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Biosynthesis of UDP-sugars from 32P-labelled precursors in various biological materials
Comp. Bioehem. Physiol., 1969, Vol. 30, pp. 561 to 567. Pergamon Press. Printed in Great Britain
B I O S Y N T H E S I S OF UDP-SUGARS FROM asP-LABELLED PRECURSORS IN VARIOUS BIOLOGICAL MATERIALS TERESA SAWICKA Institute of Biochemistry and Biophysics, Polish Academy of Science, Warsaw, Poland (Received 9 January 1969)
A b s t r a c t - - 1 . The crude extracts from liver, brain, sublingual gland of rat and from hog and rabbit gastric mucosa, as well as from posterior silk glands of Bombyx mori catalyse the following sequence of reactions : N-acetylglucosamine6-phosphate -~ N-acetylglucosamine-l-phosphate ~ UDP-N-acetylglucosamine. Glucose-l,6-diphosphate stimulated this process. Glucosamine-6phosphate, galactosamine-6-phosphate and N-acetylgalactosamine-6-phosphate did not enter this sequence. 2. Considerable incorporation of [a2P]glucose-l-phosphate into uridine diphosphate sugars was found in hog and rabbit gastric mucosa, in rat tissues and in human and cow's milk. 3. The formation of UDP-galactose from UDP-glucose and [~zP]galactose1-phosphate was detected in rat tissues and in rabbit gastric mucosa. INTRODUCTION IN THE biological systems U D P - N - a c e t y l g l u c o s a m i n e * is formed as a result of pyrophosphorylase action on U T P and N-acetylglucosamine-l-phosphate. A similar enzyme isolated f r o m mast cells has been reported to catalyse the formation of U D P - g l u c o s a m i n e f r o m U T P and g l u c o s a m i n e - l - p h o s p h a t e (Silbert & Brown, 1961). Also U D P - N - a c e t y l g a l a c t o s a m i n e was shown to result f r o m the reaction of U T P with N - a c e t y l g a l a c t o s a m i n e - l - p h o s p h a t e in the presence of a rat liver extract (Maley et al., 1968). Whereas U D P - N - a c e t y l g l u c o s a m i n e and U D P - N - a c e t y l g a l a c t o s a m i n e take part in the biosynthesis of glycoproteins and some mucopolysaccharides, the biological significance of U D P - g l u c o s a m i n e is not yet clear. T h e aim of the present p a p e r was to compare the enzymatic systems leading to the formation of U D P - d e r i v a t i v e s of hexosamines f r o m their -6-phosphates and U T P in various biological materials. T h e data on the formation of U D P glucose and UDP-galactose in these materials are also included. M A T E R I A L S AND M E T H O D S Hog (Sussus) gastric mucosa was scraped from pig stomachs obtained at the local slaughter-house. Rabbit (Oryctolagus cuneatus) gastric mucosa was taken from the stomachs of commercial market animals. Male Wister rats (Rattus rattus), weighing 180--200 g, were * Abbreviations used: UDP, uridine diphosphate; UTP, uridine triphosphate. 561
562
TERESA SA'~VICKA
used for obtaining enzyme preparations from liver, brain and sublingual glands. The posterior silk glands of Bombyx mori were kindly provided by the Department of Protein Biosynthesis of this Institute. The tissues to be studied were homogenized with 9 vol. of physiological saline in a glass homogenizer. The homogenate of silk gland was centrifuged for 10 rain at 15,000 g. The homogenates from other tissues were centrifuged for 1 hr at 105,000g. The clear supernatants were used as the source of enzyme. Milk from women during the first days of lactation was kindly provided by the Warsa'~ Lactarium. Cow's colostrum and cow's milk on the third day after delivery was obtained from the Institute of Animal Physiology and Nutrition, Polish Academy of Science, Jabionna, Warsaw, Poland. The milk samples were defatted by centrifugation at 5000 g for 10 rain at 4°C. The defatted samples were used as the source of enzyme. Chemicals
n-Glucosamine HC1 was from E. Hoffman-La Roche & Cie, France. Galactosamine HC1 and glucose-l,6-diphosphate were from L. Light & Co. Ltd., Colnbrook, England. U T P , U D P G , hexokinase, charcoal and [3~P]glucose-l-pbosphate were the same as in a previous paper (Sawicka & Chojnacki, 1968). [~2P]galactose-l-phosphate was prepared as described elsewhere (Sawicka & Chojnacki, 1969). [z~P]glucosamine-6-phosphate and [32P]galactosamine-6-phosphate were prepared from glucosamine or galactosamine and [32P]-ATP using yeast hexokinase, according to the procedure described for obtaining glucose-6-phosphate (Risse et al., 1967). The isolation of hexosamine-6-phosphates (10 #moles) from the reaction mixtures was performed by column chromatography on Dowex 