2 Uridine Diphosphoryl Glucose Pyrophosphorylase

Uridine Diphosphory1 Glucose Pyrophos9horylase RICHARD L . TURNQUIST

R . GAURTH HANSEN

I . Introduction . . . . . . . . . A . Measurement of Activity . . . . B . Purification . . . . . . . C . Analytical and Synthetic Applications I1. Metabolic Function . . . . . . . A . Cytology . . . . . . . B . Metabolism . . . . . . . C . Metabolic Regulation . . . . I11. Properties . . . . . . . . . A . Optima . . . . . . . . B . Structure . . . . . . . C . Kinetics . . . . . . . D . Specificity . . . . . . . E . Mechanism . . . . . . .

. . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . .

. . . . . . . . . . . . . . .

. . . . . . . . . . . . . . .

51 52 53 54 55 55 57 59 62 62 62 65 68 69

.

I Introduction

Uridine diphosphoryl glucose pyrophosphorylase (UTP:a-wglucose-lphosphate uridylyltransferase. EC 2.7.7.9) catalyzes the formation of nucleoside diphosphate sugars from nucleoside triphosphates and sugar1-phosphates [Eq. ( 1 ) ] . NTP

+ sugar-1-phosphate 2 NDP-sugar + PP. 51

(1)

52

RICHARD L. TURNQUIST AND R. GAURTH HANSEK

The enzyme appears to be ubiquitous in nature, and, since its discovery (I, d ) , it has been detected and purified from a variety of sources (3-9). Its universal occurrence is not surprising since a major product of the reaction that it catalyzes, UDPglucose, has a central role as a glucosyl donor in numerous cellular transformations (10). A. MEASUREMENT OF ACTIVITY Several different assay procedures have been developed to detect and quantify UDPglucose pyrophosphorylase. The rate of reaction (1) can be determined in either direction using a variety of substrates. I n the direction of pyrophosphorolysis of the nucleoside diphosphate sugar when the sugar is glucose, the quantity of glucose 1-P produced can be determined using coupling enzymes which convert it to glucose 6-P and 6-phosphogluconate (11). In addition to the appropriate enzymes, phosphoglucomutase and glucose 6-phosphate dehydrogenase, the assay mixture contains NADP', pyrophosphate, and the nucleoside diphosphate glucose. The reduction of NADP' is followed a t 340 nm. If the nucleoside diphosphate sugar contains uridine, guanine, adenine, or inosine, the nucleoside triphosphate formed in the reaction may be used to phosphorylate 3-P-glyceric acid, which can be reduced by NADH (12 ) . Glyceraldehyde-3-P dehydrogenase and 3-P-glycerate kinase are used as coupling enzymes, and the oxidation of NADH is followed spectrophotometrically. 1. H. M. Kalckar and E. Cutolo, Proc. Znt. Congr. Biochem., 2nd, 1962 p. 260 (1953). 2. A. Munch-Petersen, H. M. Kalckar, E. Cutolo, and E. E. B. Smith, Nature (London) 172, 1036 (1953). 3. E. E. B. Smith, G . T. Mills, and E. M. Harper, J. Gen. Microbiol. 16, 426 ( 1957). 4. E. F. Neufeld, V. Ginsburg, E. Putman, E. W. Fanshier, and W. Z. Hassid, ABB 69, 602 (1957). 5. C. Villar-Palasi and J. Larner, BBA 30, 449 (1958). 6. J. H. Pazur and E. W. Shuey, Fed. Proc., Fed. Amer. Soc. Ezp. Biol. 20, 216 (1961). 7. M. Axelos and C. Peaud-Lenoil, Bull. SOC.Chirn. Biol. 51, 261 (1969). 8. P. N. Viswanathan, Zndiun J. Biochem. 6, 124 (1969). 9. I. J. Russell and D. R. Lineback, Carbohyd. Res. 15, 123 (1970). 10. V. Ginsburg, Advan. Enzymol. 26, 35 (1964). 11. A. Munch-Petersen, Acta Chem. Scand. 9, 1523 (1955). 12. H. Verachtert, S. T. Bass, L. L. Seifert, and R. G. Hansen, Anrtl. Biochern. 13, 259 (1965).

2.

URIDISE DIPHOSPHORTL GLUCOSE PTROPHOSPHORYLASE

53

The synthesis of UDPglucose may be quantified with UDPglucose dehydrogenase (IS), which oxidizes the nucleoside sugar to UDPglucuronate and concomitantly reduces the NAD'. Two moles of NAD' are reduced for every mole of UDPglucose formed, increasing the sensitivity of the assay. A more time consuming but also more sensitive assay for UDPglucose formation involves incubating the enzyme with U T P and radioactive sugar-1-phosphates. After the reaction is stopped, the radioactive nucleoside diphosphate sugars are adsorbed on charcoal, then eluted, and the radioactivity measured (14, 16). B. PURIFICATION Uridine diphosphoryl glucose pyrophosphorylase was successfully crystallized from calf (16), human, (17),lamb, goat, and rabbit (18) livers. The procedure utilized an alkaline extraction of homogenized liver followed by protamine sulfate treatment. The enzyme was precipitated from the supernate fraction with ammonium sulfate, and, after dialysis, was treated with calcium phosphate gel and adsorbed on a DEAEcellulose column. After elution, the enzyme was again concentrated with ammonium sulfate, dissolved, and crystallized from ammonium sulfate solution. Various procedures have been used successfully in partially purifying the enzyme from other sources. Ginsburg (19) purified the enzyme from mung bean acetone powder using ammonium sulfate fractionation, alumina C y treatment and cellulose chromatography. Bovine mammary pyrophosphorylase was purified from acetone powder using essentially the same procedure as used for human liver ( 2 0 ) . Tsuboi et al. (21) purified human erythrocyte enzyme using calcium phosphate gel, ammonium sulfate, cold ethanol, DEAE-cellulose and Sephadex G-200 fractionations. Although the specific activity of the enzyme was quite 13. J. 1,. Strominger, H. M. Kalckar, J. Axelrod, and E. S. Maxwell, JACS 76, 6411 (1954). 14. D. M. Carlson and R. G. Hansen, JBC 237, 1260 (1962). 15. A. Munch-Petersen, Acta Chem. Scand. 11, 1079 (1957). 16. G. J. Albrecht, S. T. Bass, L. L. Seifert, and R. G. Hansen, JBC 241, 2968 (1966). 17. J. Knop and R. G. Hansen, JBC 245, 2499 (1970). 18. J. Knop, Master's Thesis, Michigan State University, 1969. 19. V. Ginsburg, JBC 232, 55 (1958). 20. V. S. Steelman and K. E. Ebner, BBA 128, 92 (1966). 21. K. K. Tsuboi, K. Fukunaga, and J. C. Petricciani, JBC 244, 1008 (1969).

