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Isolation of adenosine diphosphate D-glucose from corn graiws
BIOCHEMICAL
Vol. 12, No. 3, 1963
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ISOLA!PION OF ADETOSlXE DIPEOSPEATE D-GLUCOSE FROMCOBB GEAIllS Eduardo Becondo: Marcel0 Dankercand I&s F. Leloir Inetituto de Inveetigaclonee Bioq&lcaa Vuudaoi~n CampomarW and Faoultad de Cienolas Exaetas y Baturalee, Obligado 2490, Buenos Aires (281, Argentlzm
Received
June
12,
1%3
Adenoeine dlphosphate D-gltaoose (ADP6) has been found to reaof about ten-fold faefer than uridlne dlphoephate D-glucose (UDPG) in the eyutheals of etaroh (Reoondo and Leloir, 1961). Subeequently some evidenoe was obtained indicating that ADPG IS a natural compound. Thus a epeoffio emyme which catalyzes ADPG formation from adenoeiue triphoephate and gluoose 1-phoephate was ieolated from wheat flour (Eepada, 1962). Furthermore Kauee and Kandler (1962) obeerved that after adminietering 14c02 to Chlorella a radioaotive compound which cochromatographed with synthetic ADPf3 could be deteoted. Beoently, a phoephorylase which aote preferentially on ADPG and catalyzes lte phoaphorolyeis to adenoelne diphoephate and gluooee l-phosphate ha8 been found in wheat germ (Dankert, Gon9alree and Beoondo, 1963). The present communioation report6 the isolation of ADPG from an alcoholic extraot of corn gralne. Five kilogrru~e of eweet corn graine in the milky eta&e were disintegrated in 10 liters of ethanol (95s) with a blendor and filtered. The extraot wa6 preoipitated with mercuric aoetate ae deacrlbed by Caputto, Delolr, Cardini and Paladini (1950). After deoompoelng the meroury salt8 with H2S the nuoleotldes were adm Fellows of the Coneejo yaclonal y Tkicas. 204
de Inveetigaciones
Cientifioas
Vol. 12, No. 3, 1963
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
and eluted with ethanol-ammonia-water (2% 0.50:75). Aliquote were then submitted to paper chromatography in ethanol-amonitn acetate of pH 7.5 (Paladin1 and Leloir,1952) (Whatman 17 paper). A band with the 8ame mobility of a synthetic ADPG was eluted and rechromatographed in ethanol-ammonium acetate of pH 3.8 (Paladini and Leloir, 1952). After a final chronstoa single band with the mobility of graphy in the pH 7.5 solvent, ADPG was isolated. About 8 )rroles of ADPG were obtained per kilogram of corn grain. The ultraviolet epectrum of the mmpound was identical to that of adeaoaine in acid, neutral or alkaline medium. !The subatauoe moved like ADPG during paper eleotrophoreeie in sodium carbonateeodium bicarbonate buffer of pH 9.2 and sodium phoephate buffer of pH 7.5. Chromatography in ethanol-ammonia-water (Paladini and Leloir, 1952) produced adenoeine monophosphate and a cyclic sugar phosphate a8 does synthetic ADPG. After aofd hydrolysis at pH 2 and 1000 for 10 minutes, the eubetanoe gave ademoelae diphoephate, Borne adeaoeiae monophoephate and traces of adeaiue, as wae shown by cbraaatography ia the neutral ethaaol-glmnoalum aoetate eolvent. Paper obroaafography of the hydrolysis produet in butanol-pyridine-water (deanea, Mae and Dialer, 1951) and paper eleotrophoreeie An potassium tetraborate followed by treatneat with the ellrer aitrate-sodium hydroxide reagent (Trevelyan, Procter