ENZYMATIC FORMATION OF URIDINE DIPHOSPHATE GLUCOSE WITH PREPARATIONS FROM IMPATIENS HOLSTII* N. C. GANGULIt

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1 ENZYMATIC FORMATION OF URIDINE DIPHOSPHATE GLUCOSE WITH PREPARATIONS FROM IMPATIENS HOLSTII* BY N. C. GANGULIt (From the Department of Agricultural Biochemistry, College of Agriculture, University of California, Berkeley, California) (Received for publication, November 19, 1957) Recent investigations have revealed that uridine diphosphate glucose is an important intermediate in carbohydrate metabolism of various living cells (1). Its presence has been reported in microorganisms (2) and in animal (3) and plant tissues (4). Evidence for the reversible formation of UDPG and PP from UTP and G-l-P by pyrophosphorylase present in glucose-6-phosphate dehydrogenase preparations was first presented by Munch-Petersen et al. (5). Later, a pyrophosphorylase capable of catalyzing the formation of UDPG from the same substrates was also found by several others in sugar beet leaf (6), in mung beans and in a number of other higher plants (7), and in animal tissues (3). Berg and Joklik (8) showed the reversible synthesis of UTP plus ADP from ATP plus UDP by an enzyme, nucleoside diphosphate kinase ( nudiki ), found in muscle and yeast. The present paper is concerned with conditions affecting the enzymatic formation of UDPG with homogenates from the plant Imp&ens ho&ii. By using G-l-P containing uniformly labeled n-glucose-c4 and UTP as substrates, UDPG-Cl4 could be readily obtained. Materials and Methods Radioactive Sugars and NucleotidesThe uniformly labeled n-glucose with Cl4 (specific activity, 3 PC. per mg.) was prepared photosynthetically by Dr. Putman in this laboratory by methods previously described (9, 1). The n-glucose-c l4 l-phosphate (specific activity,.5 PC. per mg.) was * This investigation was supported in part by a research grant (No. A-1418) from the National Institutes of Health, United States Public Health Service, and by a research contract with the United States Atomic Energy Commission. t Fellow under the India Wheat Loan Educational Exchange Program from the University of Calcutta, Calcutta, India. 1 The following abbreviations are used: UDPG = uridine diphosphate glucose; I P = pyrophosphate; UTP = uridine triphosphate; UDP = uridine diphosphate; UMP = uridine monophosphate; ATP = adenosine triphosphate; ADP = adenosine diphosphate; AMP = adenosine monophosphate; G-l-P = or-n-glucose l-phosphate; DPN = diphosphopyridine nucleotide; Tris = tris(hydroxymethyl)aminomethane. 337 Downloaded from by guest on December 28, 218

2 338 URIDINE DIPHOSPHATE GLUCOSE prepared from biosynthetic sucrose uniformly labeled with 4 by the method of phosphorolysis by the same worker (11). UTP, UDP, UMP, UDPG, ATP, ADP, AMP, DPN, and UDPG dehydrogenase were purchased from the Sigma Chemical Company. G-l-P was prepared according to McCready and Hassid (12). Preparation of Plant Leaf Homogenate-The I. holstii plant was chosen for this study because it is an efficient sucrose producer. It exudes a considerable quantity of sucrose which appears at the edges of the leaves and leaf petioles as large crystals. Since UDPG is known to be a precursor in the synthesis of sucrose (13), it was anticipated that this plant possesses an efficient enzymatic mechanism for the formation of this nucleotide which serves as a substrate for the subsequent formation of sucrose. The leaf homogenate was prepared as follows: Leaf blades weighing between.8 and 1. gm. were washed in cold tap water, wrapped in aluminum foil, and dipped for 1 minute in a dry ice-acetone mixture. The leaf blades were then unwrapped and macerated in the presence of.1 M Tris buffer, ph 7.5, with a volume of buffer calculated to contain 5 mg. of leaf material per 1 ml. of homogenate. Procedure of Incubation-The reaction mixture in the various experiments consisted of 1. pmole of G-l-P,.5 PC. of uniformly labeled D- glucose-cl4 l-phosphate, 1. pmole of UTP, and.2 ml. of leaf homogenate (containing 1 mg. of leaf material) in a total volume of.3 ml. of.1 M Tris buffer, ph 7.5. In the event KF was required to inactivate the phosphatases, 1 pmole of this salt was added. Incubation was carried out for a period of 3 minutes. The reaction was stopped by the addition of 2.5 ml. of 8 per cent ethanol and subsequent heating in a water bath at 1 for 1 minute. The mixture was then centrifuged, the supernatant liquid was collected, and, after washing the residue with 1 ml. of 8 per cent ethanol and subsequent centrifugation, the washings were combined with the supernatant liquid. The whole ethanol extract was evaporated to dryness in a vacuum desiccator over KOH. The dried residue was then dissolved in.15 ml. of water and subjected to paper electrophoresis for the separation of the uridine nucleotides. Paper Electrophoresis-The nucleotides were separated by paper electrophoresis (4), in which an apparatus similar to that described by Crestfield and Allen (14), with Whatman No. 1 acid-washed filter paper with formate buffer of ph 3.5 and.1 ionic strength, was employed. Picric acid was used as a visible reference compound and caffein as an indicator of electroosmosis. Phosphoric acid ester spots were detected with the molybdate spray reagent used by Bandurski and Axelrod (15). The nucleotides were located on the paper by ultraviolet radiation contact printing (16). The Downloaded from by guest on December 28, 218