54

RICHARD L. TURNQUIST AND R. GAURTH H A N S E N

high, crystallization was not achieved. Franke and Sussman (22) purified the enzyme from the slime mold, Dictyostelium discoideum, to apparent physical and immunochemical homogeneity by ammonium sulfate fractionation followed by elution through Sephadex G-100, Hypatite C, and polyacrylamide gel. Using various combinations of these procedures, the enzyme has been purified from human brain ( 2 3 ) , rat mammary tissue (241, rabbit muscle ( 2 5 ) , maize (26), slime mold (27), bacteria (28-30),and yeast ( 3 1 ) .

C. ANALYTICALAND SYNTHETIC APPLICATIONS Several synthetic and analytical techniques have been designed which utilize UDPglucose pyrophosphorylase and the reaction it catalyzes. The enzyme has been used to effect the synthesis of ["C]UDPglucose, using labeled glucose, glucose 6-P or glucose-fructose mixtures as starting materials ( 3 2 . 3 4 ) .The enzymic synthesis of UDPglucosamine has also been described (35) which probably utilizes UDPglucose pyrophosphorylase. The enzyme was also used to determine PPi with an application to tissue extracts (36).Whenever PPi is produced in a reaction, the potential is there to use the pyrophosphorylase as an analytical coupling reagent; for example, the procedure was used to determine RNA polym22. J. Franke and M. Sussman, JBC 248, 6381 (1971). 23. D.K. Basu and B. K. Buchhawat, J . Neurochem. 7, 174 (1961). 24. R. S. Emery and R. L. Baldwin, BBA 1 3 , 223 (1967). 25. C. Villar-Palasi and J. Larner, ABB 86, 61 (1960). 26. J. D.Vidra and J. D. Loerch, BBA 159, 551 (1968). 27. G. Gustafson and B. E. Wright, Fed. Proc., Fed. Amer. SOC.Exp. Biol. 30, 1069 (1971). 28. A. Kamogawa and K. Kurahashi, J . Biochem. (Tokyo) 57, 758 (1965). 29. T. Chojnacki, T. Sawicka, and T. Korzybski, Acta Bbchim. Pol. 15, 293 (1968). 30. H. Nikaido and T. Nakae, Fed. Proc., Fed. Amer. SOC.Exp. Biol. 29, A598 (1970). 31. A. Munch-Petersen and H. M. Kalckar, "Methods in Enzymology," Vol. 2, p. 675, 1955. 32. E. R. Trucco, Nature (London) 174, 1103 (1954). 33. L. Glaser, JBC 232, 627 (1958). 34. A. Wright and P. W. Robbins, BBA 104, 594 (1965). 35. F. Malay, G. F. Malay, and H. A. Lardy, JACS 78, 5303 (1956). 36. J. C. Johnson, M. Shanoff, 8. T. Bass, J. A. Boeai, and R. C. Hansen, Anal. Biochem. 26, 137 (1968).

2.

URIDIKE DIPHOSPHORTL GLUCOSE PYROPHOSPHORYLASE

55

erase activity in biological materials. With radioactive tracers the method can be made sensitive to as little as 0.2 pmoles of PPi (37). II. Metabolic Function

A. CYTOLOGY While UDPglucose pyrophosphorylase is present in nearly all tissues, it is usually most abundant in those that display active polysaccharide synthesis. The enzyme may account for 0.2-0.3% of the extractable protein of calf liver (16), and as much as 1% of the protein in slime mold cells ( 3 8 ) .The amount of enzyme in tissue may also vary greatly with the age and physiological state of the organism. These factors probably determine the ultimate success of enzyme purification procedures. In higher animals, liver most often has the highest concentration of UDPglucose pyrophosphorylase. Skeletal muscle, heart, and kidney have intermediate enzyme levels, while spleen, lung, brain, testis, and fatty tissues have relatively low activity levels (39, 40). In most animal tissues, the pyrophosphorylase activity generally corresponds to the glycogenic activity or glycogen content of the cells. In sheep (41), chickens (.do), rats (@), pigeons, and humans ( 4 3 ) , both pyrophosphorylase activity and glycogenolysis rise during fetal life, reaching a peak near birth or hatching. Activity then falls as much as 60% as the animal ages. Silkworm ovary tissue shows high pyrophosphorylase activity near the middle of the pupal stage, which corresponds to rapid enzymic conversion of blood trehalose to glycogen (44). In tumor tissue, which characteristically has high glycolytic activity and decreased glycogen content, the pyrophosphorylase activity values are as much as 50-60% less than those of normal tissues (46). Brain tissue is the exception to the rule in that it has a low specific activity of UDPglucose pyrophosphorylase but a high glycogen content (39). 37. H. Flodgaard, Eur. J . Biochem. 15, 273 (1970). 38. P. Newel1 and M. Sussman, J M B 49, 627 (1970). 39. C. Villar-Palasi and J. Lamer, ABB 86, 270 (1960). 40. M. T. Rinaudo, C. Giunta, M. L. Boazi, and R. Bruno, Enzymologia 36, 321 (1969). 41. F. J. Ballard and I. T. Oliver, BJ 95, 191 (1965). 42. F. J. Ballard and I. T. Oliver, BBA 71, 578 (1963). 43. K. J. Isselbacher, Science 126, 652 (1957). 44. 0. Yamashita, J . Sericult. SOC.Jup. 38, 329 (1969). 45. V. N. Nigam, H. L. MacDonald, and A. Cantero, Cancer Res. 22, 131 (1962).