50 H + (1 × 30 cm). The hexosamine-6-phosphate was retained by the resin and subsequently eluted with 0"3 N HCI according to Wheat (1966). N-acetylation of hexosamine-6-phosphate (10#moles) was performed according to Distler et al. (1954). The reaction mixture was passed through a Dowex 50 H + (0"7 × 5 cm) column to remove Na +. The effluents were evaporated to dryness on a rotatory evaporator and redissolved in water. The specific activity of all radioactive compounds used for experiments with enzyme preparations was 8 x 106 counts/rain per #mole. The purity of hexosamine-6-phosphates and N-acetylhexosamine-6-phosphates was checked by paper chromatography in isopropanol :0"2 N HC1 (9: 1), according to McGarrahan & Maley (1962). R I = 0"08 for the former and 0'55 for the latter. Assay systems
The assay depended on the estimation of radioactive nucleotide sugars adsorbed on charcoal (Sawicka & Chojnacki, 1968). Complete reaction systems can be seen in Table 1. The radioactive nucleotide adsorbed on charcoal which was formed from [a2P]glucose-1phosphate and U T P was identified as UDP-glucose by paper chromatography with authentic substance in solvents I and II (Sawicka & Chojnacki, 1969). The one formed from Nacetylglucosamine-6-phosphate and U T P had a similar R I value. Protein was estimated according to Lowry et al. (1951), other analytical methods were the same as previously described (Sawicka & Chojnacki, 1968). RESULTS AND DISCUSSION F i g u r e 1 r e p r e s e n t s the effect of t h e a d d e d g l u c o s e - l , 6 - d i p h o s p h a t e o n t h e rate of f o r m a t i o n of ~ P - l a b e l l e d n u c l e o t i d e f r o m N - a c e t y l g l u c o s a m i n e - 6 - p h o s p h a t e w i t h t h e e n z y m e p r e p a r a t i o n f r o m r a b b i t gastric m u c o s a . T h e effect of glucose-1,6d i p h o s p h a t e c a n be seen o n l y w i t h a low a m o u n t of e n z y m e p r e p a r a t i o n . O n i n c r e a s i n g the a m o u n t of t h e latter the effect c a n n o t be o b s e r v e d a n y longer,
BIOSYNTHESIS OF U D P - s u G A R S
FROM 32P-LABELLED PRECURSORS
563
probably due to the presence of some glucose-l,6-diphosphate in the enzyme preparation itself. In typical experiments with ca. 200-600 tzg of protein in enzyme preparation the percentage of incorporation of the 32p-labelled substrate was very high and ranged from 5 to 50 per cent. 20
8
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0.6
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FIo. 1. The effect of glucose-l,6-diphosphate on the formation of [32p]UDP-Nacetylhexosamines using the assay system described in Table 1 with (A) or without (B) glucose- 1,6-diphosphate. Table 1 summarizes the enzyme activities responsible for synthesis of UDPsugars in various tissues of different animals. The results presented indicate that in the crude extracts of all the tissues studied very active N-acetylglucosamine phosphomutase (E.C. 2.7.5.2) and UDP-N-acetylglucosamine pyrophosphorylase (E.C. 2.7.7.23) are present. N-acetylglucosamine-6-phosphate can be converted by a mutase to N-acetylglucosamine-l-phosphate which reacts with U T P in the presence of a pyrophosphorylase to form UDP-N-acetylglucosamine. These results are in accord with the results of McGarrahan & Maley (1962), who found that the major pathway of glucosamine metabolism in rat liver proceeds via acetylation. In contrast to the good labelling of nueleotides from N-acetylglucosamine-6-P, glucosamine-6-P was a poor substrate. In our experiments only the extracts from rat liver and from hog and rabbit mucosa catalysed the incorporation of trace amounts of radioactivity from glucosamine-6-phosphate into nucleotide material. It is not clear whether the low labelling of nucleotide material in this case was due to the inability of phosphoglucomutase to form glucosamine-1phosphate or to the lack of the respective pyrophosphorylase. The formation of UDP-glucosamine from U T P and glueosamine-l-phosphate by enzyme from rat liver nuclei and from yeast has been reported by Maley et al. (1956). It is not known whether formation of this nucleotide sugar is of biological significance, as
-
-
264"0 264-0 180"0 160.0 130"0 86"0 60"0 60"0 60"0 60"0 60"0 75"0 31"0 23.2 11"7 5.6 --
Glucose-l-P
-
-
7"0 5"0 0-56 0.12 1-1 1.2 0"0 0"0 4"2 5-2 0"0 0"0 0"0 0"0 0"0 0.0 --
Galaetose-l-P 55"0 55"0 22"0 20"0 40"0 30'0 45"0 45"0 61"0 60"5 0"0 0"0 0"0 0-0 . . 6"5 4-1
N-acetylglucosamine-6-P
. . 0"0 0"0
0-17 -1"2 1-2 0"0 0"0 1"0 -1-8 0"8 0'0 0.0 0"0 0'0 . .
