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Enzymatic synthesis of uridine diphosphate glucosaminuronic acid
SHORT COMMUNICATIONS
355
sc 83o07
Enzymic synthesis of uridine diphmphate 91ucosaminuronlcacid Several enzymes which catalyze reactions involving glucose can also be shown to act on glucosamine, although at lower rates. Glucosamine-6-P is formed from glucosamine in the presence of bexokinase (ATP:v-hexose 6-phosphotransferase, EC 2.7.Lx ) and ATP (see ref. x) ; glucosamine-x-P can be formed from glucosamine-6-P in the presence of phosphoglucomutase (D-glucose z,6-diphosphate: D-glucose-x-phosphate phosphotransferase, EC 2.7.5.x ) and Glc-I,6-P s (ref. 2) ; UDP-glucosamine can be formed from glucosamine-I-P and UTP in the presence of an enzyme which has not been distinguisbed from UDPG pyrophosphorylase (UTP:--D-glucose-x-phosphate uridyltransferase, EC 2.7.7.9)s,4; and UDP-galactosamine can be formed from UDPglucosamine in the presence of an enzyme which has not been separated from UDPG 4-epimerase (EC 5.x.3.2) 5. It was thought to be of interest to see if UDP-giucosaminuronic acid could be synthesized from UDP-glucosamine in the presence of NAD + and UDPG dehydrogenase (UDPG: NAD oxidoreductase, EC I.I.X.22) in a manner analogous to the above reactions. We wish to report this synthesis. UDP-glucosamine and UDP-Ex-14CJglucosamine were prepared as previously described 4. NAD +, UDPG, and UDPG dehydrogenase (calf liver) were purchased from Sigma Chemical Company. Glucosaminuronic acid was a generous gift of Dr. K. HEY~S. Results of a typical incubation of UDPG and UDP-glucosamine at room temperature in the presence of NAD + and UDPG dehydrogenase are shown in Fig. z. UDPG was completely oxidized to UDP-glucuronic acid as measured by NADH formation (assayed by absorption at 34o m~) e. When UDP-glucosamine was used as substrate there was a much slower reduction of NAD + and the reaction was not carried to completion. If more enzyme were added at this point, additional reduction of NAD + occurred, but the reaction still was not carried to completion. Larger amounts of UDP-glucosamine and UDP-[x-l*C]glucosamine were reacted with xooo-2ooo units of UDPG dehydrogenase in other experiments with similar results of from Io % to 30 % yield of the new nucleotide sugar.
0.~ p •Ipu 0.,1 E
a~ Z 0
~ 0.1 e
1
2 Time(h)
e
??
3
:
?
4
Fig. z. Incubation of UDPG and UDP-glucosamine with UDPG dehydrogenase and NAD+. The reaction mixture contained, in a volume of 2.5 ml, o.x M glycine (pH 8.7); 2.5/Jmoles NAD+; zoo units UDPG dehydrogenase; and either UDPG (O--O), or UDP-glucceamine ( 0 - - 0 ) , o.25 ~umole. N A D H formation at room temperature was ~ - ~ y e d by absorption at 34 o rap. Biockim. Biophy$. Acta, 83 (z964) 355-357
356
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UDP-[I-t4C]glucosarninuronic acid was isolated by logarithmic gradient elution of the reaction products from a I cm x 5 cm column of Dowex-i X8 (formate form, 2oo-4oo mesh). A mixing volume of 2oo ml of water and a reservoir containing 4 N formic acid was used. Fractions of 5 ml were collected. Two radioactive peaks with absorption at 260 mt~ were located. The first, at Fractions I8-23 was identified as UDP-glucosamine. The second, appearing at Fractions 44-51 was evaporated in vacu.o at room temperature and then chromatographed on Whatman No. I paper with ethanol-ammonium acetate (pH 7.8) 7. A single ultraviolet-absorbing spot which contained all the radioactivity was found with RVMp = o.95. The area of the paper conraining this spot was cut out, washed with methanol, and eluted with water. The nucleotide isolated had a typical uridine spectrum at neutral and alkaline pH. Analysis showed uridine:total phosphate = I:2.o 9. Only 3o-5o % of the