15 Nucleotide Phosphomonoesterases

Nucleotid e Phosphomonoesterases GEORGE I. DRUMMOND

MASANOBU YAMAMOTO

I . 5'-Nucleotidase . . . . . . . . . . . . . A . Bacterial 5'-Nucleotidase . . . . . . . . . B . Yeast iY-Nucleotidase . . . . . . . . . . C . Snake Venom 5'-Nucleotidase . . . . . . . . D. Bull Seminal Plasma 5'-Nucleotidase . . . . . . . . . . . . . . . E . Liver 5'-Nucleotidase F. Intestinal 5'-Nucleotidase . . . . . . . . . . . . . . . . G . 5'-Nucleotidase from Pituitary H . 5'-Nucleotidase from Nerve Tissue . . . . . . I . 5'-Nucleotidase from Cardiac Tissue . . . . . . J . 5'-Nucleotidase from Other Vertebrate Tissues . . . K . 5'-Nucleotidase from Ehrlich Ascites Tumor Cells . . . L. 5'-Nucleotidase from Potatoes . . . . . . . M . Comparison of the Enzymes . . . . . . . . 11. 3'-Nucleotida~e . . . . . . . . . . . . . . . . . . . . . A . Rye Grass 3'-Nucleotidase B. M u g Bean 3'-Nucleotidase . . . . . . . . C . 3'-Nucleotidase from Wheat Seedlings . . . . . . D . 3'-Nucleotidase from Microorganisms . . . . . .

337 338 341 342 342 343 345 346 346 347 348 348 349 349 352 353 353 353 354

.

I S'-Nucleotidase

5'-Nucleotidase (5'-ribonucleotide phosphohydrolase. EC 3.1.3.5) is widely distributed in nature and a voluminous literature has appeared in the past decade on the enzyme from vertebrate tissues. seminal fluid. snake venoms. yeasts. and bacteria . Studies regarding the discovery and early investigations of the enzyme have been reviewed by Heppel ( 1 ) and

.

1 . L . A . Heppel. "The Enzymes. " 2nd ed., Vol . 5. p 49. 1961.

337

338

G. I. DRUMMOND AND M. YAMAMOTO

more recently by Bodansky and Schwartz ( 2 ) . The enzymes from these various sources have some properties in common ; however, numerous differences exist with respect to substrates and physical and chemical properties. Because of the diversity of studies each enzyme will be reviewed according to its source; this will be followed by a synopsis of the main similarities and contrasting features. A. BACTERIAL 5'-NUCLEOTIDASE In 1963, Kohn and Reis (3) first drew attention to the fact that extracts from many species of bacteria-Proteus, Hemophilus, Staphylococcus, Escherichia, and Clostridium-were capable of hydrolyzing both ribonucleoside 3'- and 5'-monophosphates. From their studies on relative activities with the two substrates, pH optima, and the effects of metal ions, they concluded that bacterial 3'- and 5'-nucleotidases were distinct and separate enzymes. Since that time both activities have been examined closely. Neu and Heppel ( 4 ) found that the 5'-nucleotidase of E . wltwas released into solution when spheroplasts were prepared with ethylenediaminetetraacetate (EDTA)-1ysozyme (6). The enzyme is also released from E. coli cells by osmotic shock. In this procedure, cells, preferably in the exponential growth phase suspended in hypertonic sucrose, are centrifuged and rapidly dispersed in a medium of low ionic strength (6). A number of degradative enzymes including 5'-nucleotidase, alkaline phosphatase, and cyclic phosphate diesterase are released into solution (7, 8) (see also Chapter 16 by Drummond and Yamamoto, this volume). Using the osmotic shock technique, Neu (9) has achieved a 5000-fold purification of 5'-nucleotidase from E. coli. The enzyme was pure as judged by molecular sieve chromatography, gel electrophoresis, and ultracentrifugation. A molecular weight of 52,000 was determined. The purified preparation hydrolyzes all 5'-ribo- and 5'-deoxyribonucleotides with preference for 5'-AMP. It does not attack 2'-AMP, 3'-AMP, cyclic 2',3'-AMP, or inorganic pyrophosphate. It appears unusual in that in addition to 5'nucleotides, adenosine triphosphate (ATP) , uridine diphosphate glucose, and bis (p-nitrophenyl) phosphate are hydrolyzed. The ratio of activities 2. 3. 4. 5. 6. 7. 8. 9.