Glucosamine-6-P
. . -
-
--
0"4 ---0"0 0"0 ----0'0 0-0 ---
N-acetylgalactosamine-6-P
0"0 0"0
0"0 0'0 0"0 0"0 0"0 0"0 ----0"0 0"0 ---
Galactosamine-6-P
0-0 lack of activity; - - , n o t studied. F o r t h e tests with [szP]glucose-l-P c o m p l e t e s y s t e m c o n t a i n e d : 0 " 0 2 5 / z m o l e of [3~P]glucose-l-P, 0"1 /zmole U T P , 10/~moles of T r i s - H C 1 buffer, p H 7'5, 6/~moles MgC12 a n d 50/zl of e n z y m e p r e p a r a t i o n . F i n a l v o l u m e was 0"35 ml, i n c u b a t e d at 37°C for 15 rain. F o r t h e tests w i t h [s2P]galactose-l-P c o m p l e t e s y s t e m c o n t a i n e d : 0"025/~mole of [32p]galactose-1-P, 0"1/Lmole U D P G , 20/zmoles of glycine buffer, p H 8"1, c o n t a i n i n g 3 / z m o l e s of E D T A , a n d 50/zl of e n z y m e p r e p a r a t i o n in a final v o l u m e of 0-35 ml, i n c u b a t e d at 37°C for 15 rain. F o r t h e tests with [ a z p ] h e x o s a m i n e - 6 - p h o s p h a t e s a n d [ a 2 p ] N - a c e t y l h e x o s a m i n e - 6 - p h o s p h a t e s t h e c o m p l e t e s y s t e m c o n t a i n e d : 0'025/~mole of radioactive substrate, 0 ' 1 / z m o l e U T P , 10 ttmoles of T r i s - H C 1 buffer, p H 7-5, 6 / z m o l e s MgCI~, 0"2/~mole g l u c o s e - l , 6 d i p h o s p h a t e , 50/zl of e n z y m e p r e p a r a t i o n . F i n a l v o l u m e was 0"45 ml, i n c u b a t e d at 37°C for 1 hr. T h e reactions were s t o p p e d b y a d d i n g an e q u a l v o l u m e of 10~o trichloroacetic acid. A f t e r c e n t r i f u g a t i o n t h e s u p e m a t a n t s were treated w i t h 50 m g of charcoal in 1 m l of water. T h e n charcoal was w a s h e d t h r e e t i m e s w i t h w a t e r a n d t r a n s f e r r e d o n t o the planchets. T h e radioactivity was m e a s u r e d u s i n g a Geiger-M~iller mica e n d - w i n d o w c o u n t e r a n d c o n v e n t i o n a l ancillary eouit~ment.
Bombyx mori posterior silk glands
Cow's c o l o s t r u m
Cow's milk
H u m a n milk
R a b b i t gastric m u c o s a
H o g gastric m u c o s a
Rat sublingual gland
Rat b r a i n
Rat liver
Source of e n z y m e s
Radioactive precursors
TABLE 1--FORMATION OF UDP-suGARS FROM [32P]LABELLED PRECURSORS BY VARIOUS ENZYME PREPARATIONS EXPRESSED IN r e # m o l e s OF [3~P]UDP-suGARs/mg OF PROTEIN PER h r
BIOSYNTHESIS OF UDP-suGARS FROM 3SP-LABELLED PRECURSORS
565
UDP-glucosamine, which could be synthesized by extracts from the mast cell tumour (Silbert & Brown, 1961), was not active as an intermediate in the formation of mucopolysaccharides (Silbert, 1963). In rat liver UDP-glucosamine may theoretically be a precursor in the formation of galactosamine containing heteropolysaccharides via UDP-galactosamine. On the other hand, the investigations of McGarrahan & Maley (1962) suggest that glucosamine in rat liver is first Nacetylated and subsequently converted into the 6-phosphate. Sinohara & Asano (1967) found that the carbohydrate moiety of fibroin from the silkworm Bombyx mori contains a detectable amount of glucosamine (0.16 per cent) and mannose (0.17 per cent) and that the major pathway for the metabolism of glucosamine and N-acetylglucosamine in the silkworm is that involving the conversion to fructose-6-phosphate (Sinohara & Asano, 1968). The results presented in Table 1 show that acetylation of glucosamine-6-phosphate in extracts of silk glands of Bombyx mori is required for introducing the compound into the uridine coenzyme pathway of metabolism. This experiment points at the universal nature of the necessity of acetylation in both mammalian and non-mammalian tissues producing glycoproteins. It may therefore be assumed that in the uridine activation mechanism as well as in the transglycosylation step the amino sugar with the N-acetylated amino group takes a part. It is worth mentioning that N-acetylated derivatives of amino acids play an important role in the initiation and termination steps of polypeptides synthesis (Lucas-Lenard & Lipmann, 1967). On ion-exchange paper chromatography, using polyethyleneimine paper (Sawicka & Chojnacki, 1969), the labelled nucleotide material formed from U T P and N-acetylglucosamine phosphate resembled a general pattern of nucleoside diphosphate sugars. The method did not enable distinction between the glucosamine and galactosamine types of uridine coenzymes. As one can see in Table 1, with galactosamine-6-phosphate and N-acetylgalactosamine-6-P a negligible labelling of nucleotide material was observed only in rat liver and brain. In the other tissues which were tested these compounds had no biological activity. In fact there are no data in the literature suggesting the presence of enzymes converting the galactosamine-6-phosphate and N-acetylgalactosamine-6-phosphate into the sugar- 1-phosphates that are required substrates for pyrophosphorylases. They are supplied in rat liver via the direct phosphorylation of sugar at C-1 (Cardini & Leloir, 1953). The studies of the synthesis of nucleoside diphosphate sugars in milk were undertaken as this material was known to catalyse the blood group specific transglycosylations (Kobata et al., 1968a, b). In our studies, however, no labelling of nucleotide material was detected on using hexosamine-6-phosphates and Nacetylhexosamines-6-phosphates and UTP. This might indicate the absence of the respective uridylyltransferases other than that responsible for the activation of glucose-l-phosphate. The absence of galactose-l-phosphate uridylyltransferase from milk is in accord with the results obtained with mammary glands where UDPgalactose is produced mainly via UDP-glucose 4-epimerization.
