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426 Studies on Uridine-Diphosphate-Glucose By A. C. PALADINI AND L. F. LELOIR Instituto de Inve8tigacione&s Bioquimicas, Fundacion Campomar, J. Alvarez 1719, Buenos Aires, Argentina (Received 18 September 1951) A previous paper (Caputto, Leloir, Cardini & found that the substance supposed to be uridine-2'- Paladini, 1950) reported the isolation of the co- phosphate was uridine-5'-phosphate. The hydrolysis enzyme of the galactose -1- phosphate --glucose - 1 - product of UDPG has now been compared with a phosphate transformation, and presented a tenta- synthetic specimen of uridine-5'-phosphate. Both tive structure for the substance. This paper deals substances were found to be identical as judged by with: (a) studies by paper chromatography of puri- chromatographic behaviour (Fig. 1) and by the rate fied preparations of uridine-diphosphate-glucose (UDPG); (b) the identification of uridine-5'-phos- 12A UDPG phate as a product of hydrolysis; (c) studies on the ~~~~~~~~~~~~~(a) alkaline degradation of UDPG, and (d) a substance similar to UDPG which will be referred to as UDPX. UMP Adenosine UDPG preparation8 8tudied by chromatography. 0 UjDPX Paper chromatography with appropriate solvents 0 has shown that some of the purest preparations of UDP UDPG which had been obtained previously contain two other compounds, uridinemonophosphate 0 4 (UMP) and a substance which appears to have the same constitution as UDPG except that it contains an unidentified component instead of glucose. This Xj 128 IUMP Glucose substance will be provisionally referred to as UDPX (Fig. la). The three components have been tested for co- enzymic activity in the galactose-1-phosphate-- 0-4 -J UDPX glucose-l-phosphate transformation, and it has been confirmed that UDPG is the active substance. Slow ester Fast ester For each mole of uridine of UDPG in a sample ex- 0-8 - ll (d) tracted from the paper the total phosphate was UMP (synthetic) 2-04, the labile phosphate (15 min. in N-acid at 0-4 Glucose-I-phosphate 1000) 1-04, and the reducing power (calc. as glucose) after hydrolysis. (10 min. in 0-1N-acid at 1000) or 10 20 30 1-03 moles. Distance (cm.) When UDPG is hydrolysed at pH 2 during 10 min. Fig. 1. Chromatograms of UDPG preparations. Samples at 1000 glucose is liberated and, as shown in Fig. 1 b, run simultaneously at 300. Solvent: ethanol-ms-ammo- the UDPG and UTDPX peaks are replaced by a nium acetate, pH 7-5. Adenosine was added as reference slow-moving component which is uridinediphos- substance. The log IO/I values were measured at 260 m,u. phate. a, partially purified UDPG; b, same after heating 15 min. at 1000 at pH 2; c, heated 5 min. at 100° with Fig. 1c shows the results obtained after inacti- excess NH4OH; d, synthetic uridine-5'-phosphate plus vatingUDPGwith alkali. Besidesuridine phosphate glucose-l-phosphate. Glucose and its esters were located a fast- and/or a slow-moving sugar ester are formed. after removing the paraffin by ether extraction followed Identification ofuridine-5'-phosphate. The product by spraying with aniline phthalate. obtained by hydrolysing off with acid the glucose and one phosphate group from UDPG was pre- of acid hydrolysis (Table 1). The crystalline barium viously (Caputto et al. 1950) postulated to be salts of the two substances were prepared, and after uridine-5'-monophosphate. However, the hydro- recrystallization from water it was found that the lysis curves of this compound resembled more those microscopic aspect. of both samples was the same. given by Gulland & Smith (1947) for uridine-2'-phos- The X-ray diffraction patterns obtained by Prof. phate. Since thenBrown, Haynes& Todd (1950) have Galloni were identical for both samples. Vol. 5I URIDINE-DIPHOSPHATE-GLUCOSE 427 Table 1. Acid hydrolysis of uridine phosphates Treatment with acid would yield the same products, but since glucose-l-phosphate is hydrolysed im- (Samples heated at 100° in 0-1 N-H2SO4.) mediately only glucose-2-phosphate would remain. P hydrolysed (%) Thus the 'Slow Ester' prepared with alkali should be a mixture of glucose-l- and glucose-2-phos- UMP from phates, while that prepared with acid should be Synthetic UMP from UDPG by Time uridine-5'- UDPG by acid alkaline glucose-2-phosphate. (hr.) phosphate hydrolysis hydrolysis The exact conditions under which UDPG is 8-2 12-5 13-7 13-7 degraded with alkali have not been determined. It 20-4 26-5 28-2 29-2 is decomposed rapidly during chromatography with 36-5 44-4 46-0 43-9 the ethanol-ammonia solvent. Under these condi- 59-5 57-0 59-7 58-7 tions the formation of the 'Fast Ester' apparently occurs in less than 20 min., since the latter appears The alkaline degradation of