3 N. C. GANGULI 339 separated nucleotides were eluted from the electrophoretogram with water and analyzed. Paper Chromatography-Whatman No. 1 filter paper and ethanol-ammonium acetate solvent were used for the separation of the nucleotides by paper chromatography (17). The nucleotides were detected by the same method as that used in paper electrophoresis. The sugars were separated after hydrolysis, with n-propanol-ethyl acetate-water (7 : 1: 2) as solvent (18), and detected by alkaline silver nitrate (19). Uridine Diphosphate Glucose Assay-This nucleotide was identified by both chemical and enzymatic methods. Chemical identification was made according to the method of Caputto et al. (2), from the appearance of the n-glucose, phosphorus, and uridine after acid hydrolysis of the com- pound. The n-glucose was also assayed for Cl4 activity. The enzymatic assay was carried out by means of two different reactions: (a) by the pyrophosphorylase reaction, by use of the unknown compound in the presence of pyrophosphate and mung bean enzyme preparation (6), and estimation of the UTP formed, and (b) by the UDPG dehydrogenase reaction in which the reduction of DPN was measured in the presence of the compound in question in a Beckman spectrophotometer at 34 rnp (21). Quantitative estimation of UDPG was done by determining the ultraviolet absorption at 262 rnp by using a molar extinction coefficient of 982 (22> * Estimation of Cl4 Activity-Samples were counted directly on Whatman No. 1 filter paper with a Tracerlab rate meter (SU-3A) supplied with a Geiger tube, as previously described (23). Unless otherwise stated the radioactivity measurements were expressed as mean values of duplicate samples. After subjecting the samples to paper electrophoresis or paper chromatography, radioautographs of the separated compounds were made by placing the papers in contact with 14 X 17 inch Eastman medical no-screen x-ray films. The standard exposure period was arbitrarily fixed to a period of 4 days for a sample with 4 c.p.m. Downloaded from by guest on December 28, 218 Results Synthesis of Uridine Diphosphate Glucose-(F-Leaf homogenate was incubated in the presence of UTP and n-glucose-cl4 l-phosphate. The reaction mixture was extracted with ethanol, evaporatored to dryness, and subjected to electrophoresis. After separation of the uridine derivatives, they were detected by ultraviolet absorption, by their Cl4 activity determined with a Geiger counter, and by radioautography of the paper electrophoretograms. The ultraviolet prints and the radioautographs obtained from the paper electrophoretograms were then superimposed on