566
TERESA SAWICKA
T h e formation of U D P - g l u c o s e from [saP]glucose-l-phosphate and U T P was observed in all tested tissues even in h u m a n and cow's milk and in cow's colostrum. A distinct labelling was observed in rat tissues and in hog and rabbit gastric mucosa. T h e incorporation of a*P from glucose-l-P into nucleotides was about 10-100 times higher than the formation of labelled nucleotides from galactose-l-phosphate and U D P - g l u c o s e in rat tissues and in rabbit gastric mucosa extracts. T h e formation of UDP-galactose by this pathway in the extracts from hog gastric mucosa was not observed. T h e biological materials investigated in this paper are widely used in studies on the formation of glycoproteins, thus knowledge of the mechanisms of activation of sugars for this synthesis may be important for this problem. REFERENCES CARDINI C. E. & LELOIR L. F. (1953) Enzymic phosphorylation of galactosamine and galactose. Nrchs Biochem. Biophys. 45, 55-64. DXSTLEI~J. ]., MERmCK J. M. & ROSEMANS. (1954) Glucosamine metabolism--III. Preparation and N-acetylation of crystalline D-glucosamine- and D-galactosamine-6phosphoric acids. J. biol. Chem. 230, 497-509. KOBATAA., GROLLMANE. F. & GINSBIJRGV. (1968a) An enzymatic basis for blood type A in humans. Archs Biochem. Biophys. 124, 609-612. KOBATAA., GROLLMANE, F. & GINSBURGV. (1968b) An enzymatic basis for blood type B in humans. Biochem. biophys. Res. Commun. 32, 272-277. LowRY O. H., ROSEBROUGHN. J., FARRA. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. LUCAS-LENARDJ. & LIeMANN F. (1967) Initiation of polyphenylalanine synthesis by Nacetylphenylalanyl-SRNA. Proc. natn. Acad. Sci. U.S.A. 57, 1050-1057. McGARRArlAN J. F. & MALE¥ F. (1962) Hexosamine metabolism--I. The metabolism in vivo and in vitro of D-glucosamine-l-C'4 and N-acetyl-D-glucosamine-l-C14 in rat liver. .~. biol. Chem. 237, 2458-2465. MALEY F., MALEY G. F. & LAI~DYH. A. (1956) The synthesis of c~-D-glucosamine-1phosphate and N-acetyl-~-D-glucosamine-l-phosphate. Enzymatic formation of uridine diphosphoglucosamine. J. Am. Chem. Soc. 78, 5303-5307. MALEY F., TAI~NTINOA. L., McGARRAHANJ. F. & DEL GIACCOR. (1968) The metabolism of D-galactosamine and N-acetyl-D-galactosamine in rat liver. Biochem.J. 107, 637-644. RlSSE H. J., DROGE W., RUSCHMANN E., LUDERITZ O., WESTPHAL O. • SCHLOSSARDTJ. (1967) Eine neue Gruppe yon Salmonella R-Mutanten. Eur. J. Biochem. 1, 216-232. SAWlCKAT. & CHOJNACKIT. (1968) Formation of galactogen from glucose phosphates in albumen gland of Helix pomatia. Comp. Biochem. Physiol. 26, 707-713. SAWlCKAT. & CHOJNACKIT. (1969) Formation of uridine diphosphate sugars from ~2p. labelled hexose phosphates in human red blood cells, Clin. chim. Jtcta 23, 463-468. SINOHARAH. & AsANoY. (1967) Carbohydrate content of fibroin and sericin of the silkworm, Bombyx mori. ~. Biochem. 62, 129-130. SINOHARAH. & ASANOY. (1968) Amino sugar metabolism in the silkworm, Bombyx mori. J. Biochem. 63, 8-13. SILBERT J. E. (1963) Incorporation of t4C and 3H from nucleotide sugars into a polysaccharide in the presence of a cell-free preparation from mouse mast cell tumors. J. biol. Chem. 238, 3542-3546. SILBERT]. E. & BROWND. H. (1961) Enzymic synthesis of uridine diphosphate glucosamine and heparin from [14C]-glucosamine by mouse mast-cell tumor. Biochim. biophys. Acta $4, 590-592.
BIOSYNTHESIS OF UDP-sUGARS FROM 32P-LABELLED PRECURSORS
567
WHEAT W. (1966) Analysis of hexosamines in bacterial polysaccharides by chromatographic procedures. In Methods in Enzymology (Edited b y COLOWICK S. P. ~ KAPLAN N. O.), Vol. 8, pp. 60-75. Academic Press, New York and London.
Key Word Index--UDP-sugars; UDP-galactose; UDP-glucose; gastric mucosa; silk glands; Bombyx mori; rat; pig; rabbit.