nucleotide was hydrolyzed by boiling in I N HE1 for 3 h. Hydrolysis under more severe conditions was attempted, but was even less successful in yielding quantitative amounts of labile phosphate and hexosaminuronic acid, presumably due to other degradation of the nucleotide sugar. A hydrolysate of the radioactive nucleotide sugar (I N HCI for 2 h at Ioo °) was chromatographed on Dowex-5o according to the method of GARDELL8. Radioactivity was found in the fractions containing unhydrolyzed nucleotide sugar and in the same fractions that contained added carrier glucosaminuronic acid. There was no radioactivity in fractions containing carrier glucosamine or galactosamine. The fractions containing glucosaminuronic acid were evaporated i , vacuo at room temperature and chromatographed on Whatman No. x paper in n - b u t a n o l - p y r i d i n e water (6:4: 3, v/v). The glucosaminuronic acid was located °, and this spot contained all the radioactivity. The results of the separation of glucosaminuronic acid by column and paper chromatography agree with those of CRUMFrON et al. 1°. It was also thought to be of interest to attempt the N-acetylation of UDPglucosaminuronic acid. Excess acetic anhydride was added to a solution containing 0.2 #mole of UDP-[I-t4C]glucosaminuronic acid (Iooo counts/min) and enough Tris was added to the mixture to make it alkaline to phenolphthalein. After x5 min at room temperature, the nucleotides in the reaction mixture were adsorbed on charcoal, .,o ammoniacal alcohol, dried in vac~w, and chromatographed on eluted with 5o o/ Whatman No. t paper with ethanol-ammonium acetate (pH 7.8). A single ultravioletabsorbing spot which contained all the radioactivity was found with RVMP : LIO. The area of this spot was cut out, washed with methanol, and eluted with water. An aliquot of this eluate containing 2oo counts/min was chromatographed on a I cm ,: r5 cm column of Dowex-I X8 (formate form, 2oo-400 mesh). After first eluting the column with 5o ml of 4 N formic acid, logarithmic gradient elution was carried out with 2oo ml of 4 N formic acid in the mixing flask and 4 N formic acid-o.2 M ammonium formate in the reservoir flask. Fractions of 5 ml were collected. A single peak of radioactivity was found in Fractions 32-36. Carrier UDP-N-acetylglucosamine was eluted from this column at Fractions x2-i6, and carrier UDP-glucuronic acid was eluted at Fractions 34-39- The radioactive compound, presumed to be UDP-N-acetylglucosaminuronic acid, was not characterized further. The pathway of synthesis of UDP-glucosaminuronic acid described in this report may not be of physiological significance. However, glucosaminuronic acid has been isolated from bacterial polysaccharides n,l*, and it is probable that nucleotide sugars Biochim. Biophys..4eta, 83 (I964) 355 357
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containing glucosaminuronic acid or N-acetylglucosaminuronic acid are intermediates in the synthesis of these polysaccharides. This is Publication No. 372 of the Robert W. Lovett Memorial for the Study of Diseases Causing Deformities, Harvard Medical School, Massachusetts General Hospital. This investigation was supported by Research Grant A-3564-O of the National Institute of Arthritis and Metabolic Disease, National Institutes of Health and was carried out while J.S. was supported by the Charles King Trust of the Massachusetts General Hospital, and E . H . was supported b y a U.S. Public Health Service Medical Student Research Training Program summer fellowship through Harvard Medical School. The authors wish to thank Dr. S. M. KRANE for helpful advice and encouragement in all phases of this work. Department o f M M i c i n e , Harvard M M i c a l School, Massachusea~s General Hospital, Boston, Mass. (U.S.A.)
