders of magnitude. A Hill plot of the velocity consists of two straight lines with a slope=2 at lumazine levels below 6.0×10^-5M and with a slope=1 at the levels beyond this value. When the lumazines were bound to the enzyme in a random order, Ka'+Kd' and Ka'Kd (or KaKd'), where Ka' and Kd' denote the dissociation constants of a ternary enzyme-substrate complex and Ka and Kd are the constants of the two binary complexes present, were calculated as 4.5×10^-5M and 2.7×10^-9M², respectively, while, when an ordered mechanism held, Kd and Ka' (or Ka and Kd') corresponded to the respective values of 6.0×10^-5 and 4.5×10^-5M. A reaction mechanism without a kinetically significant ternary complex was not the case for E. ashbyii enzyme.
A simplified method for purification of riboflavin synthetase from E. ashbyii is reported. The specific enzyme activity of the purified preparation was 6, 370mμmoles of the vitamin produced for one hour per mg of protein at 37°C.

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The initial velocity of xanthine oxidase reaction catalyzed by milk xanthine oxidase is determined over the range of substrate concentrations from 1 to 0.005mM by following the consumption of molecular oxygen by a polarographic method using a Clark oxygen electrode. The Michaelis constant determined is 1.3×10^-5M. The activity is blocked by the substrate levels higher than 40μM. The constant for the substrate inhibition is calculated to be 1.3×10^-3M. The strong inhibition of the enzyme by 2-amino-4-hydroxy-6-formylpteridine develops with time: the constant for dissociation of the enzyme-inhibitor complex, K_i, is determined to be 8.0×10^-8M when the enzyme is not treated with the inhibitor prior to the start of the reaction by addition of the substrate, but 7.0×10^-9M when pretreatment for 10min is made. This potency, however, is markedly decreased by the reduction of a 7, 8-double bond or a formyl group at position 6 of the pyrazine ring of the antagonist. 2-Amino-4-hydroxy-6-formyl-7, 8-dihydropteridine and 2-amino-4-hydroxy-6-hydroxymethylpteridine inhibit the enzyme activity with the respective Ki values of 2.5×10^-5M and 10^-5M by competing with the substrate at the active center on the enzyme. These findings suggest that the strong affinity of the formylpteridine for the enzyme correlates profoundly with an oxidized structure of the pyrazine ring substituted with a formyl group at the position 6.

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