B I O S Y N T H E S I S OF UDP-SUGARS FROM asP-LABELLED PRECURSORS IN VARIOUS BIOLOGICAL MATERIALS TERESA SAWICKA Institute of Biochemistry and Biophysics, Polish Academy of Science, Warsaw, Poland (Received 9 January 1969)
A b s t r a c t - - 1 . The crude extracts from liver, brain, sublingual gland of rat and from hog and rabbit gastric mucosa, as well as from posterior silk glands of Bombyx mori catalyse the following sequence of reactions : N-acetylglucosamine6-phosphate -~ N-acetylglucosamine-l-phosphate ~ UDP-N-acetylglucosamine. Glucose-l,6-diphosphate stimulated this process. Glucosamine-6phosphate, galactosamine-6-phosphate and N-acetylgalactosamine-6-phosphate did not enter this sequence. 2. Considerable incorporation of [a2P]glucose-l-phosphate into uridine diphosphate sugars was found in hog and rabbit gastric mucosa, in rat tissues and in human and cow's milk. 3. The formation of UDP-galactose from UDP-glucose and [~zP]galactose1-phosphate was detected in rat tissues and in rabbit gastric mucosa. INTRODUCTION IN THE biological systems U D P - N - a c e t y l g l u c o s a m i n e * is formed as a result of pyrophosphorylase action on U T P and N-acetylglucosamine-l-phosphate. A similar enzyme isolated f r o m mast cells has been reported to catalyse the formation of U D P - g l u c o s a m i n e f r o m U T P and g l u c o s a m i n e - l - p h o s p h a t e (Silbert & Brown, 1961). Also U D P - N - a c e t y l g a l a c t o s a m i n e was shown to result f r o m the reaction of U T P with N - a c e t y l g a l a c t o s a m i n e - l - p h o s p h a t e in the presence of a rat liver extract (Maley et al., 1968). Whereas U D P - N - a c e t y l g l u c o s a m i n e and U D P - N - a c e t y l g a l a c t o s a m i n e take part in the biosynthesis of glycoproteins and some mucopolysaccharides, the biological significance of U D P - g l u c o s a m i n e is not yet clear. T h e aim of the present p a p e r was to compare the enzymatic systems leading to the formation of U D P - d e r i v a t i v e s of hexosamines f r o m their -6-phosphates and U T P in various biological materials. T h e data on the formation of U D P glucose and UDP-galactose in these materials are also included. M A T E R I A L S AND M E T H O D S Hog (Sussus) gastric mucosa was scraped from pig stomachs obtained at the local slaughter-house. Rabbit (Oryctolagus cuneatus) gastric mucosa was taken from the stomachs of commercial market animals. Male Wister rats (Rattus rattus), weighing 180--200 g, were * Abbreviations used: UDP, uridine diphosphate; UTP, uridine triphosphate. 561
562
TERESA SA'~VICKA
used for obtaining enzyme preparations from liver, brain and sublingual glands. The posterior silk glands of Bombyx mori were kindly provided by the Department of Protein Biosynthesis of this Institute. The tissues to be studied were homogenized with 9 vol. of physiological saline in a glass homogenizer. The homogenate of silk gland was centrifuged for 10 rain at 15,000 g. The homogenates from other tissues were centrifuged for 1 hr at 105,000g. The clear supernatants were used as the source of enzyme. Milk from women during the first days of lactation was kindly provided by the Warsa'~ Lactarium. Cow's colostrum and cow's milk on the third day after delivery was obtained from the Institute of Animal Physiology and Nutrition, Polish Academy of Science, Jabionna, Warsaw, Poland. The milk samples were defatted by centrifugation at 5000 g for 10 rain at 4°C. The defatted samples were used as the source of enzyme. Chemicals
n-Glucosamine HC1 was from E. Hoffman-La Roche & Cie, France. Galactosamine HC1 and glucose-l,6-diphosphate were from L. Light & Co. Ltd., Colnbrook, England. U T P , U D P G , hexokinase, charcoal and [3~P]glucose-l-pbosphate were the same as in a previous paper (Sawicka & Chojnacki, 1968). [~2P]galactose-l-phosphate was prepared as described elsewhere (Sawicka & Chojnacki, 1969). [z~P]glucosamine-6-phosphate and [32P]galactosamine-6-phosphate were prepared from glucosamine or galactosamine and [32P]-ATP using yeast hexokinase, according to the procedure described for obtaining glucose-6-phosphate (Risse et al., 1967). The isolation of hexosamine-6-phosphates (10 #moles) from the reaction mixtures was performed by column chromatography on Dowex 50 H + (1 × 30 cm). The hexosamine-6-phosphate was retained by the resin and subsequently eluted with 