56

RICHARD L. TURNQUIST AND R. GAURTH HANSEN

While most of the UDPglucose pyrophosphorylase in animal cells is found in solution in the cytoplasm (25, 44, @), about 5-10% is bound to the microsomal fraction. It has therefore been concluded that glucose 1-P is constantly being recycled into glycogen and that this mechanism may help regulate glycogen storage ( 4 7 ) . In higher plants, enzyme level variations are usually associated with a change in capacity for starch and sucrose synthesis ( 4 8 4 2 ) . In lower plants, the enzymic activity may vary with cellulose or trehalose formation (bS, 6 4 ) . In plant cells, too, UDPglucose pyrophosphorylase has been found fully (65) or mostly (56, 57) dissolved in the cytoplasm. The enzyme may be especially concentrated in chloroplasts and starch granules (8, 68, 5 9 ) . Pyrophosphorylase activity may be markedly changed by physiological manipulation. Sugar beets synthesize increased amounts of the enzyme when pyrocatechol or vanadyl sulfate is applied to the foliage (60, 61). Tri-iodothyronine will significantly increase the pyrophosphorylase activity in hypothyroid rat muscle (62) while 1311 will lower enzyme levels in the same tissue. Riboflavin deficiency will also lower pyrophosphorylase levels in rats (6s).Meal fed rats have higher enzymic activities in muscle (50%) and adipose (300%) tissues than do nibbling rats (64, 6 5 ) . 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.

E. Reid, BBA 32, 251

(1959).

V. T. Maddaiah and N. B. Madsen, Can. J . Biochem. 46, 521 (1968). J. F. Turner, Awt. J . Biol. Sci. 22, 1321 (1969). J. F. Turner, A w t . J . Biol. Sci. 22, 1145 (1969). R. Pressey, Plant Physiol. 44, 759 (1969). C. Y. Tsai, F. Salamini, and 0. E. Nelson, Plnnt Physiol. 46, 299 (1970). K. C. Tovey and R.M. Roberts, P h n t Soil 48, 406 (1970). B. E. Wright and M. L. Anderson, BBA 31, 310 (1959). K. Zetsche, 2. Naturjorsch. B 23, 369 (1968). A. E. S. Gussin and J. H.McCormnck, Phytochemistry 9, 1915 (1970). M. A. Hall and L. Ordin, Plant Physiol. 40, Suppl. XXXVIII (1965). M. A. Hall and L. Ordin, Physiol. Plant 20, 624 (1967). I. F. Bird, H. K. Porter, and C. R. Stocking, BBA 100, 366 (1965). W. A. Huber, M. A. R. de Fekete, and H. Zieglcr, Planta 87, 360 (1969). 60.B. Singh and D. J. Wort, Plant Physiol. 44, 1321 (1969). 61. B. Singh and D. J. Wort, Physiol. Plnnt 23, 920 (1970). 62. C. Pitra, E. G . Krause, and A. Wollenberger, Endokrinologie 54, 225 (1969). 63. H. B. Burch, 0. H. Lowry, M. E. Bradley, and P. F. Max, Jr., Anzei. J . Phyaiol. 219, 409 (1970). 64. J. H. Wiley and G . A. Leveille. Fed. Proc., F e d . Amer. SOC.Exp. B i d . 28, 625 (1969).

65. J. H. Wiley and G . A. Leveille, J . N u t i . 100, 85 (1970).

2.

DRIDINE DIPHOSPHORYL GLUCOSE PYROPHOSPHORYLASE

57

B. METABOLISM A prime function of UDPglucose pyrophosphorylase in most animal cells is to activate glucosyl residues for the synthesis of glycogen. Although glycogen synthesis is obviously not solely dependent upon the activity of pyrophosphorylase, changes in pyrophosphorylase activity may affect glycogen levels ( 6 6 ) . Pathological conditions that foster abnormally high concentrations of pyrophosphorylase may result in increased levels of stored glycogen ( 6 7 ) . Since UDPglucose inhibits glycogen phosphorylase, the increase in glycogen content probably results from a combination of enhanced synthesis and decreased catabolism (68). The role of UDPglucose pyrophosphorylase in the synthesis of cellulose in higher plants has been the subject of considerable controversy. While UDPglucose is the principal sugar nucleotide involved in the production of most plant polysaccharides, GDPglucose has generally been considered to be the glucosyl donor in cellulose production (69). Evidence now indicates, however, that in some plants, cellulose is produced either totally or in part from UDPglucose (57, 70-73). In lower plants and microorganisms cellulose is synthesized with UDPglucose as the glucosyl donor (33, 7 4 ) , but the identity of the ultimate donor in higher plants awaits further clarification. A similar controversy exists in relation to the biosynthesis of starch. Both UDPglucose and ADPglucose have been implicated as the glucosyl donor (8, 75-77). While differences exist between plants and even within the same plant (78) both nucleotides and their respective pyrophosphorylases are probably involved. Sucrose, which is the primary source for starch production, is converted by sucrose synthetase to UDPglucose 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78.

R. Kornfield and D. H. Brown, JBC 238, 1604 (1963). G. Okuno, S. Hisukuri, and M . Nishikawa, Nature (London) 212, 1490 (1966). N. B. Madsen, BBRC 6, 310 (1961).

W. Z. Hassid, Science 165, 137 (1969). D. 0. Briimmond and A. P. Gibbons, Biochem. 2. 342, 308 (1965). L. Ordin and M. A. Hall, Plant Physwl. 42, 205 (1967). L. Ordin and M. A. Hall, Plant Physiol. 43, 473 (1968). G. Franz, Phytochemistry 8, 737 (1969). C. Ward and B. E . Wright, Biochemistry 4, 2021 (1965). L. F. Leloir, M. A. R. de Fekete, and C. E. Cardini, JBC 236, 636 (1961). L. F. Leloir, BJ 91, 1 (1964). T. Murata, T. Sugiyama, and T. Akasawa, A B B 107, 92 (1964). Y. Tanaka and T. Akazawa, Plant Cell Physiol. 9, 405 (1968).