uridine-diphosphate- as a well defined spot with practically no tailing. glucose. It has been reported previously (Caputto At pH 8 at 18° UDPG remained unchanged during et al. 1950) that UDPG loses its catalytic activity 18 hr. At pH 8-5 in 2 min. at 1000 a mixture of after a mild treatment with alkali. It was found that UMP, and 'Fast' and 'Slow' ester was formed. In this inactivation was accompanied by a stabilization concentrated ammonia at 00 during 30 min. UMP of the glucose residue and by the liberation of a and 'Fast Ester' were formed. secondary acid group of phosphoric acid. Further work on this point has shown that mild alkaline 2 I treatment of UDPG leads to the formation of UMP and a glucose ester in which the phosphate is doubly esterified. This substance ('Fast Ester') moves faster than any of the known glucose esters during paper chromatography. With a more drastic 9~~~~~~~ alkaline treatment or with acid the 'Fast Ester' is transformned into another substance or substances which move more slowly. These are referred to as pH6 ; 'Slow Ester(s)'. H20H 0 10 20 H H Quantity of base (1u-equiv.) Fig. 3. Titration curve of the 'Fast Ester'. The substance OH H was passed through a column of cation exchange resin in the hydrogen form. One sample was titrated directly 11|~~a (curve A) and another sample (curve B) was heated H O a 15 min. to 100° before titration. After this treatment b D 10 % of inorganic phosphate and 10% ofthe glucose were 11 liberated. The arrow marked P shows the pamoles ofphos- 0 phate in the sample. Fig. 2. Properties of the 'Fast Ester'. Table 2 shows the The experiments which will be described can be B. values ofthe 'Fast Ester' compared with glucose interpreted by assigning to the 'Fast Ester' the and glucose-l-phosphate. With the solvents which structure of a 1:2-monophosphoric ester of glucose were used the RF values are grossly inversely pro- (Fig. 2). Further treatment with alkali would yield portional to the number of acid groups in the mole- a mixture of glucose-2- and glucose-1-phosphate by cule: thus hexosediphosphates move slower than the hydrolysis of the links marked a and b respectively. monophosphates. For the 'Fast Ester' the values Table 2. Paper chromatography of the 'Fast' and 'Slow' ester8 (Whatman no. 1 paper.) Rp values 'Fast 'Slow Glucose-l- Solvent Ester' Ester' phosphate Glucose Ethanol (77 % v/v) 0-29 0-10 0-42 Ethanol ammonia 0-53 0-17 0-14 0-58 Ethanol ammonium acetate, pH 7-5 0-55 0-22 0-20 0-71 428 A. C. PALADINI AND L. F. LELOIR I952 are nearly as high as those of free glucose. This fact follows (% liberation of P): glucose- 1 -phosphate, 0; was the first indication that the substance contains glucose-2-phosphate, 94 %; 'Slow Ester', 100%. fewer acid groups than any of the known hexose- Liberation of phosphate during osazone forma- monophosphates. This was confirmed by electro- tion would appear in theory to be specific for sugars metric titration (Fig. 3) which shows the presence of with a free carbonyl containing phosphate in the 1 a primary but no secondary acid group. Acid or 2 positions. However, it has been observed that hydrolysis of the 'Fast Ester' yielded a sugar which glucose-3-phosphate (Raymond & Levene, 1929) was identified as glucose by paper chromatography and fructose-3-phosphate (Levene, Raymond & in several solvents. The same result was obtained Walti, 1929) also lose phosphate during osazone after hydrolysis with alkaline phosphatase. formation. The structure proposed in Fig. 2 for the 'Fast Ester' is consistent with experiments described in a Glucose-1-phosphate previous paper (Leloir, 1951), in which a 'Fast 80 _ 0 * 0 0 ~e K=0026--# 0 0 I- 20 Glucose-2-phosphate Fast ester (corr.) /1 I jII 0 20 40 60 80 100 120 60 120 180 0 60 120 180 Time (min.) Time (min.) Fig. 4. Acid hydrolysis of the ' Fast Ester '. Samples heated Fig. 5. Alkaline hydrolysis of the 'Fast Ester'. Inorganic at 1000 in 0-1N-H5S04. Reducing power measured with phosphate was estimated after the samples (1-1 jM) were Somogyi's (Somogyi, 1945) copper reagent followed by heated in 2 ml. of0.1 N-NaOH to 1000 in stoppered bronze arsenomolybdlic acid (Nelson, 1944). *-, phosphate; tubes. In a parallel experiment glucose-i-phosphate was 0-0, reducing power as glucose. estimated with yeast phosphoglucomutase (Cardini, Paladini, Caputto, Leloir & Trucco, 1949) activated with The curve of hydrolysis of the 'Fast Ester' in glucose diphosphate. Glucose- I-phosphate standards were 011N-acid is shown in Fig. 4. The curve shows a run at the same time. The samples of the 'Fast Ester' break at about 26,% hydrolysis as if it were the gave 26 % of glucose-i-phosphate after heating 5 min.
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