0"3 N HCI according to Wheat (1966). N-acetylation of hexosamine-6-phosphate (10#moles) was performed according to Distler et al. (1954). The reaction mixture was passed through a Dowex 50 H + (0"7 × 5 cm) column to remove Na +. The effluents were evaporated to dryness on a rotatory evaporator and redissolved in water. The specific activity of all radioactive compounds used for experiments with enzyme preparations was 8 x 106 counts/rain per #mole. The purity of hexosamine-6-phosphates and N-acetylhexosamine-6-phosphates was checked by paper chromatography in isopropanol :0"2 N HC1 (9: 1), according to McGarrahan & Maley (1962). R I = 0"08 for the former and 0'55 for the latter. Assay systems
The assay depended on the estimation of radioactive nucleotide sugars adsorbed on charcoal (Sawicka & Chojnacki, 1968). Complete reaction systems can be seen in Table 1. The radioactive nucleotide adsorbed on charcoal which was formed from [a2P]glucose-1phosphate and U T P was identified as UDP-glucose by paper chromatography with authentic substance in solvents I and II (Sawicka & Chojnacki, 1969). The one formed from Nacetylglucosamine-6-phosphate and U T P had a similar R I value. Protein was estimated according to Lowry et al. (1951), other analytical methods were the same as previously described (Sawicka & Chojnacki, 1968). RESULTS AND DISCUSSION F i g u r e 1 r e p r e s e n t s the effect of t h e a d d e d g l u c o s e - l , 6 - d i p h o s p h a t e o n t h e rate of f o r m a t i o n of ~ P - l a b e l l e d n u c l e o t i d e f r o m N - a c e t y l g l u c o s a m i n e - 6 - p h o s p h a t e w i t h t h e e n z y m e p r e p a r a t i o n f r o m r a b b i t gastric m u c o s a . T h e effect of glucose-1,6d i p h o s p h a t e c a n be seen o n l y w i t h a low a m o u n t of e n z y m e p r e p a r a t i o n . O n i n c r e a s i n g the a m o u n t of t h e latter the effect c a n n o t be o b s e r v e d a n y longer,
BIOSYNTHESIS OF U D P - s u G A R S
FROM 32P-LABELLED PRECURSORS
563
probably due to the presence of some glucose-l,6-diphosphate in the enzyme preparation itself. In typical experiments with ca. 200-600 tzg of protein in enzyme preparation the percentage of incorporation of the 32p-labelled substrate was very high and ranged from 5 to 50 per cent. 20
8
-& Z r~
E-, / •--'~/ O.J
l
0.2
I
0.3 Prot"ein,
I
0-4
I
0.5
F
0.6
I
0,7
mg
FIo. 1. The effect of glucose-l,6-diphosphate on the formation of [32p]UDP-Nacetylhexosamines using the assay system described in Table 1 with (A) or without (B) glucose- 1,6-diphosphate. Table 1 summarizes the enzyme activities responsible for synthesis of UDPsugars in various tissues of different animals. The results presented indicate that in the crude extracts of all the tissues studied very active N-acetylglucosamine phosphomutase (E.C. 2.7.5.2) and UDP-N-acetylglucosamine pyrophosphorylase (E.C. 2.7.7.23) are present. N-acetylglucosamine-6-phosphate can be converted by a mutase to N-acetylglucosamine-l-phosphate which reacts with U T P in the presence of a pyrophosphorylase to form UDP-N-acetylglucosamine. These results are in accord with the results of McGarrahan & Maley (1962), who found that the major pathway of glucosamine metabolism in rat liver proceeds via acetylation. In contrast to the good labelling of nueleotides from N-acetylglucosamine-6-P, glucosamine-6-P was a poor substrate. In our experiments only the extracts from rat liver and from hog and rabbit mucosa catalysed the incorporation of trace amounts of radioactivity from glucosamine-6-phosphate into nucleotide material. It is not clear whether the low labelling of nucleotide material in this case was due to the inability of phosphoglucomutase to form glucosamine-1phosphate or to the lack of the respective pyrophosphorylase. The formation of UDP-glucosamine from U T P and glueosamine-l-phosphate by enzyme from rat liver nuclei and from yeast has been reported by Maley et al. (1956). It is not known whether formation of this nucleotide sugar is of biological significance, as
-
-
264"0 264-0 180"0 160.0 130"0 86"0 60"0 60"0 60"0 60"0 60"0 75"0 31"0 23.2 11"7 5.6 --
Glucose-l-P
-
-
7"0 5"0 0-56 0.12 1-1 1.2 0"0 0"0 4"2 5-2 0"0 0"0 0"0 0"0 0"0 0.0 --
Galaetose-l-P 55"0 55"0 22"0 20"0 40"0 30'0 45"0 45"0 61"0 60"5 0"0 0"0 0"0 0-0 . . 6"5 4-1
N-acetylglucosamine-6-P
. . 0"0 0"0
0-17 -1"2 1-2 0"0 0"0 1"0 -1-8 0"8 0'0 0.0 0"0 0'0 . .