and Harrieoa, 1950) revealed a large glucose @pot, and smaller on88 In the soaes oorresponding to galaotoee and manuo8e. Adea%ne wa8 the only ultraviolet absorbing product produced by hydrolysis of the ADPG fraatloa in 3 f HCl at 1000 dur%ag 1 hour, as judged by ohronatography in lsopropanol-HCl-water (170:41:39) (Uyatt,1951). The ratio adeaoslna-total pheaphate-reducing sugar8 expressed a8 gluooee was 1:1.9:1.04. Total phosphate ms determined by the Fiske and SubbaRow method (1925) sad reduolng power by the Somogyi (1945) and Ieleon (1944) methods. An easymatlo assay with gluooee oxidsae (Huggett and Rixoa, 1957) after acid hydrolyele showed that 60$ to 70$ of the resorbed
on charcoal
BIOCHEMICAL
Vol. 12, No. 3, 1963
BIOCHEMCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
during sugar was gluaose. The nucleoside diphoaphate mgar ua8 tested with the starch ayntheslsingl enzyme from beam (Leloir, Fekete and Cardini, 1961) and the orthophoephate adeuylyl tramferaae (Dankert, Goqalvee and Becondo, 1963) from wheat germ. The remalte are ehoun in Table I. Apparently the subatauoe ieolated i6 rurinly ADPG aontamiaated with other nueleotidee of elmilar atimoture. Further etudiee are being married out to alarify the nature of the contaminating nuoleotldes. TAB&B1
Enqme
StarUh eynthetaee Orthophoaphate adenylyl transferas Glucoee oxidaee
Product me88ured
ADPGinn corn (100 xyxoles)l
Synthetlo (100 ~Le)l
r)laole6
yuolem
45
90
50
66
40
57
Adenoeine dlphoaphate* Adenoelne dipho*phate2
Glucose lpho6phat.3 Glucose
%almlated from abaorbaney at 260 rnp. *DetermIned with pyruvate kinaee (Cabib and I&o&r, 1958). %Aeaeured with phoaphogluoomutaae-glueoee C-phosphate dehydrogeaase (Hunoh-Peterson, 1955).
ACKROVS The authors wieh to exprese their gratitude to Drs. Carlo8 Carding and Joaquin Eepada for their help and adrioe and to Roe1 & Co. Ltd. for collaboration in the large male step.
E.
vol. 12, No. $1963
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Thia inveetlgcrtion was supported in part by a research grant (HO G-3442) from the Rational Inetitutee of Health, U. S. Public Health Service, by the Rockefeller Foundation and by the Coneejo Aaoional de Inveetigaoionee Clentffioae y Pdcnieae (Argentiaa).
Cabib, E. and Lelolr, L. F., J. Biol. Chea. a, 259 (1958). caputto, B., Leloir, Ii. F., Cardlni, C. B. and Paladini, A. C., 6. Biol. Char. a, 333 (1950). Dankert, Y., Gon*lvee, I. B. J. and Beeondo, E., Bloohfr. et Blophya. Aota, in preae. 3577 (1962). -w-s J-, J. Biol. Char. m, Flake, C. H. and SubbaRow, I., J. Biol. Chem. 66, 375 (1925). Huggett, A. St. 0. and Rlxon, D. A., Blooha. J. 66, 12P (1957). Jeanes, A., Wise, C. S. and Dimler, B. J., dnal. Chew a, 415 (1951). Kau6e, H. and Xandler, O., 2. 19aturforeoh. u, 858 (1962). Leloir, II. F., Fekete, Y. A. IL de and Cardini, C. E., iT. Bfol. Chem. a, 636 (1961). Munch-Peteraen, A., Aota Chem. Stand. 2, 1523 (1955). Aelaon, R.,, J. Biol. Chem. w, 375 (1944). Paladini, A. C. and Leloir, L. F., Bloohem. J. a, 426 (1952). Reoondo, E. and Lelolr, L. F., Bloohem. Biophye. Reeearoh C-8. 9, 85 (1961). Somoggi, Y., J. Blol. Chem. 160, 61 (1945). Trevelyan, W. X., Procter, D. P. and Harrleoa, J. S., mature 166, 444 (1950). Wyatt, 0. R., Biochem. J. & 584 (1951).