4 34 URIDINE DIPHOSPHATE GLUCOSE each other in order to detect the location of the synthesized UDPG and the incorporation of Cl4 activity. The results in Table I show that the leaf homogenate is capable of synthesizing UDPG from G-l-P and UTP. Within 3 minutes of incubation 45 per cent of the total activity was found in the UDPG. On starting with 1. pmole each of G-l-P and UTP,.41 pmole of UDPG was formed. The prints resulting from the ultraviolet radiation of the electrophoreto- TABLE Biosynthesis of UDPG with I. ho&ii Leaf Homogenate See the text. under Procedure of incubation. I Substrates G*-1-P < + UTP * Indicates radioactive material. Per cent distribution of 4 activity G*-1-P UDPG ~-Glucose* TABLE UDPG* Jmwle Effect of Fluoride on Formation of UDPG For substrates and amounts in the reaction mixture, see the text under Procedure of incubation. Substrates II Per cent distribution of Cl4 activity G*-1-P UDPG* D-GlUCOSe* G*-1-P + UTP I + I +KF I * Indicates radioactive material pmlc Downloaded from by guest on December 28, 218 grams also showed formation of UDP and UMP within this period of incubation, indicating that the homogenate preparation contained phosphatase, which was responsible for the considerable degradation of UTP. E$ect of Fluoride-Since it has been shown that during the formation of UDPG from G-l-P and UTP by leaf homogenate part of the UTP is degraded to UDP and UMP, fluoride, known to be an effective phosphatase inhibitor, was added to the reaction mixture with the expectation that the UDPG yield would be increased. The results in Table II show a significant increase in UDPG when potassium fluoride was added. Under these conditions a decrease in formation of UDP and UMP from UTP has been observed.

5 N. C. GANGULI 341 E$ect of Mwnesium-It has been previously reported that magnesium has a stimulating effect on the UDPG pyrophosphorylase reaction (7). The results of Mg++ addition to the reaction mixture are presented in Table III. Magnesium was found to have an effect of stimulating the synthesis of UDPG. A concentration of.1 pmole of MgClz enhanced the synthesis of UDPG from.45 to.49 pmole. Period of incubation--the optimal conditions for synthesis of UDPG with plant homogenate preparations with regard to time were determined by carrying out the reactions at different periods of time, ranging from 5 minutes to 4 hours. The data in Table IV indicate that the synthesis of UDPG gradually TABLE III Effect of Magnesium on Synthesis of UDPG For substrates and their concentrations in the reaction mixture, see the text under Procedure of incubation. Substrates Per cent distribution G*-1-P - G*-1-P + UTP pmole MgCL ( L * Indicates radioactive material. UDPG* of C* activity n-glucose* UDPG* ~molc increases with time, reaching a maximum at the end of the 1st hour. Subsequently, a breakdown of UDPG takes place, as evidenced from the appearance of UMP and uridine, and from the decrease in radioactivity of this sugar nucleotide. After 1 hour, the Cl4 in G-l-P did not decrease significantly, whereas there was a considerable decrease in the activity of UDPG and a corresponding increase in the activity of n-glucose, indicating a degradation of UDPG. Thus, the maximal yield of UDPG under these conditions occurs after 1 hour of incubation. SpeciJicity of UDPG P~rophosphorylase-The specificity of UDPG pyrophosphorylase of the I. holstii was tested with regard to a number of substrates. The results in Table V show that UDPG is formed by the homogenate from G-l-P in the presence of either a mixture of UDP and ATP or UTP alone. Approximately 8 per cent of radioactivity was found in UDPG when UTP was used. From a mixture of UDP and ATP the conversion to Downloaded from by guest on December 28, 218

6 342 URIDINE DIPHOSPHATE GLUCOSE UDPG was 75 per cent. ATP, a mixture of UDP and ADP, or UDP alone could not be substituted for UTP for the formation of UDPG. Neither could free n-glucose be substituted for G-l-P for the formation of the sugar nucleotide. TABLE IV Formation of UDPG after Various Periods of Incubation For substrates and their concentrations in the reaction mixture, see the text under Procedure of incubation. Period of incubation Per cent distribution of 4 activity Formation of nucleotides G*-1-P UDPG* D-GlUCOS@ UDPG* In@ Uridine min I * Indicates radioactive material TABLE V pmole pamole wde Trace Specijkitu of UDPG Pyrophosphorylase The reaction mixture contained a mixture of.2 pmole of MgClz and.2 ml. of leaf homogenate (1 mg. of tissue material), to which 1 pmole quantities of various substrates were added, in a total volume of.3 ml. of.1 M Tris buffer, ph 7.5. After a 6 minute period of incubation, the reaction was stopped, and the components were separated and assayed. Substrates UDPG JTP disformed lppeared - - I er cent C activity of reaction products G*-1-P ~-Glucose UDPG* Downloaded from by guest on December 28, 218 G*-1-P UTP I + UDP + ATP.. I + UMP +. + UDP + ADP.. I + ATP UDP ATP+UMP.. UTP Glucose* + UTP ATP + UTP pnwle.75.7 p??wle l Indicates radioactive material.