58

RICHARD L. TURNQUIST AND R. GAURTH HANSEN

and fructose. The UDPglucose is converted by pyrophosphorylase to glucose 1-P, and then to ADP glucose, which seems to be the immediate glucosyl donor for starch formation (48,49, 79,80). Since in some plants there is evidence that UDPglucose is the only nucleotide involved in starch synthesis, two different mechanisms may be operating. I n maize endosperm (61), sucrose is hydrolyzed by invertase to glucose and fructose. The glucose is converted to glucose 6-P and then to glucose 1-P which in turn is converted by UDPglucose pyrophosphorylase to UDPglucose. In this endosperm, the UDPglucose can be used directly in the synthesis of starch. In other plants, sucrose may be converted by sucrose synthetase to UDPglucose which is then converted to glucose 1-P by pyrophosphorylase as in normal starch production. Here, however, the glucose 1-P is converted directly into starch by starch phosphorylase without first being incorporated into ADPglucose (60, 81). Both of these latter pathways occur in young plants and seem to be temporary until ADPglucose pyrophosphorylase is present. Uridine diphosphoryl glucose is also the glucosyl donor for sucrose production which utilizes essentially the reverse pathway of starch synthesis (82, 83).The glucose 1-P resulting from starch phosphorolysis is coupled with fructose through intermediate formation of UDPglucose via the pyrophosphorylase pathway (84, 86). Uridine diphosphoryl glucose pyrophosphorylase is also necessary for the metabolism of galactose. In the normal galactose metabolism pathway, the enzyme galactose 1-P uridylyltransferase (EC 2.7.7.12) utilizes UDPglucose to convert galactose 1-P to UDPgalactose. However, the (a)

Galactose

(b) Gal-1-P (r)

+ ATP

+ UDP-Glc TJDP-Gal

kinase

transferase

+ ADP TJDP-Gal + Glr-1-P Gal-1-P

epimersse

(2)

UDP-Glr

pyrophosphorylase pathway has the capacity to synthesize UDPgalactose even in the absence of the transferase (86). Without UDPglucose 79. M. A. R . de Fekete and C. E. Cardini, A B B 104, 173 (1964). 80. T. Murata, T. Sugiyama, T. Minamikawa, and T. Akanawa, ABB 113, 34 ( 1966). 81. M. A. R. de Fekete, Planta 87, 311 (1969). 82. C. E. Cardini, L. F. Leloir, and J. Chiriboga, JBC 214, 149 (1955). 83. M. A. R . de Fekete, Planta 87, 324 (1969). 84. D. P. Burma and D. C. Mortimer, ABB 62, 16 (1956). 85. M. D. Hatch, J. A. Sacher, and K. T. Glasaiou, Plant Physiol. 38, 338 (1963). 86. T. Sawicka m d T. Chojnachi, Clin. Chim. Acta 23, 463 (1969).

2.

URIDINE DIPHOSPHORYL GLUCOSE PYROPHOSPHORYLASE

+ ATP kinaae Gal-1-P + ADP pyrophoephorylase Gal-1-P + UTP , ’ UDP-Gal + PPi

59

(a) Galact,ose (b) (c)

UDP-Gal

(d) UDP-Glc

.

epimerase

’ UDP-Glc

pyrophoephorylase

+ PPi ,

(3)

’G l e l - P

+ UTP

pyrophosphorylase, galactose will not be incorporated into microbial cell walls (87-89). Uridine diphosphoryl glucose pyrophosphorylase participates in the synthesis of numerous other compounds including various cell wall po1y.mers in both higher plants (90-92) and microorganisms (88,93), trehalose (94), glycosides (96),glycolipids (96), heparin (97), microbial antigens (98), lactose (20), glucuronides (99, IOO), and rhamnose (87, 101).

C. METABOLIC REGULATION Since UDPglucose participates in numerous metabolic pathways, the enzyme that catalyzes its synthesis may not be subject to extensive metabolic control. Experimental results tend to bear this out. While UDPglucose pyrophosphorylase is subject to product inhibition like many other enzymes, additional controls on either its synthesis or the reaction it catalyzes are minimal. I n plants, the administration of indole acetic acid (auxin) may cause 87. T. A. Sundararajan, A. Rapin, and H. M. Kalckar, Proc. Nut. Acad. Sci. U . S. 48, 2187 (1962). 88. T. Fukasawa, K. Jokura, and K. Kurahashi, BBRC 7, 121 (1962). 89. T. Fukasawa, K. Jokura, and K. Kurahashi, BBA 74, 608 (1963). 90. D. S. Feingold, E. F. Neufeld, and W. Z. Hassid, JBC 233, 783 (1958). 91. S. H. Goldemberg and L. R. Marechal, BBA 71, 743 (1963). 92. 0. A. Pavlinova and M. F. Prasblova, Fiziol Rast. 17, 295 (1970). 93. M. Lieberman, C. Buchanan, and A. Markovitz, Proc. Nut. Acad. Sci. U. S.

65, 625 (1970). 94. R. Roth and M. Sussman, JBC 243, 5081 (1968). 95. G. Franz and H. Meier, Planta Med. 17, 396 (1969). 96. J. A. Curtino, R. 0. Calderon, and R. Caputto, Fed. Proc., Fed. Amer. SOC. E z p Biol. 27, 346 (1968). 97. I. Danishefsky and 0. Heritier-Watkins, BBA 139, 349 (1957). 98. R. L. Bernstein and P. W. Robbins, JBC 240, 391 (1965). 99. M. Shikamnra and K. K. Tsuboi, Amer. J . Dis. Child. 102, 600 (1961). 100. R. M. Roberts and K. M. K.Rao, Fed. Proc., Fed. Amer. SOC.Exp. Bid. 30, 1117 (1971). 101. G. A. Barber, ABB 103, 276 (1963).

