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Enzymic phosphorylation of d -ribulose in guinea-pig liver
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SHORT COMMUNICATIONS SC 11006
Enzymic phosphorylation of D-ribulose in guinea-pig liver HOLLMANN et al. 1 reported the formation of D-ribulose from ribitol in animal tissues. The work from this laboratory ~ has shown the enzymic oxidative decarboxylation of n-gluconic acid to D-ribulose in guinea-pig-liver preparation. Although the existence of a phosphokinase for D-ribulose was reported by FROMM3 in Aerobacter aerogenes, little is known about the fate of D-ribulose in animal body. In this communication, the isolation of ATP-dependent D-ribulokinase from guinea-pig liver is described, together with the identification of D-ribulose 5-phosphate as the reaction product. D-Ribulose was prepared by decomposing the o-nitrophenylhydrazone according to the method of GLATTHAAR AND REICHSTEIN4. The 5-phosphate was prepared according to the method of HURWITZ et al. 5, using D-ribose 5-phosphate and spinach ribosephosphate isomerase (EC 5.3.I.6), and was isolated with a Dowex-I-formate column e. The enzyme was purified as follows. The supernatant fraction of guinea-pig liver KC1 homogenate was subjected to heat treatment (5o°, 5 min), fractionation with (NH~)2SO4 (0.2-0.35 satn.7), dialysis against o.o154 M phosphate buffer (pH 6.7), negative adsorption on carboxymethyl-cellulose (previously buffered with o.o154 M phosphate buffer; p H 6.7); precipitation at p H 6.0 (the pH of the solution was adjusted at first to about 5.0 with I N CHsCOOH and then gradually adjusted to 6.0 with 2 N NH4OH ), and again fractionation with (NH4)2SO 4 (0.2-0.35 satn.7). The last two steps were repeated twice. The final preparation had scarcely any ribosephosphate isomerase activity 8. The D-ribulokinase activity was assayed in the reaction mixture containing 8/,moles of MgClz, 4.5/,moles of ATP, 2.6/~moles of D-ribulose, 0.2 ml of the enzyme solution and I5.4/~moles of phosphate buffer (pH 7.5), in a total volume of I.O ml. Incubation was carried out for 15 rain at 37 °, and the reaction was stopped b y the addition of 2.0 ml of 5% ZnSO 4.7H~0 and 2.0 ml of 0.3 N Ba(OH)~. The mixture was centrifuged, and I.O ml of the supernatant was assayed by the orcinol reaction. The activity was measured by the decrease of D-ribulose. The final preparation showed a specific activity about 30 times that of the original supernatant fraction. Isolation of the reaction product was carried out as follows. A reaction mixture containing a large amount of u-ribulose and the enzyme preparation was incubated for IOO min at 37 °. The reaction was stopped with glacial acetic acid, and the solution was treated with acid-treated Norit to remove nucleotides. The mixture was filtered in the cold, and the filtrate was finally adjusted to p H 7.4-7.5 with Ba(OH)v The resulting precipitate was discarded after centrifugation, and to the clear supernatant were added about 4 vol. of ethanol. The mixture was allowed to stand overnight at 2 °, and the resulting precipitate was collected by centrifugation at 2 °, washed twice with 80% ethanol and dried over KOH. The aqueous solution of the dried material was treated with Amberlite IR-I2O H-form to decompose the barium salt and the eluate was lyophilized. Identification of the isolated reaction product was performed as follows. (I) Paper chromatography by using two solvent systems: (a) o.o8 M acetate buffer (pH 3.5)-95% ethanol (i : 4, v/v), and (b) isopropanol-trichloroacetic acidBiochim. Biophys. Acta, 64 (1962) 18o-181
SHORT COMMUNICATIONS
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water-28% aq. ammonia (75 mh 5 g: 25 ml: 0.3 ml). The chromatograms obtained showed the same RF values as those of the authentic 9-ribulose 5-phosphate. (2) A positive cysteine-carbazole reaction observed after the incubation of the reaction product and the reagent for 30 min at 37 °, with absorption maximum at 540 m/z. (3) The absorption spectrum of orcinol reaction, which was characteristic of that given by ribulose 5-phosphate (A540m~,/A6~o ma approx. 0.3). (4) The colour of the orcinol reaction after dephosphorylation of the reaction product with crude prostatic acid phosphatase resembled that given by ribulose, although it was not quite typical. An aliquot of the solution reacted positively for inorganic phosphate by using the Fiske and Subba Row reagent. From these various tests, it was concluded that the reaction product was o-ribulose 5-phosphate. When ATP was omitted from the standard reaction mixture, the orcinol reaction of the solution obtained after incubation showed the characteristic absorption spectrum of ribulose. This suggests that the direct phosphorylation of D-ribulose takes place in the standard reaction mixture without the intermediary formation of other sugars. Thanks are due to Dr. K. WAKABAYASHI, Dr. K. YAMADA, Dr. S. ISHIKAWA, Dr. Y. KAGAWAand Dr. Y. MANO in this Department, and Dr. T. HASHIMOTO in the Department of Physiological Chemistry and Nutrition for their kind help.