JEREMIAH E. SILBERT* EDWARD F. X. HUGHES
t D. H. BnowN, Biochim. Biophys. ,4c/a, 7 (I95 I) 487• s D. H. BROWN,f . Biol. Chem., 204 (1953) 877. s F. MALSV,G. F. MALE','AI~DH. A. LARDY,J. Am. Chem. So¢., 78 (x956) 5303• 4 j. E. SILBERTAND D. H. BROWN,Biochim. Biophys. Acta, 54 (x961) 59o. t F. MALgV AND G. F. MALBV, Biochim. Biophys. Acta, 3 x (x959) 577e j. L. ST~OMINGeR,E. S. MAXWELLAN]>H. M. KALCKAR,in S. P. COLOWICKANDN. O. KAPLAN, Methods in Er.:ymology, Vol. 3, Academic Press, New York, x957, p. 974. • A. C. PALADIN!ANDL. F. LELOIR,Biochem. f . , 5 ! (I952) 426. s S. GAnDELL,Acta Chem. Scand., 7 (I953) 207. t S. M. PAItTRIDGE, Biochem. f . , 42 (1948) 238. to M. J. CRUMPTON,Biochem.J., 72 (I959) 479. 11 A. R. WILLXAMSONAND S. ZAMENHOF, f . Biol. Chem., 238 (I963) 2255. ts T . H .
HASt:ELLANDS. HANI~SSlAN,Biochim. Biophys. Acta, 83 (I964) 35-
Received June 8th, z96 4 " Present address: V.A. Hospital, x5o S. Huntington Ave., Boston, Mass. (U.S.A.). Biochim. Biophys. Acta, 83 (x964) 355-357
sc 83ox 3
Heterogeneity of sialomucopolysaccharides prepared from whole rat brain The isolation of sialomucopolysaccharides from crude mitochondrial fractions prepared from dog brain has been reported from this laboratory'. Subsequent work s established that this substance is present in crude mitochondrial and microsomal fractions prepared from rat brain homogenates. Although the mitochondrial fraction contained the larger share of the total brain siaiomucopolysaccharide, the microsomal fraction had a higher concentration of this substance when expressed in terms of the amount of sialomucopolysaccharide present per mg of protein. The method previously used to isolate sialomucopolysacchaxide from crude subcellular particulate fractions prepared from rat brain s was applied to whole rat brains in the present work. After removal of the brain from the animal, it was homogenized in x9 volumes of chloroform-methanol (2:x, v/v) and centrifuged. The precipitate was dried and subsequently processed as reported earlier I. Briefly, this involved Bi~.llim. Biophys. Acta, 83 (z964) 357-360
355
sc 83o07
Enzymic synthesis of uridine diphmphate 91ucosaminuronlcacid Several enzymes which catalyze reactions involving glucose can also be shown to act on glucosamine, although at lower rates. Glucosamine-6-P is formed from glucosamine in the presence of bexokinase (ATP:v-hexose 6-phosphotransferase, EC 2.7.Lx ) and ATP (see ref. x) ; glucosamine-x-P can be formed from glucosamine-6-P in the presence of phosphoglucomutase (D-glucose z,6-diphosphate: D-glucose-x-phosphate phosphotransferase, EC 2.7.5.x ) and Glc-I,6-P s (ref. 2) ; UDP-glucosamine can be formed from glucosamine-I-P and UTP in the presence of an enzyme which has not been distinguisbed from UDPG pyrophosphorylase (UTP:--D-glucose-x-phosphate uridyltransferase, EC 2.7.7.9)s,4; and UDP-galactosamine can be formed from UDPglucosamine in the presence of an enzyme which has not been separated from UDPG 4-epimerase (EC 5.x.3.2) 5. It was thought to be of interest to see if UDP-giucosaminuronic acid could be synthesized from UDP-glucosamine in the presence of NAD + and UDPG dehydrogenase (UDPG: NAD oxidoreductase, EC I.I.X.22) in a manner analogous to the above reactions. We wish to report this synthesis. UDP-glucosamine and UDP-Ex-14CJglucosamine were prepared as previously described 4. NAD +, UDPG, and UDPG dehydrogenase (calf liver) were purchased from Sigma Chemical Company. Glucosaminuronic acid was a generous gift of Dr. K. HEY~S. Results of a typical incubation of UDPG and UDP-glucosamine at room temperature in the presence of NAD + and UDPG dehydrogenase are shown in Fig. z. UDPG was completely oxidized to UDP-glucuronic acid as measured by NADH formation (assayed by absorption at 34o m~) e. When UDP-glucosamine was used as substrate there was a much slower reduction of NAD + and the reaction was not carried to completion. If more enzyme were added at this point, additional reduction of NAD + occurred, but the reaction still was not carried to completion. Larger amounts of UDP-glucosamine and UDP-[x-l*C]glucosamine were reacted with xooo-2ooo units of UDPG dehydrogenase in other experiments with similar results of from Io % to 30 % yield of the new nucleotide sugar.