0. Bodansky and M. K. Schwartz, Advan. Clin. Chem. 11, 277 (1968). J. Kohn and J. L. Reis, J Bacterial. 86, 713 (1963). H. C. Neu and L. A. Heppel, BBRC 17, 215 (1964). R. Repaske, BBA 30, 225 (1958). L. A. Heppel, Science 156, 1451 (1967). H. C. Neu and L. A. Heppel, JBC 240, 3685 (1965). N. G. Nossal and L. A. Heppel, JBC 241, 3055 (1968). H. C. Neu, JBC 242, 3896 (1967).

15.

339

NUCLEOTIDE PHOSPHOMONOEGTERASES

for these unrelated substrates remained constant during purification to apparent homogeneity; heat inactivation curves with respect to each substrate were parallel. These facts suggested that the various hydrolytic activities were associated with a single protein (9). This would seem to be a rather unusual enzyme since the substrates are quite unrelated and hydrolysis of bis (p-nitrophenyl) phosphate is usually indicative of diesterase activity. The 5’-AMP hydrolytic activity is stimulated 100-fold by 0.5 mM Co2+;it is inhibited by Zn2+and chelating agents. For stimulation of 5’-AMP and ATP hydrolysis, MnZ+can replace Co2+but Coz+is not needed for UDPG hydrolysis. Glaser et al. ( 9 a ) have studied extensively the nucleoside diphosphosugar hydrolase activity of E. coli and have also concluded that this activity and 5’-nucleotidase are associated with the same protein. Thus, the ratio of 5’-nucleotidase to UDP-sugar hydrolase activity remained constant over a 1000-fold range of purification and both were inactivated equally by heat treatment. These investigators showed that 14C-uridine-labeled UDP-D-glucose was hydrolyzed to uridine and inorganic phosphate without extensive mixing with a pool of nonlabeled 5’-UMP. This suggested an enzyme-bound complex of 5‘UMP as intermediate. Based on these findings they concluded that UDPG is hydrolyzed by the following sequence: UDP-D-glucose

+E

a-glucose-1-P

UDP-D-glucose-E

+

E-UMP

-+

It E + 5’-UMP

E

+ uridine + Pi

The enzyme also cleaves UDP-D-galactose and UDP-N-acetyl-D-galactosamine; other nucleosidediphosphate sugars containing adenine, guanine, and cytosine as the base are hydrolyzed at less than 5% of the rate of uridine nucleotides. The authors suggested (9a) that the enzyme is likely concerned with intracellular (rather than extracellular) UDPG, acting to maintain suitable levels of nucleotides in the cell. With such a wide range of specificity, the name 5’-nucleotidase for this bacterial enzyme seems inappropriate. When E. coli were grown in the presence of low concentrations of EDTA, a striking reduction in the activity of 5’-nucleotidase (also alkaline phosphatase and cyclic phosphate diesterase) occurred (10) and the data suggest that EDTA acts by binding a trace metal essential for activity. To investigate this further, E . coli were grown in the presence of s5Zn2+and subjected to osmotic shock (11).5’-Nucleotidase was purified from the shock fluid to apparent homogeneity. The purification was ac9a. L. Glaser, A. Melo, and R. Paul, JBC 242, 1944 (1967). 10. H. F. Dvorak, JBC 243, 2640 (1968). 11. H. F. Dvorak and L. A. Heppel, JBC 243, 2647 (1968).