Department of Biochemistry, Faculty of Medicine, University of Tokyo, Tokyo (Japan)
TSUNEO KAMEYAMA NORIO SHIMAZONO
1 S. HOLLMANN AND O. TOUSTER, J. Biol. Chem., 225 (1957) 87. 2 y . KAGAWA, T. KAMEYAMA, Y. MANO AND N. SHIMAZONO, Biochim. Biophys. Acta, 44 (196o) 205. 8 H. J. FROM~t, J. Biol. Chem., 234 (1959) 3o97 . 4 C. GLATTHAAR AND T. REICHSTEIN, Helv. Chim. Acta, 18 (1935) 80. 5 j . HURWlTZ, A. WEISSBACH, B . L . HORECKER AND P. Z. SMYRNIOTIS, J. Biol. Chem., 218 (1956 ) 769. s B. L. HORECKER, in S. P. COLOWICK AND m. O. ]5~APLAN, Methods in Enzymology, Vol. 3, Academic Press, Inc., N e w York, 1957, p. t9o. T. B. OSBORNE AND E. STRAUSS, in E. ABDERHALDEN, Handbuch der biologischen Arbeitsmethoden, Teil 8, U r b a n & Schwarzenberg, Abt. I, 1922, p. 387 . a B. AXELROD, in S. P. COLOWICK AND N. O. I~APLAN, Methods in Enzvmology, Vol. i, Academic Press, I n c , New York, 1955, p. 363.
Received March 22nd, 1962 Biochim. Biophys. Acta, 64 (1962) i 8 o - i 8 i
SHORT COMMUNICATIONS SC 11006
Enzymic phosphorylation of D-ribulose in guinea-pig liver HOLLMANN et al. 1 reported the formation of D-ribulose from ribitol in animal tissues. The work from this laboratory ~ has shown the enzymic oxidative decarboxylation of n-gluconic acid to D-ribulose in guinea-pig-liver preparation. Although the existence of a phosphokinase for D-ribulose was reported by FROMM3 in Aerobacter aerogenes, little is known about the fate of D-ribulose in animal body. In this communication, the isolation of ATP-dependent D-ribulokinase from guinea-pig liver is described, together with the identification of D-ribulose 5-phosphate as the reaction product. D-Ribulose was prepared by decomposing the o-nitrophenylhydrazone according to the method of GLATTHAAR AND REICHSTEIN4. The 5-phosphate was prepared according to the method of HURWITZ et al. 5, using D-ribose 5-phosphate and spinach ribosephosphate isomerase (EC 5.3.I.6), and was isolated with a Dowex-I-formate column e. The enzyme was purified as follows. The supernatant fraction of guinea-pig liver KC1 homogenate was subjected to heat treatment (5o°, 5 min), fractionation with (NH~)2SO4 (0.2-0.35 satn.7), dialysis against o.o154 M phosphate buffer (pH 6.7), negative adsorption on carboxymethyl-cellulose (previously buffered with o.o154 M phosphate buffer; p H 6.7); precipitation at p H 6.0 (the pH of the solution was adjusted at first to about 5.0 with I N CHsCOOH and then gradually adjusted to 6.0 with 2 N NH4OH ), and again fractionation with (NH4)2SO 4 (0.2-0.35 satn.7). The last two steps were repeated twice. The final preparation had scarcely any ribosephosphate isomerase activity 8. The D-ribulokinase activity was assayed in the reaction mixture containing 8/,moles of MgClz, 4.5/,moles of ATP, 2.6/~moles of D-ribulose, 0.2 ml of the enzyme solution and I5.4/~moles of phosphate buffer (pH 7.5), in a total volume of I.O ml. Incubation was carried out for 15 rain at 37 °, and the reaction was stopped b y the addition of 2.0 ml of 5% ZnSO 4.7H~0 and 2.0 ml of 0.3 N Ba(OH)~. The mixture was centrifuged, and I.O ml of the supernatant was assayed by the orcinol reaction. The activity was measured by the decrease of D-ribulose. The final preparation showed a specific activity about 30 times that of the original supernatant fraction. Isolation of the reaction product was carried out as follows. A reaction mixture containing a large amount of u-ribulose