207
Vol. 12, No. 3, 1963
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ISOLA!PION OF ADETOSlXE DIPEOSPEATE D-GLUCOSE FROMCOBB GEAIllS Eduardo Becondo: Marcel0 Dankercand I&s F. Leloir Inetituto de Inveetigaclonee Bioq&lcaa Vuudaoi~n CampomarW and Faoultad de Cienolas Exaetas y Baturalee, Obligado 2490, Buenos Aires (281, Argentlzm
Received
June
12,
1%3
Adenoeine dlphosphate D-gltaoose (ADP6) has been found to reaof about ten-fold faefer than uridlne dlphoephate D-glucose (UDPG) in the eyutheals of etaroh (Reoondo and Leloir, 1961). Subeequently some evidenoe was obtained indicating that ADPG IS a natural compound. Thus a epeoffio emyme which catalyzes ADPG formation from adenoeiue triphoephate and gluoose 1-phoephate was ieolated from wheat flour (Eepada, 1962). Furthermore Kauee and Kandler (1962) obeerved that after adminietering 14c02 to Chlorella a radioaotive compound which cochromatographed with synthetic ADPf3 could be deteoted. Beoently, a phoephorylase which aote preferentially on ADPG and catalyzes lte phoaphorolyeis to adenoelne diphoephate and gluooee l-phosphate ha8 been found in wheat germ (Dankert, Gon9alree and Beoondo, 1963). The present communioation report6 the isolation of ADPG from an alcoholic extraot of corn gralne. Five kilogrru~e of eweet corn graine in the milky eta&e were disintegrated in 10 liters of ethanol (95s) with a blendor and filtered. The extraot wa6 preoipitated with mercuric aoetate ae deacrlbed by Caputto, Delolr, Cardini and Paladini (1950). After deoompoelng the meroury salt8 with H2S the nuoleotldes were adm Fellows of the Coneejo yaclonal y Tkicas. 204
de Inveetigaciones
Cientifioas
Vol. 12, No. 3, 1963
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
and eluted with ethanol-ammonia-water (2% 0.50:75). Aliquote were then submitted to paper chromatography in ethanol-amonitn acetate of pH 7.5 (Paladin1 and Leloir,1952) (Whatman 17 paper). A band with the 8ame mobility of a synthetic ADPG was eluted and rechromatographed in ethanol-ammonium acetate of pH 3.8 (Paladini and Leloir, 1952). After a final chronstoa single band with the mobility of graphy in the pH 7.5 solvent, ADPG was isolated. About 8 )rroles of ADPG were obtained per kilogram of corn grain. The ultraviolet epectrum of the mmpound was identical to that of adeaoaine in acid, neutral or alkaline medium. !The subatauoe moved like ADPG during paper eleotrophoreeie in sodium carbonateeodium bicarbonate buffer of pH 9.2 and sodium phoephate buffer of pH 7.5. Chromatography in ethanol-ammonia-water (Paladini and Leloir, 1952) produced adenoeine monophosphate and a cyclic sugar phosphate a8 does synthetic ADPG. After aofd hydrolysis at pH 2 and 1000 for 10 minutes, the eubetanoe gave ademoelae diphoephate, Borne adeaoeiae monophoephate and traces of adeaiue, as wae shown by cbraaatography ia the neutral ethaaol-glmnoalum aoetate eolvent. Paper obroaafography of the hydrolysis produet in butanol-pyridine-water (deanea, Mae and Dialer, 1951) and paper eleotrophoreeie An potassium tetraborate followed by treatneat with the ellrer aitrate-sodium hydroxide reagent (Trevelyan, Procter and Harrieoa, 1950) revealed a large glucose @pot, and