340

G. I. DRUMMOND AND M. YAMAMOTO

companied by an enrichment with respect to 65Zn which could not be removed by dialysis. This and other evidence suggest that 5’-nucleotidase (and nucleoside cyclic phosphate diesterase) are metalloproteins, possibly zinc metalloproteins. The fact that 5’-nucleotidase is released into solution during spheroplast formation and during osmotic shock suggests a surface localization of the enzyme. Other evidence as to surface localization has also been provided. Thus, when an E . coli mutant (U-7a) which lacks alkaline phosphatase was grown on 2’-AMP as a carbon and phosphate source a severe lag in growth occurred (12). I n the presence of 5’-AMP growth took place rapidly. The inference was that 5‘-nucleotidase, being a surface enzyme, was capable of cleaving 5’-AMP to provide a carbon and phosphorus source for the cell. The possible location of these enzymes between the cell wall and cell membrane in the “periplasmic” space has been considered by Heppel ( 6 ) and is also discussed in Chapter 16 by Drummond and Yamamoto, this volume. Osmotic shock has also been used to release the enzyme from various Enterobacteriaceae: Shigella sonnei, Salmonella heidelberg, and Proteus vulgaris (13, 14). The enzyme from all these organisms exhibit properties similar to the E . coli enzyme with regard to the p H optimum, ion stimulation, substrate specificity, and physical properties. Mauck and Glaser (15) have recently purified a periplasmic enzyme from Bacillus subtilis which catalyzes the hydrolysis of several nucleosidediphosphate sugars ( ADP-glucose, GDP-glucose, GDP-mannose, CDP-glucose, etc.) to the corresponding nucleoside, inorganic phosphate and sugar phosphate. The enzyme shows 5’-nucleotidase activity and both activities seem to be catalyzed by the same protein. Unlike the E . coli enzyme, no divalent cations are required and it does not hydrolyze ATP. A specific protein inhibitor for 5’-nucleotidase has been purified from E. coli cell cytoplasm (10, 16). It prevents the action of the enzyme on 5’-AMP, ATP, and UDPG. It also inhibits the hydrolysis of 5’-AMP by the 5‘-nucleotidases from A . aerogenes, S. sonnei, and S. typhimurium (10). Other Enterobacteriaceae also possess similar intracellular protein inhibitors (13) which inhibit all hydrolytic activities of the 5’-nucleotidase of these organisms. The relevance of this inhibitor protein to the action of the enzyme in vivo is not known. 12. H.C.Neu, JBC 242, 3905 (1967). 13. H.C.Neu, Biochemistry 7, 3766 (1968). 14. H.C.Neu, J . Bacterbl. 95, 1732 (1968). 15. J. Mauck and L. Glaser, BiochemGtry 9, 1140 (1970). 16. H.F.Dvorak, Y. Anraku, and L. A. Heppel, BBRC 24,628 (1966).

15.

NUCLWTIDE

PHOSPHOMONOESTERASES

341

B. YEAST 5’-NUCLEOTIDASE A 5’-nucleotidase from yeast (Saccharomyces oviformis) has been purified to electrophoretic homogeneity and studied kinetically in detail by Takei (17-21). The enzyme hydrolyzes all ribo- and deoxyribonucleoside 5’-phosphates. It does not hydrolyze nicotinamide mononucleotide, 3‘- or 2’-AMP, sugar phosphates, or P-glycerol phosphate. Like the bacterial enzyme, yeast 5’-nucleotidase is markedly activated by Co2+and Ni2+ (18) and inhibited by EDTA. It was found that purified preparations of the enzyme possessed nucleotide pyrophosphatase (EC 3.6.1.9) activity. Substrates for the nucleotide pyrophosphatase are NAD, NADH2, FAD, ATP, and to a lesser degree, NADP and inorganic pyrophosphate (21). This activity could not be eliminated during purification of the 5’-nucleotidase to electrophoretic homogeneity (19). The pH profile of both activities were the same (pH 6.3-6.5) ; both activities were equally labile on heating a t temperatures between 40” and 60” for 5 min; their stabilities to UV irradiation and urea treatments were identical ; and both were similarly inhibited by N-bromosuccinimide, iodine, Zn2+, Ag+, and Cu2+ ( 2 1 ) . These data indicate that both 5’-nucleotidase and nucleotide pyrophosphatase reside in the same enzyme protein in S . oviformis. The enzyme seems to catalyze the catabolism of NAD in this organism as follows:

+ + adenosine + Pi + NhlN

(1) NAD -+ 5’-AMP NMS (2) 5’-AMP -+ adenosine I’,

(3) NAD

-+

The K , values for 5’-nucleotides are in the range of 0.2 mM for purine ribonucleotides and higher (2 mM range) for pyrimidine ribonucleotides (20). The enzyme is inhibited competitively by purine and pyrimidine bases, nucleosides, 2’- and 3’-mononucleotides, and NMN; NAD and NADP display a mixed type of inhibition against 5’-AMP hydrolysis ( K i values of 1 and 7 mM, respectively). Takei has concluded that the active sites for the two activities although residing in the same protein are not identical. It seems possible that the enzyme may be composed of two protein subunits each with a separate activity and with active centers 17. 9. Takei, Agr. Bwl. Chem. ( T o k ~ o 29, ) 372 (1965). 18. S.Takei, Agr. Bwl. Chem. ( T o k y o ) 30, 1215 (1966). 19. S.Takei, Agr. Biol. Chem. ( T o k y o ) 31, 917 (1967). 20. S.Takei, Agr. Bwl. Chem. (Tokyo) 31, 1251 (1967). 21. S. Takei, J. Totsu, and K . Nakanishi, Agr. BWZ. Chem. (Tokyo) 33, 1251 (1969).

342

G. I. DRUMMOND AND M. YAMAMOTO

in part common to each other. In general, this enzyme seems remarkably similar to the bacterial 5’-nucleotidase.

c. SNAKE VENOM

5’-NUCLEOTIDASE

That various snake venoms contain potent 5’-nucleotidase activity has been known for over 30 years. Until fairly recently only relatively crude preparations have been available ( 2 2 ) .Sulkowski et al. have purified the enzyme 1000-fold from Bothrops atroz venom ( 2 3 ) .The preparation was free of alkaline phosphatase and phosphodiesterase activities which are rich in venom of several species (24). The enzyme hydrolyzes all riboand deoxyribo-5’-nucleotideswith greatest reactivity for 5’-AMP. It does not attack 3’-nucleotides, ATP, ribose-5-phosphate1 inorganic pyrophosphate, p-nitrophenyl phosphate, nor dinucleotides of the type d-pXpY or pXpYp ( 2 3 ) .Specificity studies indicate a requirement that C-1 of ribose must contain a nitrogenous base and that the hydroxyl group on C-3 must be free. The enzyme is strongly inhibited by EDTA (0.1-1 mM) Mg2+,and Ni2+.A similar enzyme and this inhibition is reversed by CW+, has been purified from venoms of Crotalus adamanteus, Hemachatus haemachates, and Vipera m s e l l i (26, 2 6 ) . The H . haemachates enzyme is activated by 2’- and 3‘-mononucleotides and by 0-amino acids (26) which seem to do so by increasing the dissociation of enzyme and product. This enzyme is activated by Co2+and Mg2+,and activation is increased in the presence of 3’-AMP (26). 5‘-Nucleotidase has been purified to electrophoretic homogeneity from cobra (Naja naja atra) venom ($7). Properties of this enzyme are again similar to the B . atroz venom enzyme with regard to substrate specificity, activation by Mg2+ and Mn2+, and inhibition by Zn2+and Ni2+.It differs in that the pH optimum is 6.5-7.0.

D. BULL SEMINALPLASMA B’-NUCLEOTIDASE Some of the properties of the enzyme from this source were reviewed by Heppel ( 1 ) . More recently, attention has been focused on certain kinetic properties of the enzyme, especially its double pH optimum. Using 22. 23. 24. 25. 26. 27.

W. Bjork, BBA 49, 195 (1961). E. Sulkowski, W. Bjork, and M. Laskowski, JBC 238,2477 (1903). G. M. Richards, G. du Vair, and M. Laskowski, Sr.,Biochemiatry 4, 501 (1965). W. Bjork, BBA 89, 483 (1964). W. Bjork, Arkiv Kemi 27, 555 (1967). Y. Chen and T. Lo, J . Chinese Chem. SOC.(Taiwan) 15,M (1988).

15.

