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Contribution of Inosinic Acid to the Activation of Diphosphothiamine in Liver-Pyruvate Oxidase System1

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Contribution of Inosinic Acid to the Activation of Diphosphothiamine in Liver-Pyruvate Oxidase System1 THEJOURNAL OFVITAMINOLOGY 7, 271-275 (1961) CONTRIBUTION OF INOSINIC ACID TO THE ACTIVATION OF DIPHOSPHOTHIAMINE IN LIVER-PYRUVATE OXIDASE SYSTEM1 AKIOMAEKAWA, JUNZO HAYASHI, TAKEICHI OSHIMA, TAKAO SUZUKI ANDYOSHIKAZU SAHASHI Laboratoryof Biochemistryand Nutrition,Department of Agricultural Chemistry,Tokyo Agricultural University, Setagaya, Chitose, Tokyo (ReceivedSeptember 8, 1961) It is well known that the enzymatic formation of diphosphothiamine (DPT) takes place with adenosine triphosphate (ATP) as a phosphorylating agent. K. Lohmann and P. Schuster (1) first found that the extracts of the brain and other tissues could serve as the sources of the specific transphosphorylating enzymes. F. Leuthardt and H. Nielsen (2) repeated the experiments and demonstrated the conversion of thia mine to DPT in many animal tissues and microorganisms with participation of ATP. K. H. Kiessling (3) observed yeast cells to convert thiamine to mono- and tripho sphothiamine, neither of which alone had cocarboxylase activity However, Rossi Fanelli (4) showed the pure triphosphothiamine isolated from yeast to be able to activate rat-liver pyruvate oxidase system similarly to DPT. N. Shimazono (5) de monstrated more enzymatic formation of DPT by yeast enzyme at certain pH levels when inosine or guanosine-triphosphate was added in place of ATP. He also Liver 4g cold 0.25M sucrose (9 parts) was added found the latter triphosphates to be under stirring replaceable by inosine-monophosphate Homogenate (IMP) plus ATP or guanosine-monophos centrifuged at 600•~g for 10min. Supernatant phate plus ATP. 30ml of 0.04M MgCl2 solution was added No further investigation on the acti and centrifuged at 2200•~g for 5min. Precipitate vation of DPT in pyruvate-oxidase sys 30ml of cold 0.04M MgCl2 solution was added tem seems to have appeared. The au and centrifuged at 2200•~g for 5min. thors attempted to elucidate the existence Precipitate 30ml of cold 0.04M MgCl2 solution was of an additive effect of inosinic acid on added and centrifuged as above. the activation of DPT in liver-pyruvate Precipitate oxidase system. The activity increase cold 0.04M MgCl2 solution was added. of DPT in liver-pyruvate oxidase system Washed liver homogenate, 28mla a The content of nitrogen determined by was confirmed in vitro by addition of micro-Kjeldahl method was found to be IMP or ATP. Further, feeding experi 1 to 2mg/ml. ments were performed with thiamine FIG. 1 Preparation of Washed Liver deficient rats and the response was tested Homogenate 1 Biochemical Studies on Inosinic Acid. III. Partly reported at the Tokyo Meeting of Agricultural Chemical Society, April 22, 1961. 271 272 MAEKAWA, HAYASHI, OSHIMA, SUZUKI AND SAHASHI 1961 with oral administration of IMP. The growth curves did not indicate a positive effect as seen Fig. 1. After 28 day-feeding with thiamine-deficient diet, the animals were killed, and the liver homogenate was tested for the increase of liver-pyruvate oxidase activity after addition of IMP. The marked increase was recognized in the animals receiving thiamine and IMP orally, whereas the activity was low after addi tion of thiamine alone. These findings seem to suggest that IMP may contribute to the activation of DPT in liver-pyruvate oxidase system. Further investigations for the mechanism are now in progress. EXPERIMENTAL 1. Animals (a) The albino rats weighing ca. 100g fed on an ordinary ration were used. (b) Young rats weighing ca. 50g were used. The animals were divided at random into two groups, one of which was fed on a completely thiamine-deficient diet . The other group receiving orally 25ƒÊg of the vitamin per head daily served as a control. The thiamine-deficient diet was composed of, in g, granular sucrose, 56; purified casein, 20; soy bean oil plus cod-liver oil (10:1), 10; and McCollum salt mixture, No. 185, 4. The vitamin mixture, 0.05g, was orally supplemented per head daily . The vitamin mixture (1ml) consisted of, in mg, riboflavin, 0.5; pyridoxine, 0.5; niacin, 2; calcium pantothenate, 2; p-aminobenzoic acid, 10; inositol, 10; choline chloride, 100; folic acid, 2 ; and vitamin B12, 0.01. 