and the enzyme preparation was incubated for IOO min at 37 °. The reaction was stopped with glacial acetic acid, and the solution was treated with acid-treated Norit to remove nucleotides. The mixture was filtered in the cold, and the filtrate was finally adjusted to p H 7.4-7.5 with Ba(OH)v The resulting precipitate was discarded after centrifugation, and to the clear supernatant were added about 4 vol. of ethanol. The mixture was allowed to stand overnight at 2 °, and the resulting precipitate was collected by centrifugation at 2 °, washed twice with 80% ethanol and dried over KOH. The aqueous solution of the dried material was treated with Amberlite IR-I2O H-form to decompose the barium salt and the eluate was lyophilized. Identification of the isolated reaction product was performed as follows. (I) Paper chromatography by using two solvent systems: (a) o.o8 M acetate buffer (pH 3.5)-95% ethanol (i : 4, v/v), and (b) isopropanol-trichloroacetic acidBiochim. Biophys. Acta, 64 (1962) 18o-181
SHORT COMMUNICATIONS
181
water-28% aq. ammonia (75 mh 5 g: 25 ml: 0.3 ml). The chromatograms obtained showed the same RF values as those of the authentic 9-ribulose 5-phosphate. (2) A positive cysteine-carbazole reaction observed after the incubation of the reaction product and the reagent for 30 min at 37 °, with absorption maximum at 540 m/z. (3) The absorption spectrum of orcinol reaction, which was characteristic of that given by ribulose 5-phosphate (A540m~,/A6~o ma approx. 0.3). (4) The colour of the orcinol reaction after dephosphorylation of the reaction product with crude prostatic acid phosphatase resembled that given by ribulose, although it was not quite typical. An aliquot of the solution reacted positively for inorganic phosphate by using the Fiske and Subba Row reagent. From these various tests, it was concluded that the reaction product was o-ribulose 5-phosphate. When ATP was omitted from the standard reaction mixture, the orcinol reaction of the solution obtained after incubation showed the characteristic absorption spectrum of ribulose. This suggests that the direct phosphorylation of D-ribulose takes place in the standard reaction mixture without the intermediary formation of other sugars. Thanks are due to Dr. K. WAKABAYASHI, Dr. K. YAMADA, Dr. S. ISHIKAWA, Dr. Y. KAGAWAand Dr. Y. MANO in this Department, and Dr. T. HASHIMOTO in the Department of Physiological Chemistry and Nutrition for their kind help.
Department of Biochemistry, Faculty of Medicine, University of Tokyo, Tokyo (Japan)
TSUNEO KAMEYAMA NORIO SHIMAZONO
1 S. HOLLMANN AND O. TOUSTER, J. Biol. Chem., 225 (1957) 87. 2 y . KAGAWA, T. KAMEYAMA, Y. MANO AND N. SHIMAZONO, Biochim. Biophys. Acta, 44 (196o) 205. 8 H. J. FROM~t, J. Biol. Chem., 234 (1959) 3o97 . 4 C. GLATTHAAR AND T. REICHSTEIN, Helv. Chim. Acta, 18 (1935) 80. 5 j . HURWlTZ, A. WEISSBACH, B . L . HORECKER AND P. Z. SMYRNIOTIS, J. Biol. Chem., 218 (1956 ) 769. s B. L. HORECKER, in S. P. COLOWICK AND m. O. ]5~APLAN, Methods in Enzymology, Vol. 3, Academic Press, Inc., N e w York, 1957, p. t9o. T. B. OSBORNE AND E. STRAUSS, in E. ABDERHALDEN, Handbuch der biologischen Arbeitsmethoden, Teil 8, U r b a n & Schwarzenberg, Abt. I, 1922, p. 387 . a B. AXELROD, in S. P. COLOWICK AND N. O. I~APLAN, Methods in Enzvmology, Vol. i, Academic Press, I n c , New York, 1955, p. 363.
Received March 22nd, 1962 Biochim. Biophys. Acta, 64 (1962) i 8 o - i 8 i