NUCLEOTIDE PHOSPHOMONOEST~ASES

343

a partially purified enzyme, Bodansky and Schwartz (68)found that in the presence of MgZ+,L-histidine inhibited the enzyme below pH 7.5 but activated i t above this pH value, shifting the optimum from 7.5 to 9.3. In the absence of MgZ+,L-histidine produced inhibition below p H 9. The second pH optimum was independent of buffer, but was Mg2+ and temperature dependent (29). It was also dependent upon the nature of the substrate since the phenomena was exhibited only with 5’-AMP, 5’-GMP, and 5’-IMP. From these studies, Levin and Bodansky (29) have proposed a model to explain the role of Mg2+ in producing a second optimum a t pH 9. The model involves four binding sites: one for the C-2 hydroxyl of ribose, one for water, another for phosphate, and one for Mg2+.The model proposed is contingent upon the absence of isozymes, and none was detected by starch gel electrophoresis. Pilcher and Scott (SO), however, have resolved bull seminal plasma 5’-nucleotidase into three active components by electrophoresis on polyacrylamide gels. It is thus possible that the double pH optimum is a reflection of heterogeneity.

E. LIVER5’-NUCLEOTIDASE 5’-Nucleotidases have been studied in liver from various species and activity has been identified in lysosomes, cytoplasmic supernatants and plasma membrane preparations. Arsenis and Touster (31) have purified a 5’-nucleotidase from rat liver lysosomes to apparent homogeneity. The enzyme is unusual in that it hydrolyzes 2’-, 3’-, and 5’-mononucleotides equally well with preference for 5‘-dAMP. It also hydrolyzes FMN, p nitrophenyl phosphate, and ,&glycerol phosphate, but not inorganic pyrophosphate or bis (p-nitrophenyl) phosphate. Unlike the 5’-nucleotidases described thus far, divalent cations such as Co2+,Mn2+,and Mg2+have no activating effect, but EDTA is inhibitory. In spite of the broad substrate specificity kinetic experiments indicate that a single enzyme is involved. Because of its broad substrate specificity it has been suggested (31) that it may play a key role in lysosomal catabolism of nucleic acids. An apparently different 5’-nucleotidase has been partially purified (50-fold) from acetone powder preparations of chicken liver (32): 5’-IMP and 5’-GMP are hydrolyzed by this preparation more rapidly than other 5‘-nucleotides ; 5‘-AMP, 5’-UMP, and 5’-CMP are hydro28. 0. Bodansky and M. K. Schwartz, JBC 238, 3420 (1963). 29. S. J. Levin and 0. Bodansky, JBC 241, 51 (1966). 30. C. W. Pilcher and T. G. Scott, BJ 104, 41c (1967). 31. C. Arsenis and 0. Touster, JBC 243, 5702 (1968). 32. R. Itoh, A. Mitsui, and K. Tsushima, BBA 146, 151 (1967).