60

RICHARD L. TURNQUIST AND R. GAURTH HANSEN

a large increase in the rate of glucose incorporation into cell wall polysaccharides, but the effect seems to be on enzymes other than UDPglucose pyrophosphorylase (102, 103). Stimulation of the autonomic nervous system, either sympathetic or parasympathetic, will affect glycogen metabolism in the mammalian liver, but it is glycogen synthetase which is affected and not UDPglucose pyrophosphorylase (104, 106). Insulin was tested in mammals with contradictory results. The hormone was reported to have no effect on pyrophosphorylase levels (106), but the lack of it, caused by alloxan treatment, decreased UDPglucose pyrophosphorylase activity in rat salivary glands by as much as 70% (107).It was speculated that insulin might exert a regulatory effect on uridylyltransferases. Generally, however, the control on UDPglucose pyrophosphorylase activity is genetic, regulating the amount of enzyme synthesized. Most of the genetic studies were carried out in microorganisms, especially Escherichia coli, and the location of the UDPglucose pyrophosphorylase structural gene was precisely determined (93, 108). The gene is not located in the gal operon (89, 109). Uridine diphosphoryl glucose pyrophosphorylase activity increases dramatically but unequally during cap formation in Acetabularia. Enzymic activity appears to be concentrated in the apical cells and decreases basally. The gradient is not the result of activators or inhibitors but rather of actual variations in the total amount of enzyme produced by differential synthesis. This is true even in anucleate cells, indicating that the synthesis may be regulated in the cytoplasm a t the level of translation of long-lived messenger RNA (641. The unequal distribution of the pyrophosphorylase may be the result of a messenger RNA gradient toward the apex of the stalk, implying a migration of the RNA from the nucleus in the rhizoid (110). Investigations into the metabolic control of UDPglucose pyrophosphorylase in the slime mold, Dictyostelium discoideum, have generated considerable controversy. As the organism differentiates to the plasmodium stage, large amounts of protein disappear, carbohydrate synthesis 102.. A. Abdul-Baki and P. M. Ray, Plant Physiol. 42, Suppl., S-4 (1967). 103. A. Abdul-Baki and P. M. Ray, Plant Physiol. 47, 537 (1971). 104. T. Shimazu and T. Fujimoto, BBA 252, 18 (1971). 105. T. Shimazu, BBA 252, 28 (1971). 106. C. Villar-Palasi and J. Larner, ABB 94, 436 (1961). 107. T. Szymczek, B. Swiatkowska, and M. Jachimowicz, Acta Biochim. Pol. 18, 177 (1971). 108. J. A. Shapiro, J . Bacteriol. 92, 518 (1966). 109. F. Jacob and J. Monod, J M B 3, 318 (1961). 110. K. Zetsche, Planta 89, 244 (1969).

2.

URIDINE DIPHOSPHORYL GLUCOSE PYROPHOSPHORYLASE

61

increases, and there is an apparent increase in the specific activity of UDPglucose pyrophosphorylase (53,111). In the 18-hr maturation process, the specific activity of the enzyme was reported to increase as much as tenfold (112, 113). A large increase in enzymic activity resulting in elevated carbohydrate metabolism was reported (74, 114). Serious questions were subsequently raised as to whether increased pyrophosphorylase activity exerts an effect on carbohydrate metabolism or whether the pyrophosphorylase activity increases a t all. Experiments indicated that the specific activity of the enzyme increases only slightly (11 5 ) , and reports of large increases measured in vitro resulted from artifacts such as enzyme instability and the effects of different substrate concentrations (116, 117). Kinetic considerations indicate that even if the pyrophosphorylase levels did increase, this alone could not cause the observed increase in UDPglucose synthesis (118), which may be simply the result of increases in the levels of UTP and glucose 1-P which are substrates for the pyrophosphorylase (119). By contrast it has been argued that the increases in enzyme levels are real and not resulting from differential stability. The kinetic models are somewhat suspect in that substrate molecules do not occur randomly throughout the cytoplasm but are compartmentalized and concentrated (1go). Cycloheximide and actinomycin D prevent the enzyme increase, showing that it is dependent upon new protein synthesis (112). Uridine diphosphorylglucose pyrophosphorylase accumulation, controlled at the level of genetic transcription, is linked with and necessary for the morphological changes that occur during differentiation (38, 121). Enzyme concentrations, however, are rarely the limiting factors in metabolic reactions; substrate availability usually determines reaction rates. Computer kinetic models of slime mold differentiation have indicated that changes in the pyrophosphorylase concentration over a wide range would have little or no effect on UDPglucose synthesis. If enzyme is not limiting, the reaction will 111. J. M. Ashworth, BJ 106, 28p (1968). 112. J. M. Ashworth and M. Sussman, JBC 242, 1696 (1967). 113. P. C.Newell, J. S. Ellingson, and M. Sussman, BBA 177, 610 (1969). 114. R. Roth, J. M. Ashworth, and M. Sussman, Proc. Nut. Acad. Sm’. U. S. 59, 1235 (1968). 115. R. G. Pannbacker, Biochemistry 6, 1287 (1967). 116. B. Wright and D . Dahlberg, J. Bacteriol. 95, 983 (1968). 117. R. Marchall, D.Sargent, and B. E. Wright, Biochemistry 9, 3087 (1970). 118. B. Wright, W. Simon, and B. T. Walsh, Proc. Nut. Acud. Sci. U . S. 80, 644 (1968). 119. B. E. Wright, J . Cell. Physiol. 72, Suppl. 1, 145 (1968). 120. P. C. Newell and M. Sussman, JBC 244, 2990 (1969). 121. P. C. Newell, M. Longlands, and M. Sussman, J M B 58, 541 (1971).

62

RICHARD L. TURNQUIST AND R. GAURTH HANSEN

only go as fast as the rate a t which substrate is made available (122). However, the enzyme increase may be necessary to overcome UDPglucose product inhibition and allow the observed changes in glucose 1-P levels ( 2 7 ) . The value of these models has been questioned since many parameters may not be accurately known and their estimation might lead to serious error in the results. It is now generally agreed that UDPglucose pyrophosphorylase activity does increase in the slime mold during differentiation, but the effect that this increase has on carbohydrate metabolism is still the subject of debate. 111. Properties

A. OPTIMA All of the UDPglucose pyrophosphorylases that have been studied show an absolute requirement for a divalent cation. Magnesium a t 1-3 mM seems to satisfy this requirement best, while Mnz+, Coz+,and NiZ+ have about one-fourth the optimal activity. The cations become inhibitory a t high concentrations. Mercaptoethanol or dithiothreitol is required for human liver ( 1 7 ) , erythrocyte ( 2 1 ) , and mung bean (19) enzymes to protect them from oxidation. While most UDPglucose pyrophosphorylases can be isolated and stored a t 4", the enzyme from E . coli was found to be labile a t 0" (98). The optimal pH range for activity of the various UDPglucose pyrophosphorylases is broad and generally is slightly alkaline, as follows: yeast, 6.5-8.0 (31) ; E . coli, 7.5-9.0 (28); slime mold, 7.8 (22); pea seedlings, 7.5-9.0 (123); mung bean, approximately 8 (19) ; bovine mammary tissue, 8-9 (20); rat mammary tissue, 7.5-9 (124); human erythrocytes, 8-9 (21); human liver, 7.6-9.2 (17); rabbit muscle, 6.5-8 (26); and calf liver, 8.5 (16).