7 N. C. GANGULI 343 Reversibility of Pyrophosphorylase Reaction-The reversibility of this reaction was observed when a mixture of 1 pmole of UDPG, 1 pmole of PP, 1 pmole of KF, and.2 Kmole of MgClz was incubated for 3 minutes with.2 ml. of leaf homogenate. After chromatographing the mixture, a new ultraviolet-absorbing spot was formed on the paper having an electrophoretic mobility equal to that of authentic UTP. When the spot was eluted from the paper and incubated with G-l-P and DPN in the presence of mung bean pyrophosphorylase (7) and UDPG dehydrogenase (21), by the method of Kalckar and Anderson (24), a reduction of DPN was observed at 34 rnp in a Beckman spectrophotometer. This confirms the fact that the ultraviolet-absorbing compound was UTP. Isolation of Radioactive Uridine Diphosphate Glucose on Preparative Scale-With the availability of n-glucose-c4 l-phosphate and UTP it was possible to prepare C14-labeled UDPG. The procedure adapted for preparation of this sugar nucleotide was as follows: A mixture consisting of 13 pmoles of G-l-P and 5 NC. of n-glucose-cl4 l-phosphate, 12 pmoles of UTP, 12 pmoles of MgCL, and 1 ml. of leaf homogenate in a volume of 15 ml. of.1 M Tris buffer, ph 7.5, was incubated for 1 hour. The reaction was stopped by the addition of 1 ml. of 95 per cent ethanol and subsequent heating of the mixture in a water bath to boiling. The precipitated protein was then centrifuged, the residue was washed twice with 2 ml. portions of 85 per cent ethanol, and the washings were combined with the original supernatant ethanolic extract. The combined extract was evaporated to dryness in a vacuum desiccator over potassium hydroxide, and the residue was taken up in.5 ml. of water. The uridine compounds and the unchanged G-l-P in the extract were separated as follows: The solution (.5 ml.) was applied on ten different filter papers,.5 ml. on each as a band, and subjected to paper electrophoresis in formate buffer, ph 3.5, at 75 volts. After 3 hours the paper bands corresponding to UDPG were cut out and eluted with water (25). The combined water eluate was then analyzed for Cl4 activity and for uridine content. The analysis showed the formation of 7.5 pmoles of UDPG containing 3.5 PC. of Cl4 activity. The compound had a characteristic spectrum of a uridine nucleotide (Emax at 262 rnp in.1 N HCl, E&Esao =.77, Ezso/Ezeo =.34). After hydrolysis with.1 N HCl for 5 minutes and subsequent paper chromatographic analysis in ethanol-ammonium acetate, a radioactive glucose spot and inactive UDP were produced. Evidence for Presence of Nucleotide Diphosphate Kinase in Leaf Homogenate-The I. holstii was tested for its ability to form UTP from ATP and UDP with the results presented in Table VI. The data show that when a mixture of 1 pmole each of ATP and UDP was incubated in the presence of leaf homogenate,.45 hmole of UTP was Downloaded from by guest on December 28, 218

8 344 URIDINE DIPHOSPHATE GLUCOSE formed, indicating that the homogenate contains nucleotide diphosphate kinase. This enzyme, capable of catalyzing the reaotion ATP -I- UDP ti ADP + UTP, was previously found in muscle and yeast (8). Since in addition to pyrophosphorylase the homogenate contains nucleoside diphosphate kinase, it would be expected that a mixture of ATP and UDP could be substituted for UTP in the reaction with G-l-P for the formation of UDPG. Data showing that UDPG is actually formed from these substrates are presented in Table VII. TABLE Synthesis of UTP from ATP and UDP by Leaf Homogenate VI Substrates present (1 WK+C each) ATP disappeared UDP disappeared UTP formed /mole pm& &mole UDP ATP. t ADP..2 + AMP...22 UMP + ATP TABLE Synthesis of UDPG from G-i-P, UDP, and ATP by Leaf Homogenate Substrates present (1 JUII& each) UDPG formed (~-D-G-~-P UDP ATP I + UMP + + UDP + ADP VII.72 -I- I Per cent radioactivity T G-1-P DGlUCOSe IJDPG Downloaded from by guest on December 28, 218 SUMMARY Homogenates from the plant Impatzkns holstii contain a pyrophosphorylase capable of catalyzing the reversible formation of uridine diphosphate glucose and inorganic pyrophosphate from uridine triphosphate and a-n-glucose l-phosphate. Mg++ ion was found to stimulate synthesis of the sugar nucleotide. By using a-n-glucose-c4 l-phosphate in the pyrophosphorylase reaction uridine diphosphate glucose-cl4 was prepared. The plant homogenate also contains the enzyme, nucleotide diphosphate kinase, capable of catalyzing the reversible formation of uridine triphos-