smaller on88 In the soaes oorresponding to galaotoee and manuo8e. Adea%ne wa8 the only ultraviolet absorbing product produced by hydrolysis of the ADPG fraatloa in 3 f HCl at 1000 dur%ag 1 hour, as judged by ohronatography in lsopropanol-HCl-water (170:41:39) (Uyatt,1951). The ratio adeaoslna-total pheaphate-reducing sugar8 expressed a8 gluooee was 1:1.9:1.04. Total phosphate ms determined by the Fiske and SubbaRow method (1925) sad reduolng power by the Somogyi (1945) and Ieleon (1944) methods. An easymatlo assay with gluooee oxidsae (Huggett and Rixoa, 1957) after acid hydrolyele showed that 60$ to 70$ of the resorbed
on charcoal
BIOCHEMICAL
Vol. 12, No. 3, 1963
BIOCHEMCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
during sugar was gluaose. The nucleoside diphoaphate mgar ua8 tested with the starch ayntheslsingl enzyme from beam (Leloir, Fekete and Cardini, 1961) and the orthophoephate adeuylyl tramferaae (Dankert, Goqalvee and Becondo, 1963) from wheat germ. The remalte are ehoun in Table I. Apparently the subatauoe ieolated i6 rurinly ADPG aontamiaated with other nueleotidee of elmilar atimoture. Further etudiee are being married out to alarify the nature of the contaminating nuoleotldes. TAB&B1
Enqme
StarUh eynthetaee Orthophoaphate adenylyl transferas Glucoee oxidaee
Product me88ured
ADPGinn corn (100 xyxoles)l
Synthetlo (100 ~Le)l
r)laole6
yuolem
45
90
50
66
40
57
Adenoeine dlphoaphate* Adenoelne dipho*phate2
Glucose lpho6phat.3 Glucose
%almlated from abaorbaney at 260 rnp. *DetermIned with pyruvate kinaee (Cabib and I&o&r, 1958). %Aeaeured with phoaphogluoomutaae-glueoee C-phosphate dehydrogeaase (Hunoh-Peterson, 1955).
ACKROVS The authors wieh to exprese their gratitude to Drs. Carlo8 Carding and Joaquin Eepada for their help and adrioe and to Roe1 & Co. Ltd. for collaboration in the large male step.
E.
vol. 12, No. $1963
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Thia inveetlgcrtion was supported in part by a research grant (HO G-3442) from the Rational Inetitutee of Health, U. S. Public Health Service, by the Rockefeller Foundation and by the Coneejo Aaoional de Inveetigaoionee Clentffioae y Pdcnieae (Argentiaa).
Cabib, E. and Lelolr, L. F., J. Biol. Chea. a, 259 (1958). caputto, B., Leloir, Ii. F., Cardlni, C. B. and Paladini, A. C., 6. Biol. Char. a, 333 (1950). Dankert, Y., Gon*lvee, I. B. J. and Beeondo, E., Bloohfr. et Blophya. Aota, in preae. 3577 (1962). -w-s J-, J. Biol. Char. m, Flake, C. H. and SubbaRow, I., J. Biol. Chem. 66, 375 (1925). Huggett, A. St. 0. and Rlxon, D. A., Blooha. J. 66, 12P (1957). Jeanes, A., Wise, C. S. and Dimler, B. J., dnal. Chew a, 415 (1951). Kau6e, H. and Xandler, O., 2. 19aturforeoh. u, 858 (1962). Leloir, II. F., Fekete, Y. A. IL de and Cardini, C. E., iT. Bfol. Chem. a, 636 (1961). Munch-Peteraen, A., Aota Chem. Stand. 2, 1523 (1955). Aelaon, R.,, J. Biol. Chem. w, 375 (1944). Paladini, A. C. and Leloir, L. F., Bloohem. J. a, 426 (1952). Reoondo, E. and Lelolr, L. F., Bloohem. Biophye. Reeearoh C-8. 9, 85 (1961). Somoggi, Y., J. Blol. Chem. 160, 61 (1945). Trevelyan, W. X., Procter, D. P. and Harrleoa, J. S., mature 166, 444 (1950). Wyatt, 0. R., Biochem. J. & 584 (1951).
207