0.~ p •Ipu 0.,1 E
a~ Z 0
~ 0.1 e
1
2 Time(h)
e
??
3
:
?
4
Fig. z. Incubation of UDPG and UDP-glucosamine with UDPG dehydrogenase and NAD+. The reaction mixture contained, in a volume of 2.5 ml, o.x M glycine (pH 8.7); 2.5/Jmoles NAD+; zoo units UDPG dehydrogenase; and either UDPG (O--O), or UDP-glucceamine ( 0 - - 0 ) , o.25 ~umole. N A D H formation at room temperature was ~ - ~ y e d by absorption at 34 o rap. Biockim. Biophy$. Acta, 83 (z964) 355-357
356
SHORT COMMUNICATIONS
UDP-[I-t4C]glucosarninuronic acid was isolated by logarithmic gradient elution of the reaction products from a I cm x 5 cm column of Dowex-i X8 (formate form, 2oo-4oo mesh). A mixing volume of 2oo ml of water and a reservoir containing 4 N formic acid was used. Fractions of 5 ml were collected. Two radioactive peaks with absorption at 260 mt~ were located. The first, at Fractions I8-23 was identified as UDP-glucosamine. The second, appearing at Fractions 44-51 was evaporated in vacu.o at room temperature and then chromatographed on Whatman No. I paper with ethanol-ammonium acetate (pH 7.8) 7. A single ultraviolet-absorbing spot which contained all the radioactivity was found with RVMp = o.95. The area of the paper conraining this spot was cut out, washed with methanol, and eluted with water. The nucleotide isolated had a typical uridine spectrum at neutral and alkaline pH. Analysis showed uridine:total phosphate = I:2.o 9. Only 3o-5o % of the nucleotide was hydrolyzed by boiling in I N HE1 for 3 h. Hydrolysis under more severe conditions was attempted, but was even less successful in yielding quantitative amounts of labile phosphate and hexosaminuronic acid, presumably due to other degradation of the nucleotide sugar. A hydrolysate of the radioactive nucleotide sugar (I N HCI for 2 h at Ioo °) was chromatographed on Dowex-5o according to the method of GARDELL8. Radioactivity was found in the fractions containing unhydrolyzed nucleotide sugar and in the same fractions that contained added carrier glucosaminuronic acid. There was no radioactivity in fractions containing carrier glucosamine or galactosamine. The fractions containing glucosaminuronic acid were evaporated i , vacuo at room temperature and chromatographed on Whatman No. x paper in n - b u t a n o l - p y r i d i n e water (6:4: 3, v/v). The glucosaminuronic acid was located °, and this spot contained all the radioactivity. The results of the separation of glucosaminuronic acid by column and paper chromatography agree with those of CRUMFrON et al. 1°. It was also thought to be of interest to attempt the N-acetylation of UDPglucosaminuronic acid. Excess acetic anhydride was added to a solution containing 0.2 #mole of UDP-[I-t4C]glucosaminuronic acid (Iooo counts/min) and enough Tris was added to the mixture to make it alkaline to phenolphthalein. After x5 min at room temperature, the nucleotides in the reaction mixture were adsorbed on charcoal, .,o ammoniacal alcohol, dried in vac~w, and chromatographed on eluted with 5o o/ Whatman No. t paper with ethanol-ammonium acetate (pH 7.8). A single ultravioletabsorbing spot which contained all the radioactivity was found with RVMP : LIO. The area of this spot was cut out, washed with methanol, and eluted with water. An aliquot of this eluate containing 2oo counts/min was chromatographed on a I cm ,: r5 cm column of Dowex-I X8 (formate form, 2oo-400 mesh). After first eluting the column with 5o ml of 4 N formic acid, logarithmic gradient elution was carried out with 2oo ml of 4 N formic acid in the mixing flask and 4 N formic acid-o.2 M ammonium formate in the reservoir flask. Fractions of 5 ml were collected. A