2. Preparation of Washed Liver Homogenate The animals were killed, and 4g of the fresh liver was rapidly homogenized after adding 9 parts of cold 0.25M sucrose solution. It was further washed with 0.04M MgCl2 according to Wright (6) as shown Fig. 1. TABLE I Reaction System for Pyruvate Oxidation in Rat Liver Homogenate Vol. 7 ACTIVATION OF DIPHOSPHOTHIAMINE 273 3. Activity Tests for Pyruvate Oxidase in Washed Liver Homogenate Activity test for pyruvate oxidase in the washed liver homogenate was carried out with the modified Wright's pyruvate oxidation system as shown in Table I. The uptake of O2 was estimated using the Warburg apparatus . RESULTS 1. Effect of Thiamine and IMP on the Activation of Pyruvate Oxidase System in Washed Liver Homogenate The rats weighing ca. 100g fed on an ordinary ration were used . First, the additive effect in vitro of thiamine and IMP on the activation of pyruvate oxidase system in the washed liver homogenate was tested . It was found that the uptake of O2 was markedly low when thiamine and IMP were added . (Fig. 2). 2. Additive Effect of IMP on the Activation of Diphosphothiamine in Liver Py ruvate Oxidase System Further experiments were carefully repeated for the confirmation of the addi tive effect of IMP on the activation of liver-pyruvate oxidase system . The results indicated that IMP might contribute to the activation of DPT in liver pyruvate oxidase system, whereas it was rather low when DPT alone was added . (Fig. 3). Comparative test was made to know whether the additive effect of ATP on the activation of pure DPT will be seen in the enzyme system with the results shown in Fig. 3. The test for the additive response of ADP on the same enzyme system showed that the activity was quite lower than after addition of ATP or IMP . (Fig. 4). FIG. 2 Effect of IMP on the Activation Fio. 3 Effect of IMP on the Activation of Liver-Pyruvate Oxidase System of DPT 3. Feeding Experiments of Inosinic Acid in Thiamine-Deficient Rats In the first experiment, the growth curves of thiamine-deficient rats receiving orally inasinic acid were obtained. The single administration of IMP had no growth- 274 MAEKAWA, HAYASHI, OSHIMA, SUZUKI AND SAHASHI 1961 FIG. 4 Effect of ADP on Liver-Pyruvate-Oxidase Activity promoting effect, but administering both thiamine and IMP showed a marked effect. (Fig. 5). In the second experiment, the effect of administering IMP orally on the formation of liver-pyruvate oxidase in thiamine-deficient animals was studied. The pyruvate-oxidase activity in the washed liver homogenate prepared from thiamine-deficient rats receiving IMP was tested. After 28 day-feeding on a thiamine-deficient diet, the animals were killed and the liver was rapidly homo genized as given in Fig. 1. Marked FIG. 5 Growth Curves of Thiamine-De increase of the enzyme activity was re ficient Rats Receiving Thiamine Plus IMP A, thiamine-deficient; B, IMP (1mg) injected; cognized in the rats receiving thiamine C, thiamine (25ƒÊg) orally given; D, thiamine plus IMP, whereas scarcely any response (25ƒÊg) plus IMP (1mg) orally given; E, thia was seen in the animals receiving IMP mine (25ƒÊg) orally given and IMP (1mg) injected. alone by injection (Table II). Vol. 7 ACTIVATION OF DIPHOSPHOTHIAMINE 275 TABLE II Pyruvate Oxidase Activities Expressed as Qo2 (N) in Washed Liver Homogenate of Thiamine-Deficient Rats Receiving Inosinic Acid SUMMARY 1. Additive effect of inosinic acid on the activation of diphosphothiamine in liver pyruvate oxidase system was studied, and the increase in enzyme activity was con firmed by the addition of inosinic acid or adenosine triphosphate in vitro . 2. Further, feeding experiments were carried out with thiamine-deficient rats , and the response was studied after oral administration of inosinic acid . No effect of it on the growth curve was seen, but a marked increase of liver-pyruvate oxidase system was recognized after oral administration of thiamine plus inosinic acid . 3. The results seem to suggest that inosinic acid may contribute to the activation of diphosphothiamine in liver-pyruvate oxidase system. ACKNOWLEDGMENT This work was supported in part by Vitamin B Research Committee and the expense of this research was also partly defrayed by a grant in aid from the Central Research Laboratory of Ajinomoto Co., Inc., to which the authors express their thanks . REFERENCES 1. Lohmann, K., and Schuster, P., Biochem . Z. 294, 188 (1937). 2. Leuhardt, F., and Nielsen, H., Helv. Chim. Acta 35, 1196 (1952). 3. Kiessling, K.H., Biochem. et Biophys . Acta 20, 293 (1956). 4. Rossi-Fanelli, Proc. International Symp. Enzyme Chem . (Tokyo & Kyoto) 119 (1957). 5. Shimazono, N., J. Biochem. 46, 963 (1959). 6. Wright, R. C., and Scott, E. M., J. Biol. Chem. 206, 725 (1954)..
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