B. STRUCTURE The molecular weight. and multimeric structure of calf liver UDPglucose pyrophosphorylase have been thoroughly studied. A molecular 122. B. E. Wright,

Behav. Sci. 15, 37 (1970). 123. D. H. Turner and J. F. Turner, BJ SO, 448 (1958). 124. D. K. Fitzgerald, S. Chen, and K. E. Ebner, BBA 178, 491 (1969).

2.

URIDINE DIPHOSPHORYL GLUCOSE PYROPHOSPHORYLASE

63

weight of 480,000 was reported, with eight probably identical subunits of molecular weight 60,000 (125).Electron micrographs support these figures (Fig. 1 ) . Ultracentrifugation and other studies indicate that multimers of the enzyme exist. These are probably dimers, trimers, and tetramers of the 480,000 species. The crystalline form of the enzyme is diamond-shaped (16). The enzymes isolated from human liver and human erythrocytes appear to be similar, if not identical, and not greatly different from the calf liver enzyme. The human enzymes are of slightly smaller molecular weight, about 440,000 @ I ) , and, like the calf enzyme, appear to dimerize (17).The human liver enzyme crystallizes as a long needle. R a t mammary gland pyrophosphorylase appears to be similar in size, with a reported molecular weight of 450,000 (124). This enzyme increases markedly in activity during incubation of organ explants and tissue extracts. The increase is not dependent upon hormonal activity and is not inhibited by puromycin. The molecular weight does not change, nor does the K , for UTP, although the V,,, increases markedly. The increase in activity is a function of pH, temperature, and enzyme concentration and is inhibited by urea. The protein molecule appears to undergo structural changes that lead to a more active form of the enzyme. The slime mold, D . discoideum, appears to have two forms of UDPglucose pyrophosphorylase. The largest fraction (90%) has a molecular weight of 390,000 with subunits of about 55,000 (22). Another lighter fraction comprising about 10% of the total activity has also been found (190).The two fractions are not interconvertible and the lighter is much more labile. The different forms may provide UDPglucose for different metabolic functions and thus may be separately controlled. In the microorganism SaZmoneZZa typhimurium, UDPglucose pyrophosphorylase exists in different forms, which have been designated 11, IIIa, and IIIb. A fourth form, IV, is found in certain mutants. The four enzymes differ significantly in their reaction kinetics, pH optima, and heat stability, but all have UDPglucose pyrophosphorylase activity (1266). Two genes are involved in the synthesis of the enzyme. When one gene (gal F ) is deleted, the only form that appears is IV. The second gene (gal U ) is a structural gene that codes for a polypeptide found in all forms of the enzyme. The presence of the gal F gene modifies the basic polypeptide into forms 11, IIIa, and IIIb (127).The molecular 125. S. Levine, T. A. Gillett, E. Hageman, and R. G. Hansen, JBC 244, 5729 (1969). 126. T. Nakae and H. Nikaido, JBC 246, 4386 (1971). 127. T. Nakae and H. Nikaido, JBC 248, 4397 (1971).

FIQ.1. Crystalline (top) and molecular (625,OOOX) structure of bovine liver UDPglucose pyrophosphorylase. 04

2.

URIDINE DIPHOSPHORYL GLUCOSE PYROPHOSPHORYLASE

65

FIG.2. hterconversion of enzyme monomers and dimer.

weights of isozymes 11, IIIa, IIIb, and I V are 40,000, 40,000, 8O,OOO, and 8O,OOO, respectively. Probable interconversions are shown in Fig. 2. The present thinking is that gal U codes for the basic 80,000molecular weight dimer (IV), which is stable. I n the presence of gal F , however, the enzyme is split into two molecules of monomer IIIa. Form I I I a can be converted to dimer I I I b or into monomer I1 by cell factors that are not associated with either gene and can be reproduced in vitro (128). Since all forms have different UDPglucose inhibition constants, the variety of isozymes may serve some regulatory function.

C. KINETICS The substrate affinities for UDPglucose pyrophosphorylase from various sources are indicated by the Michaelis constants shown in Table I (129-131). Turnover numbers vary from 83,000 for calf liver enzyme (16) to 8,900 for slime mold pyrophosphorylase (22). Table I1 shows inhibition constants for UDPglucose pyrophosphorylase. Uridine diphosphoryl glucose shows a highly selective product inhibition, especially in animal cells, and therefore probably exerts considerable self-regulation in metabolic control. The dissimilarity of inhibition constants between mammalian and plant enzymes suggests a difference of biological significance ( 2 1 ) . Several workers have found competitive inhibition between UDPglucose and UTP, and noncompetitive inhibition between U T P and PPi (17,21, 22) ; PPi shows noncompetitive product inhibition with both substrates in the slime mold ( 2 2 ) and is inhibitory in high concentra128. 129. 130. 131.

T. Nakae, JBC 246, 4404 (1971). I. T. Oliver, BBA 52, 75 (1961). G. L. Gustafson and J. E. Gander, JBC 247, 1387 (1972). A . Munch-Petersen, Acta Chem. Scand. 9, 1523 (1955).

66

RICHARD L. TURNQUIST AND R. GAURTH HANSEN

TABLE I SUBSTRATE AFFINITIES FOR UDPQLUCOSE PYROPHOSPHORYLASE FROM VARIOUS SOURCES K, X 106 Ensymesource Human liver Human erythrocyte Human erythrocyte Calf liver Bovine mammary Rat mammary Rat liver Guinea pig brain Rabbit muscle Dog heart Mung bean Mung bean Pea seeds Wheat Sorghum Slime mold Yeast Yeast E . wli E. w2i S . typhimurium Form I1 Form IIIa Form IIIb Form IV

Ref. K,,+UDPG i7 91 86 16 I0 94 199 i29 2% 91 19 Ii iI.9 62 130 $9 131

0.15 0.26 0.28-0134

G-1-P 9.5 17 6.7 5.5 11 39 18-28 4.5-8.6 16

UTP

PPi

4.8 33 3.3 20 14

21 48

6

10

4.5 2.8 11 11

23

8.5

100 0.25-0.33 1

4.8 26

3.0 11

4.8

2.9

3.4 2.9

7.4 14.4

3.6

15.9

5.4 44

98

98 28 126

5 2.3

8.4 100

36

0.14

UDPG

0.2

310 13

5.6 17 7 9 25 13 84 67 37 27

tions as a substrate in E. coli (98). The product inhibition by PPi of the slime mold enzyme is suggested as a possible alternate regulatory mechanism for the alleged changes in intracellular substrate concentration (28). Calf liver enzyme appears to be sensitive to Pi,which inhibits competitively with PPi at physiological concentrations. This may provide a mechanism whereby an increase in Pi,as the result of energy drain and hydrolysis of ATP,would result in a lowering of UDPglucose production and a redirection of glucose metabolism from storage to glycolysis. The inhibition by UDP, a product of glycogen synthesis, may also be significant in vivo in that it must be converted to UTP, an energy requiring reaction, for glycogen synthesis to proceed. If energy (ATP)reserves are low, glycogen synthesis would be inhibited ( 1 6 ) .