344

G. I. DRUMMOND AND M. YAMAMOTO

lyzed a t rates only 510% that of 5‘-IMP. The enzyme is inactive in the absence of divalent metal and is maximally active in the presence of 10 mM Mgz+or Co2+;MnZ+is less effective. It is competitively inhibited by nucleosides, inosine being the most potent. A similar enzyme preparation has been obtained from acetone powder preparations of rat, frog, and pig liver (33). Again 5’-IMP and 5’-GMP are the preferred substrates; the enzyme requires divalent cations and is inhibited by nucleosides. From a study of the dephosphorylation of pyrimidine nucleotides in the soluble fraction of rat liver, Fritzon (34, 35) has provided evidence that two 5’nucleotidases exist. One of the enzymes had a broader specificity for 5’-nucleotides than the other, which acted mainly on dTMP and dUMP. The former enzyme was partially purified from the 100,000 x g supernatant fraction of rat liver (36) essentially free of nonspecific phosphatases. This enzyme is activated by divalent metals, the pH optimum is 6.3 and 5’-IMP is the preferred substrate. The general properties indicate that it is identical to the one isolated from liver acetone powder preparations by Itoh et al. (33).It is entirely reasonable that the cytoplasmic enzyme alone would be extracted into solution from acetone powders. I n spite of the presence of lysosomal and cytoplasmic 5’-nucleotidases in liver, much evidence exists that most of the enzyme in this tissue is membrane bound. Using procedures for isolating subcellular structural components, Song and Bodansky have reported (37) that activity resides in membrane fragments that constitute a part of the microsomal membranes. The distribution of the enzyme between the subfractions of microsomal preparations subjected t o density gradient centrifugation suggested that most of the activity was in the heavier fraction, i.e., in those membranes with attached ribosomes (38) and therefore deriving from the endoplasmic reticulum rather than the plasma membrane. However, liver plasma membrane preparations isolated by sucrose density gradient procedures (39, .lo) show enrichment with respect to 5’-nucleotidase (41-43), and a recent modification employing CaC12 as a mem33. R. Itoh, A. Mitsui, m d K. Tsushima, J . Biochem. (Tokyo) 63, 165 (1968). 34. P. Fritzon, European J . Biochem. 1, 12 (1967). 35. P. Fritzon, BBA 151, 716 (1968). 36. P. Fritzon, BBA 178, 534 (1969). 37. C. S.Song and 0. Bodansky, JBC 242, 694 (1967). 38. C. S. Song and A. Kappas, Ann. N . Y. Acad. Sci. 166, 585 (1969). 39. P. Emmelot, C. J. Bos, E. L. Benedetti, and P. H. Rumke, BBA SO, 126 (1964). 40. D. M. Neville, Jr., BBA 154, 540 (1968). 41. R. Coleman and J. B. Finean, BBA 125, 197 (1966). 42. J. M. Graham, J. A. Higgins, and C. Green, BBA 150, 303 (1968). 43. P. Emmelot and C. J. Bos, BBA 120, 369 (1966).

15.

NUCLEOTIDE PHOSPHOMONOESTERASES

345

brane stabilizer gives especially high specific activities with respect to 5’-nucleotidase (44). The membrane-bound enzyme from both rat liver and human liver (45) has been solubilized using deoxycholate (46) and sonic oscillation ( 4 7 ) . On precipitation to remove deoxycholate, the enzyme reassociates with phospholipids and other membrane proteins to regenerate vesicular membranes ( 4 6 ) . Widnell and Unkeless (48) have obtained a highly purified 5‘-nucleotidase from rat liver microsomes and plasma membranes using classic fractionation procedures in the presence of detergent. The enzyme has been shown to be a lipoprotein containing only one phospholipid, sphingomyelin. The enzyme hydrolyzes 5’-AMP and 5’-UMP more rapidly than other 5’-nucleotides. The substrate specificity differs considerably from that of the enzyme isolated from acetone powder extracts (33) and is probably identical with the one identified in plasma membrane preparations by Song and Bodansky (37). Thus, there is likely as many as three enzymes with 5’-nucleotidase activity in liver, one lysosomal, one cytoplasmic, and one membrane bound. Their specificities and kinetic properties appear to be distinctly different. This would suggest specialized physiological functions not yet understood.

F.

INTESTINAL

5’-NUCLEOTIDASE

Center and Behal (49) have resolved 5’-nucleotidase from calf intestinal mucosa into three fractions using DEAE-cellulose chromatography. One of these was obtained free of nonspecific phosphatase. It had a pH optimum of 6-6.5, MnZ+,Mg2+,and Co’+ (1-10 mM) all enhanced activity and complete inactivation was produced with 1 mM EDTA. This enzyme hydrolyzes all 5’-ribonucleotides a t similar rates and hydrolyzes 5’deoxribonucleotides more slowly. These properties indicate that it is strikingly similar to the one obtained from acetone powder preparations of chicken and rat liver (32, 33) and from soluble supernatants of rat liver ( 3 6 ) .The other two activities (which were not fully characterized) (49) could possibly have originated from particulate material or membranes because the authors employed deoxycholate in the early phase of purification. T. K. Ray, BBA 196, 1 (1970). C. S.Song and 0. Bodansky, BJ 101, 5 C (1966). C. S. Song, B. Tandler, and 0. Bodansky, Biochem. Med. 1, 100 (1967). C. S. Song, J. 8. Nisselbaum, B. Tandler, and 0. Bodansky, BBA 150, 300 (1968). 48. C. C. Widnell and J. C. Unkeless, Proc. Natl. Acad. Sci. U.S. 61, 1050 (1968). 49. M. S.Center and F. J. Behal, ABB 114, 414 (1966). 44. 45. 46. 47.