Glucosamine-6-P
. . -
-
--
0"4 ---0"0 0"0 ----0'0 0-0 ---
N-acetylgalactosamine-6-P
0"0 0"0
0"0 0'0 0"0 0"0 0"0 0"0 ----0"0 0"0 ---
Galactosamine-6-P
0-0 lack of activity; - - , n o t studied. F o r t h e tests with [szP]glucose-l-P c o m p l e t e s y s t e m c o n t a i n e d : 0 " 0 2 5 / z m o l e of [3~P]glucose-l-P, 0"1 /zmole U T P , 10/~moles of T r i s - H C 1 buffer, p H 7'5, 6/~moles MgC12 a n d 50/zl of e n z y m e p r e p a r a t i o n . F i n a l v o l u m e was 0"35 ml, i n c u b a t e d at 37°C for 15 rain. F o r t h e tests w i t h [s2P]galactose-l-P c o m p l e t e s y s t e m c o n t a i n e d : 0"025/~mole of [32p]galactose-1-P, 0"1/Lmole U D P G , 20/zmoles of glycine buffer, p H 8"1, c o n t a i n i n g 3 / z m o l e s of E D T A , a n d 50/zl of e n z y m e p r e p a r a t i o n in a final v o l u m e of 0-35 ml, i n c u b a t e d at 37°C for 15 rain. F o r t h e tests with [ a z p ] h e x o s a m i n e - 6 - p h o s p h a t e s a n d [ a 2 p ] N - a c e t y l h e x o s a m i n e - 6 - p h o s p h a t e s t h e c o m p l e t e s y s t e m c o n t a i n e d : 0'025/~mole of radioactive substrate, 0 ' 1 / z m o l e U T P , 10 ttmoles of T r i s - H C 1 buffer, p H 7-5, 6 / z m o l e s MgCI~, 0"2/~mole g l u c o s e - l , 6 d i p h o s p h a t e , 50/zl of e n z y m e p r e p a r a t i o n . F i n a l v o l u m e was 0"45 ml, i n c u b a t e d at 37°C for 1 hr. T h e reactions were s t o p p e d b y a d d i n g an e q u a l v o l u m e of 10~o trichloroacetic acid. A f t e r c e n t r i f u g a t i o n t h e s u p e m a t a n t s were treated w i t h 50 m g of charcoal in 1 m l of water. T h e n charcoal was w a s h e d t h r e e t i m e s w i t h w a t e r a n d t r a n s f e r r e d o n t o the planchets. T h e radioactivity was m e a s u r e d u s i n g a Geiger-M~iller mica e n d - w i n d o w c o u n t e r a n d c o n v e n t i o n a l ancillary eouit~ment.
Bombyx mori posterior silk glands
Cow's c o l o s t r u m
Cow's milk
H u m a n milk
R a b b i t gastric m u c o s a
H o g gastric m u c o s a
Rat sublingual gland
Rat b r a i n
Rat liver
Source of e n z y m e s
Radioactive precursors
TABLE 1--FORMATION OF UDP-suGARS FROM [32P]LABELLED PRECURSORS BY VARIOUS ENZYME PREPARATIONS EXPRESSED IN r e # m o l e s OF [3~P]UDP-suGARs/mg OF PROTEIN PER h r
BIOSYNTHESIS OF UDP-suGARS FROM 3SP-LABELLED PRECURSORS
565
UDP-glucosamine, which could be synthesized by extracts from the mast cell tumour (Silbert & Brown, 1961), was not active as an intermediate in the formation of mucopolysaccharides (Silbert, 1963). In rat liver UDP-glucosamine may theoretically be a precursor in the formation of galactosamine containing heteropolysaccharides via UDP-galactosamine. On the other hand, the investigations of McGarrahan & Maley (1962) suggest that glucosamine in rat liver is first Nacetylated and subsequently converted into the 6-phosphate. Sinohara & Asano (1967) found that the carbohydrate moiety of fibroin from the silkworm Bombyx mori contains a detectable amount of glucosamine (0.16 per cent) and mannose (0.17 per cent) and that the major pathway for the metabolism of glucosamine and N-acetylglucosamine in the silkworm is that involving the conversion to fructose-6-phosphate (Sinohara & Asano, 1968). The results presented in Table 1 show that acetylation of glucosamine-6-phosphate in extracts of silk glands of Bombyx mori is required for introducing the compound into the uridine coenzyme pathway of metabolism. This experiment points at the universal nature of the necessity of acetylation in both mammalian and non-mammalian tissues producing glycoproteins. It may therefore be assumed that in the uridine activation mechanism as well as in the transglycosylation step the amino sugar with the N-acetylated amino group takes a part. It is worth mentioning that N-acetylated derivatives of amino acids play an important role in the initiation and termination steps of polypeptides synthesis (Lucas-Lenard & Lipmann, 1967). On ion-exchange paper chromatography, using polyethyleneimine paper (Sawicka & Chojnacki, 1969), the labelled nucleotide material formed from U T P and N-acetylglucosamine phosphate resembled a general pattern of nucleoside diphosphate sugars. The method did not enable distinction between the glucosamine and galactosamine types of uridine coenzymes. As one can see in Table 1, with galactosamine-6-phosphate and N-acetylgalactosamine-6-P a negligible labelling of nucleotide material was observed only in rat liver and brain. In the other tissues which were tested these compounds had no biological activity. In fact there are no data in the literature suggesting the presence of enzymes converting the galactosamine-6-phosphate and N-acetylgalactosamine-6-phosphate into the sugar- 1-phosphates that are required substrates for pyrophosphorylases. They are supplied in rat liver via the direct phosphorylation of sugar at C-1 (Cardini & Leloir, 1953). The studies of the synthesis of nucleoside diphosphate sugars in milk were undertaken as this material was known to catalyse the blood group specific transglycosylations (Kobata et al., 1968a, b). In our studies, however, no labelling of nucleotide material was detected on using hexosamine-6-phosphates and Nacetylhexosamines-6-phosphates and UTP. This might indicate the absence of the respective uridylyltransferases other than that responsible for the activation of glucose-l-phosphate. The absence of galactose-l-phosphate uridylyltransferase from milk is in accord with the results obtained with mammary glands where UDPgalactose is produced mainly via UDP-glucose 4-epimerization.