2.

67

URIDINE DIPHOSPHORYL GLUCOSE PYROPHOSPHORYLASE

TABLE I1 FOR UDPQLUCOSE PYROPHOSPHORYLASE INHIBITION CONSTANTS Product Ki Enzyme source

Ref.

Human liver Human erythrocyte Calf liver Dog heart Mung bean Sorghum Slime mold S. lyphimurium Form I1 Form IIIa Form IV

17 81

UTP

x

UDPG

10

1.5

8 7

2.3

81 130 97 186

10.4

Pi with PPi

UDP with UDPG 10

370

16

81

Ki X 106

106

15

16 5.0 5

10 6.3 1.3

3000-9000

400

Uridine diphosphoryl glucose is necessary for the metabolism of galactose. Galactose 1-P in high concentrations (50-fold excess over glucose 1-P) will competitively inhibit the pyrophosphorylase (129). Inhibition of the enzyme slows production of UDPglucose, which causes further accumulation of galactose 1-P, which in turn further inhibits the enzyme. Thus a cyclic process is established in which a compound inhibits its own metabolism (132, 133). D-Galactosamine is also a competitive inhibitor with glucose 1-P. It, too, requires UDPglucose for its metabolism; thus, a similar inhibition cycle is established (134, 136). The loss of UDPglucose not only affects the metabolism of the inhibitory compounds but also reduces glycogen, polysaccharide, and glucuronide metabolism, Certain pathological conditions may increase the concentrations of these inhibitors enough for them to limit UDPglucose synthesis (129). In E . coli, both UDPglucose and TDPglucose are needed for the first steps of synthesis of the polysaccharide component of antigens. Uridine diphosphoryl glucose pyrophosphorylase is inhibited by TDPglucose ( K i= 2 x The inhibition may serve a useful purpose in that blockage of later stages of polysaccharide synthesis by phages will result in the accumulation of both UDPglucose and TDPglucose. The cross 132. E. L. Talman, Physiol. Chem. Phys. 1, 131 (1969). 133. E. L. Talman, Physiol. Chem. Phys. 1, 255 (1969). 134. D. Keppler and K. Decker, Eur. J . Bbchem. 10, 219 (1969). 135. D. 0. R. Keppler, J. F. M. Rudigier, E. Bischoff, and K. Decker, Eur. J . Biochem. 17, 246 (1970).

68

RICHARD L. TURNQUIST AND R. GAURTIl HANSEN

inhibition may reinforce normal product inhibition and thus more effectively shut down early synthetic steps. The enzyme is also inhibited by TDPrhamnose (Ki = 5.2 X which is a final product of carbohydrate metabolism in the organisms (98). Chloroazanil, a triazine derivative, has also been found to be an inhibitor of UDPglucose pyrophosphorylase (136).

D. SPECIFICITY Considering its relatively high turnover number, its favorable equilibrium, and its low K,,, values for glucose 1-P and UTP, the prime function of UDPglucose pyrophosphorylase is probably to catalyze the formation of UDPglucose. However, the enzyme shows activity toward other substrates as well. Calf (16) and human (17)liver enzymes have been used to catalyze the phosphorylation of UDPglucose, TDPglucose, CDPglucose, GDPglucose, UDPgalactose, UDPxylose, and UDPmannose. Therefore, the enzyme is not absolutely specific for either the base or the sugar moiety of the substrate. The percent of initial reaction velocities compared to UDPglucose varies from 2.2% for TDPglucose to 0.1% for GDPglucose (17). Reaction rates of (‘abnormal” substrates may be increased if concentrations are raised. While UDPgalactose is pyrophosphorylated by human liver enzyme at about 2% of the rate of UDPglucose at equal substrate concentrations, the rate may be increased to as much as 10% a t higher concentrations (17). This nonspecificity of human UDPglucose pyrophosphorylase has led to postulations about its physiological importance in patients with galactosemia. Galactosemia is a molecular disease in which galactose is not metabolized because of a lack of galactose 1-P uridylyltransferase (EC 2.7.7.12). The result is an accumulation of galactose 1-P. There is, however, some conversion between glucose 1-P and UDPgalactose ( 1 S 7 ) , giving rise to the speculation that a UDPgalactose pyrophosphorylase (EC 2.7.7.10) might be present in human blood (138). Such activity has been described in microbes (139), rats, pigeons, and humans (138, 140). This enzyme might increase as a child ages, thereby lessening the symptoms of the 136. W. Kreutner and N. 0. Goldberg, Fed. Proc., Fed. Amer. SOC.E x p . Biol. 26, 508 (1967). 137. R. Gitzelmann, Pediat. Res. 3, 279 (1969). 138. H. D. Abraham and R. R . Howell, JBC 244, 545 (1969). 139. G. T. Zancan, Can. J . Microbwol. 17, 563 (1971). 140. X. J. Isselbacher, JBC 232, 429 (1958).

2.

URIDINE DIPHOSPHORYL GLUCOSE PYROPHOSPHORYLASE

69

disease (43). The existence of such an enzyme has been questioned, however, since UDPglucose pyrophosphorylase, which is present in galactosemics, will catalyze the same reaction. Further, in studies in which UDPgalactose pyrophosphorylase activity was reported, no effort was made to separate this from UDPglucose pyrophosphorylase activity (141). Additionally, the ratio of the two activities remained constant throughout purification from human liver. The ratio was also constant during sucrose gradient sedimentation and polyacrylamide gel electrophoresis, indicating that both reactions are catalyzed by the same enzyme (17). Uridine diphosphoryl galactose pyrophosphorylase activities reported for human erythrocytes have been quite low, about 1% of the UDPglucose pyrophosphorylase activity (21, 138), and some workers found no activity a t all (86, 142, 149). This is surprising since, even in the absence of a specific UDPgalactose pyrophosphorylase, UDPglucose pyrophosphorylase should show some activity. The lack of activity in these cases might result from insufficient concentrations of UDPgalactose or galactose l-P in the in vitro assay mixtures. Low concentrations of the substrates would not overcome the high K , values of UDPglucose pyrophosphorylase for these substrates. In a galactosemic victim, however, the galactose l-P concentration might be increased to a point where significant catalysis could occur. Based on the above evidence it would seem likely that UDPgalactose pyrophosphorylase is not present in human red blood cells, and the activity found is probably attributable to UDPglucose pyrophosphorylase.