346 G. 6’-NUCLEOTIDASE

G. I. DRUMMOND AND M. YAMAMOTO

FROM PITUITARY

Some of the kinetic properties of a partially purified (60-fold) 5’nucleotidase from bovine pituitary gland have been described (50-54). The specificity of this enzyme seems different from that of other tissue in that 5’-GMP and 5’-UMP are the preferred substrates (61, 5 4 ) . The enzyme is strongly inhibited by EDTA and this is reversed by Mgz+ but not MnZ+ ( 5 0 ) .It is inhibited by Zn2+and competitively inhibited by 2’- and 3’-mononucleotides and nucleosides, particularly adenosine ( 5 4 ) .The approximate molecular weight was determined to be 237,000. It cannot be determined for certain whether this is a cytoplasmic enzyme. Pituitary glands were homogenized in 0.1 M ammonium sulfate and centrifuged at low gravitational force so that membranous material could have been present in the early stages of purificatian.

H. 5’-NUCLMYTIDASE

FROM

NERVE TISSUE

The enzyme has been partially purified (70-fold) from 38,000 X g supernatant fluid from sheep brain homogenates by Ipata (55-58).The, enzyme (MW 140,000) is reported to be specific for 5’-AMP and 5’-IMP although the substrate specificity does not appear to have been examined closely. 2‘- and 3’-AMP are not hydrolyzed ( 5 6 ) . Unlike the enzyme from many sources the brain enzyme does not require divalent cations and indeed Co2+, which stimulates several other 5’-nucleotidases, was inhibitory a t 5 mM. The enzyme is strongly inhibited by very low concentrations of ATP, UTP, and CTP (50% inhibition by 0.3 & ATP) but not by GTP. 2’-AMP, 3’-AMP, and a variety of other nucleoside monophosphates, nucleosides, and sugar phosphates do not inhibit. A kinetic examination of ATP, UTP, and CTP inhibition (56-58) revealed that inhibition curves were sigmoidal, indicating cooperativity between inhibitor molecules and an allosteric type of interaction between inhibitor and protein. The metabolic significance of ATP inhibition is 50. J. Lisowski, Arch. Immunol. Therap. Exptl. 12, 542 (1964). 51. J. Lisowski, Arch. Immunol. Therap. Ezptl. 14, 195 (1966). 52. J. Lisowski, Arch. Immunol. Therap. Exptl. 14, 209 (1966). 53. J. Lisowski, Arch. Irnmunol. Therap. Exptl. 14, 217 (1966). 54. J. Lisowski, BBA 113, 321 (1966). 55. P. L.Ipata, Nature 214, 618 (1967). 56. P. L. Ipata, BBRC 27, 337 (1967). 57. P. L. Ipata, Biochemistry 7, 507 (1968). 58. P. L. Ipata, Frog. Brain Res. 29, 527 (1968).

15.

NUCLEOTIDE PHOSPHOMONOESTERASES

347

not established. Again it is not entirely clear whether this enzyme is cytoplasmic, being recovered from homogenates centrifuged a t 38,000 X g. 5’-Nucleotidase activity has been examined histochemically (6941) in brain tissue and such studies indicate that more than one 5‘-nucleotidase is present or that the enzyme exists as isozymes (62). It is certainly possible that multiple activities exist in a tissue such as brain with such a diversity of cell types. Activity is present in both white and gray matter (63, 64) and is greatest in cortical tissue, cerebellum, and spinal cord (66). 5‘-Nucleotidase activity has been reported to be greatly diminished in demyelinated areas of the brain in patients with multiple sclerosis (66).