566
TERESA SAWICKA
T h e formation of U D P - g l u c o s e from [saP]glucose-l-phosphate and U T P was observed in all tested tissues even in h u m a n and cow's milk and in cow's colostrum. A distinct labelling was observed in rat tissues and in hog and rabbit gastric mucosa. T h e incorporation of a*P from glucose-l-P into nucleotides was about 10-100 times higher than the formation of labelled nucleotides from galactose-l-phosphate and U D P - g l u c o s e in rat tissues and in rabbit gastric mucosa extracts. T h e formation of UDP-galactose by this pathway in the extracts from hog gastric mucosa was not observed. T h e biological materials investigated in this paper are widely used in studies on the formation of glycoproteins, thus knowledge of the mechanisms of activation of sugars for this synthesis may be important for this problem. REFERENCES CARDINI C. E. & LELOIR L. F. (1953) Enzymic phosphorylation of galactosamine and galactose. Nrchs Biochem. Biophys. 45, 55-64. DXSTLEI~J. ]., MERmCK J. M. & ROSEMANS. (1954) Glucosamine metabolism--III. Preparation and N-acetylation of crystalline D-glucosamine- and D-galactosamine-6phosphoric acids. J. biol. Chem. 230, 497-509. KOBATAA., GROLLMANE. F. & GINSBIJRGV. (1968a) An enzymatic basis for blood type A in humans. Archs Biochem. Biophys. 124, 609-612. KOBATAA., GROLLMANE, F. & GINSBURGV. (1968b) An enzymatic basis for blood type B in humans. Biochem. biophys. Res. Commun. 32, 272-277. LowRY O. H., ROSEBROUGHN. J., FARRA. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. LUCAS-LENARDJ. & LIeMANN F. (1967) Initiation of polyphenylalanine synthesis by Nacetylphenylalanyl-SRNA. Proc. natn. Acad. Sci. U.S.A. 57, 1050-1057. McGARRArlAN J. F. & MALE¥ F. (1962) Hexosamine metabolism--I. The metabolism in vivo and in vitro of D-glucosamine-l-C'4 and N-acetyl-D-glucosamine-l-C14 in rat liver. .~. biol. Chem. 237, 2458-2465. MALEY F., MALEY G. F. & LAI~DYH. A. (1956) The synthesis of c~-D-glucosamine-1phosphate and N-acetyl-~-D-glucosamine-l-phosphate. Enzymatic formation of uridine diphosphoglucosamine. J. Am. Chem. Soc. 78, 5303-5307. MALEY F., TAI~NTINOA. L., McGARRAHANJ. F. & DEL GIACCOR. (1968) The metabolism of D-galactosamine and N-acetyl-D-galactosamine in rat liver. Biochem.J. 107, 637-644. RlSSE H. J., DROGE W., RUSCHMANN E., LUDERITZ O., WESTPHAL O. • SCHLOSSARDTJ. (1967) Eine neue Gruppe yon Salmonella R-Mutanten. Eur. J. Biochem. 1, 216-232. SAWlCKAT. & CHOJNACKIT. (1968) Formation of galactogen from glucose phosphates in albumen gland of Helix pomatia. Comp. Biochem. Physiol. 26, 707-713. SAWlCKAT. & CHOJNACKIT. (1969) Formation of uridine diphosphate sugars from ~2p. labelled hexose phosphates in human red blood cells, Clin. chim. Jtcta 23, 463-468. SINOHARAH. & AsANoY. (1967) Carbohydrate content of fibroin and sericin of the silkworm, Bombyx mori. ~. Biochem. 62, 129-130. SINOHARAH. & ASANOY. (1968) Amino sugar metabolism in the silkworm, Bombyx mori. J. Biochem. 63, 8-13. SILBERT J. E. (1963) Incorporation of t4C and 3H from nucleotide sugars into a polysaccharide in the presence of a cell-free preparation from mouse mast cell tumors. J. biol. Chem. 238, 3542-3546. SILBERT]. E. & BROWND. H. (1961) Enzymic synthesis of uridine diphosphate glucosamine and heparin from [14C]-glucosamine by mouse mast-cell tumor. Biochim. biophys. Acta $4, 590-592.
BIOSYNTHESIS OF UDP-sUGARS FROM 32P-LABELLED PRECURSORS
567
WHEAT W. (1966) Analysis of hexosamines in bacterial polysaccharides by chromatographic procedures. In Methods in Enzymology (Edited b y COLOWICK S. P. ~ KAPLAN N. O.), Vol. 8, pp. 60-75. Academic Press, New York and London.
Key Word Index--UDP-sugars; UDP-galactose; UDP-glucose; gastric mucosa; silk glands; Bombyx mori; rat; pig; rabbit.