E. MECHANISM The most detailed study of the mechanism of UDPglucose synthesis utilized calf liver enzyme. I n one set of experiments the enzyme was incubated with substrate or substrate analogs and chromatographed through Sephadex G-25-80 (144). Uridine diphosphoryl glucose and U T P formed stable complexes with the pyrophosphorylase, while UMP, PPi, UMP plus PPi, glucose l-P and UDP did not. Since UDPglucose and U T P were doubly labeled and both labels appeared in the enzymesubstrate complex, it is likely that the entire substrate molecules were 141. 142. (1964). 143. (1967). 144.

W. K. Ting and R. G. Hansen, Proc. SOC.Exp. Biol. Med. 127, 960 (1968). W. G. Ng, W. R . Bergren, and G. N. Donnel, Nature (London) 203, 845 W. G. Ng, W. R. Bergren, and G. N. Donnel, Clin. Chim. Acta 15, 489

T. A . Gillett, S. Levine, and R. G. Hansen, JBC 2 4 , 2551 (1971).

70

RICHARD L. TURNQUIST AND R. GAURTH HANSEN

bound. Only after prior incubation with UDPglucose or UTP could PPi be complexed with the enzyme, indicating an obligatory order of substrate binding. Magnesium was not required for the binding of UDPglucose but was necessary for binding PPi, which replaced glucose 1-P from the enzyme-UDPglucose complex. Thus, it appears an ordered Bi-Bi mechanism is most likely, with the nucleoside phosphate the first substrate to be added and the last product to leave, as follows: IJTP

+E

G-1-P

E(UTP)

11

Jt

E(UDPG)

+ MgPPi

E

+ UDPG

(4)

Mg*+

Human erythrocyte pyrophosphorylase seems to have a similar mechanism ( 2 1 ) . Uridine diphosphoryl glucose and UTP inhibit one another competitively, while the other substrate-product combinations show noncompetitive inhibition. A distinguishing characteristic of the ordered Bi-Bi mechanism is competitive inhibition between the first substrate added and the last product released ( 1 4 6 ) . Calculating the equilibrium constant from experimental kinetic data according to a Haldane relationship was also consistent with an ordered Bi-Bi mechanism. Although there is considerable variation in kinetics and enzyme structure, UDPglucose pyrophosphorylases from yeast (16), mung bean (4, 19, d l ) , dog heart (.%?I), and slime mold (29) also appear to catalyze the formation of UDPglucose by the mechanism described in Eq. (4). Metal activated enzymes such as UDPglucose pyrophosphorylase may bind metal and substrate in one of four possible coordination schemes. These include metal bridge complexes (E-M-S) that may be either simple or cyclic, enzyme bridge complexes (M-E-S) , and substrate bridge complexes (E-S-M) (14.6’). Calf liver UDPglucose pyrophosphorylase appears to form a substrate bridge complex with the substrate serving as the only attachment of the metal to the enzyme. Several lines of evidence indicate this. Complexes of this type are generally limited to enzymes with nucleoside di- and triphosphate substrates that show high affinity for the metal. Since the enzyme will bind substrate equally well with or without the metal, simple and cyclic metal bridge complexes tend to be ruled out. The metals in substrate bridge complexes will not be bound to the enzyme unless the substrate is present. Studies measuring longitudinal proton magnetic relaxation rates (PRR) have demonstrated little or no enhancement unless all three complex components 145. W. W. Cleland, BBA 67, 104 (1963). 146. A. S. Mildvan, “The Enzymes,” 3rd ed., Vol. 2, p. 445, 1970.

2.

71

U R I D I N E D I P H O S P H O R Y L GLUCOSE PYROPHOSPHORYLASE

were present (147’). Calcium will often inhibit enzymes that form metal bridge complexes (E-M-S), and it can serve as an activator of substrate bridge enzymes. Calcium will serve as an alternative activator for UDPglucose pyrophosphorylase (148). Based on the above evidence, the divalent cation seems to activate the phosphorous atom of the substrate that is to be attacked by deshielding the 8- and 7-phosphorous atoms, thus making them more susceptible to substitution. The phosphate ligands probably complex with the metal by replacing its coordinated water molecules (146). Two pieces of PRR evidence, however, do not fit the above mechanism. First, there is no indication that an enzymepyrophosphate-metal complex will form. Second, when the E-S-M complex forms, free metal is released into solution (147).This indicates that more UTP is bound than metal, which makes a substrate bridge complex unlikely. Two possible explanations have been offered (149). First, an enzyme bridge complex (M-E-S) may form in which the binding of UTP weakens the metal binding by causing changes in enzyme conformation allowing the metal to be released. Such a situation, however, would be expected to allow the enzyme to bind the metal whether the substrate was present or not. Evidence indicates this does not occur. A, second, more attractive explanation is that an E-S-M complex forms in which the metal is bound more strongly to the nucleotide before the ternary complex is formed. The metal may be bound to the UTP in a tridentate (Y,p, y coordination. When the metal-UTP binds to the enzyme, the metal shifts to coordination which is much weaker than the tridentate coordination. (Y

.A/

M 0 I

b o\’I

I R-0-P-0-P-0-P-0 I I

0 f

0

f

P

+enzyme

I

-enzyme

0 Y

0

- R-0-P-0I

I

I

-P-O-P-O I

0 f

0

0 f

P

I I 0 Y

The weaker bond may allow some of the metal to be released from the complex. Neither of the above mechanisms has been proved, but the accumulated evidence would make the second more likely. ACKNOWLEDQMENT

This work was supported by National Institutes of Health Grant AM13709.

147. G. H.Reed, H. Diefenbach, and M. Cohn, JBC 247, 3066 (1972). 148. R. G. Hansen, unpublished observations. 149. A. S. Mildvan and M. Cohn, Advan. Enzymol. 33, 1 (1970).