9 N. C. GANGULI 345 phate plus adenosine diphosphate from uridine diphosphate plus adenosine triphosphate. BIBLIOGRAPHY 1. Leloir, L. F., 3rd International Congress of Biochemistry, Brussels, 154 (1955). 2. C&bib, E., Leloir, L. F., and Cardini, C. E., J. Biol. Chem., 23, 155 (1953). 3. Smith, E. E. B., and Mills, G. T., Biochim. et biophys. acta, 13, 386 (1954). 4. Ginsburg, V., Stumpf, P. K., and Hassid, W. Z., J. Biol. Chem., 223, 977 (1956). 5. Munch-Petersen, A., Kalckar, H. M., Cutolo, E., and Smith, E. E. B., Nature, 172, 136 (1953). 6. Burma, D. P., and Mortimer, D. C., Arch. Biochem. and Biophys., 62, 16 (1956). 7. Neufeld, E. F., Ginsburg, V., Putman, E. W., Fanshier, D., and Hassid, W. Z., Arch. Biochem. and Biophys., 69, 62 (1957). 8. Berg, P., and Jolik, W. K., J. Biol. Chem., 21, 657 (1954). 9. Putman, E. W., Hassid, W. Z., Krotkov, G., and Barker, H. A., J. Biol. Chem., 173, 785 (1948). 1. Putman, E. W., and Hassid, W. Z., J. Biol. Chem., 196,749 (1952). 11. Hassid, W. Z., Doudoroff, M., and Barker, H. A., J. Am. Chem. Sot., 66, 1416 (1944). 12. McCready, R. M., and Hassid, W. Z., J. Am. Chem. Sot., 66, 56 (1944). 13. Cardini, C. E., Leloir, L. F., and Chiriboga, J., J. Biol. Chem., 214, 149 (1955). 14. Crestfield, A. M., and Allen, F. W., Anal. Chem., 27, 422, 424 (1955). 15. Bandurski, R. S., and Axelrod, B., J. BioZ. Chem., 193, 45 (1951). 16. Markham, R., and Smith, J. D., Biochem. J., 46, 294 (1949). 17. Paladini, A. C., and Leloir, L. F., Biochem. J., 61, 426 (1952). 18. Baar, S., and Bull, J. P., Nature, 172,414 (1953). 19. Trevelyan, W. E., Proctor, D. P., and Harrison, J. S., Nature, 166, 444 (195). 2. Caputto, R., Leloir, L. F., Cardini, C. E., and Paladini, A. C., J. Biol. Chem., 184, 333 (195). 21. Strominger, J. L., Maxwell, E. S., Axelrod, J., and Kalckar, H. M., J. Biol. Chem., 224, 79 (1957). 22. Ploeser, J. M., and Loring, H. S., J. BioZ. Chem., 178,431 (1949). 23. Putman, E. W., and Hassid, W. Z., J. BioZ. Chem., 27,885 (1954). 24. Kalckar, H. M., and Anderson, P. E., in Colowick, S. P., and Kaplan, N. O., Methods in enzymology, New York, 3, 976 (1957). 25. Putman, E. W., in Colowick, S. P., and Kaplan, N. O., Methods in enzymology, New York, 3,62 (1957). Downloaded from by guest on December 28, 218

10 ENZYMATIC FORMATION OF URIDINE DIPHOSPHATE GLUCOSE WITH PREPARATIONS FROM IMPATIENS HOLSTII N. C. Ganguli J. Biol. Chem. 1958, 232: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites references, of which can be accessed free at tml#ref-list-1 Downloaded from by guest on December 28, 218

Enzymatic Assay of GLYCOGEN SYNTHASE (EC )

Enzymatic Assay of GLYCOGEN SYNTHASE (EC ) PRINCIPLE: UDPG + (Glycogen) n Glycogen Synthase > UDP + (Glycogen) n+1 UDP + PEP PK > UTP + Pyruvate Pyruvate + ß-NADH LDH > Lactate + ß-NAD Abbreviations: UDPG = Uridine 5'-Diphosphoglucose UDP = Uridine