single peak of radioactivity was found in Fractions 32-36. Carrier UDP-N-acetylglucosamine was eluted from this column at Fractions x2-i6, and carrier UDP-glucuronic acid was eluted at Fractions 34-39- The radioactive compound, presumed to be UDP-N-acetylglucosaminuronic acid, was not characterized further. The pathway of synthesis of UDP-glucosaminuronic acid described in this report may not be of physiological significance. However, glucosaminuronic acid has been isolated from bacterial polysaccharides n,l*, and it is probable that nucleotide sugars Biochim. Biophys..4eta, 83 (I964) 355 357
SHORT COMMUNICATIONS
357
containing glucosaminuronic acid or N-acetylglucosaminuronic acid are intermediates in the synthesis of these polysaccharides. This is Publication No. 372 of the Robert W. Lovett Memorial for the Study of Diseases Causing Deformities, Harvard Medical School, Massachusetts General Hospital. This investigation was supported by Research Grant A-3564-O of the National Institute of Arthritis and Metabolic Disease, National Institutes of Health and was carried out while J.S. was supported by the Charles King Trust of the Massachusetts General Hospital, and E . H . was supported b y a U.S. Public Health Service Medical Student Research Training Program summer fellowship through Harvard Medical School. The authors wish to thank Dr. S. M. KRANE for helpful advice and encouragement in all phases of this work. Department o f M M i c i n e , Harvard M M i c a l School, Massachusea~s General Hospital, Boston, Mass. (U.S.A.)
JEREMIAH E. SILBERT* EDWARD F. X. HUGHES
t D. H. BnowN, Biochim. Biophys. ,4c/a, 7 (I95 I) 487• s D. H. BROWN,f . Biol. Chem., 204 (1953) 877. s F. MALSV,G. F. MALE','AI~DH. A. LARDY,J. Am. Chem. So¢., 78 (x956) 5303• 4 j. E. SILBERTAND D. H. BROWN,Biochim. Biophys. Acta, 54 (x961) 59o. t F. MALgV AND G. F. MALBV, Biochim. Biophys. Acta, 3 x (x959) 577e j. L. ST~OMINGeR,E. S. MAXWELLAN]>H. M. KALCKAR,in S. P. COLOWICKANDN. O. KAPLAN, Methods in Er.:ymology, Vol. 3, Academic Press, New York, x957, p. 974. • A. C. PALADIN!ANDL. F. LELOIR,Biochem. f . , 5 ! (I952) 426. s S. GAnDELL,Acta Chem. Scand., 7 (I953) 207. t S. M. PAItTRIDGE, Biochem. f . , 42 (1948) 238. to M. J. CRUMPTON,Biochem.J., 72 (I959) 479. 11 A. R. WILLXAMSONAND S. ZAMENHOF, f . Biol. Chem., 238 (I963) 2255. ts T . H .
HASt:ELLANDS. HANI~SSlAN,Biochim. Biophys. Acta, 83 (I964) 35-
Received June 8th, z96 4 " Present address: V.A. Hospital, x5o S. Huntington Ave., Boston, Mass. (U.S.A.). Biochim. Biophys. Acta, 83 (x964) 355-357
sc 83ox 3
Heterogeneity of sialomucopolysaccharides prepared from whole rat brain The isolation of sialomucopolysaccharides from crude mitochondrial fractions prepared from dog brain has been reported from this laboratory'. Subsequent work s established that this substance is present in crude mitochondrial and microsomal fractions prepared from rat brain homogenates. Although the mitochondrial fraction contained the larger share of the total brain siaiomucopolysaccharide, the microsomal fraction had a higher concentration of this substance when expressed in terms of the amount of sialomucopolysaccharide present per mg of protein. The method previously used to isolate sialomucopolysacchaxide from crude subcellular particulate fractions prepared from rat brain s was applied to whole rat brains in the present work. After removal of the brain from the animal, it was homogenized in x9 volumes of chloroform-methanol (2:x, v/v) and centrifuged. The precipitate was dried and subsequently processed as reported earlier I. Briefly, this involved Bi~.llim. Biophys. Acta, 83 (z964) 357-360