I. 5’-NUCLEOTIDASE FROM CARDIAC TISSUE The enzyme in the myocardium has recently attracted attention because of the possibility that adenosine is a physiological regulator of coronary blood flow (67) (adenosine is a potent coronary dilator). Most of the 5’-nucleotidase activity in rat heart is membrane bound, and a partially purified preparation has been obtained by extracting acetone powder preparations with deoxycholate (68). All 5’-nucleotides are hydrolyzed. The enzyme is strongly inhibited competitively by ATP (Ki 1.8 &). Whether this provides a regulatory mechanism for adenosine formation in the heart is not known. Histochemical evidence has indicated that the enzyme resides in the sarcoplasmic reticulum and transverse tubular system of rat myocardium (69). However, other evidence indicates that enzymic activity is also localized within the walls of the coronary blood vessels (70-72). Recent histochemical studies (73) have shown that the enzyme resides not 59. T. G. Scott, J . Comp. Neurol. 122, 1 (1964). 60. T. G. Scott, J . Comp. Neuro2. 129, 97 (1967). 61. D. Naidoo, J . Histochem. Cytochem. 10, 421 (1962). 62. T. G. Scott, J . Histochem. Cytochem. 13,657 (1965). 63. H. B. Tewari and G. H. Bourne, J . Anat. 97, 65 (1963). 64. K. Nandy and G. H. Bourne, Arch. Neurol. 11, 547 (1964). 65. N. Robinson and B. M. Phillips, Clin. Chim. Acta 10, 414 (1964). 66. K. D. Barron and J. Bernsohn, Ann. ‘N. Y . Acad. Sci. 122, 369 (1965). 67. R. M. Berne, Am. J. Physwl. 204, 317 (1963). 68. H. P. Baer, G. I. Drummond, and L. Duncan, Mol. Pharmacol. 2, 67 (1966). 69. J. Rostgaard and 0. Behnke, J . Ultrastruc. Res. 12, 579 (1965). 70. J. R. Williamson and D. L. Dipietro, BJ 95, 226 (1965). 71. H. P. Baer and G. I. Drummond, Proc. Soc. Ezptl. Bwl. Med. 127, 33 (1968). 72. E. Bajusz and G. Jasmin, Acta Histochem. 18, 222 (1964). 73. K. Nakatsu, H. Clarke, and G. I. Drummond, Federation Proc. 29, 351 (1970).

348

G. I. DRUMMOND AND M. YAMAMOTO

within the cardiac cell but exists almost exclusively in the capillary endothelial cells and small blood vessels of the coronary vasculature. Location of the enzyme a t these sites could have considerable significance with regard to the role of adenosine as an autoregulator of blood flow to the heart. .J. 5’-NUCLEOTIDASE FROM OTHERVERTEBRATE TISSUES 5’-Nucleotidase present in 48,000 x g supernatant fractions of rat and guinea pig skeletal muscle extracts has been examined briefly (74).5‘UMP seems to be the preferred substrate. The enzyme from fish skeletal muscle has also been studied (75).This enzyme hydrolyzes all riboand deoxyribonucleoside 5’-phosphates (except dCMP and dTMP) with preference for 5’-IMP and 5’-UMP. The enzyme is strongly activated by Mn2+; Mg2+is a less powerful activator, and Zn2+ and EDTA are inhibitors. This enzyme thus appears similar to the soluble activity from mammalian liver (33,36). 5’-Nucleotidase in mammary gland hydrolyzes all 5’-ribonucleotides and shows a decrease from pregnancy to early lactation (76).Rats injected with glucagon show increased 5‘nucleotidase in pancreatic islet tissue (77).The enzyme in mouse kidney has been examined histochemically and electrophoretically and found to exist as isoaymes (78).Electrophoretic techniques have also provided evidence that the enzyme exists as isozymes in many other tissues of the mouse such as liver, spleen, intestine, testes, and heart (79).

K. 5‘-NUCLEOTIDASE FROM EHRLICH ASCITES TUMOR CELLS Murray and Friedrichs (80) have obtained a 5‘-nucleotidase from a particulate fraction of Ehrlich ascites tumor cells using deoxycholate. The relative rates of hydrolysis of 5’-UMP, 5’-AMP, 5’-CMP, 5’-GMP, and 5‘-IMP are 129, 100, 93, 83, and 79, respectively. Adenosine and thymidine triphosphate are competitive inhibitors of 5’-AMP hydrolysis 74. I. Cozzani, P. L. Ipata, and M.Ranieri, FEBS Letters 2, 189 (1969). 75. H. L. A. Tarr, L. J. Gardner, and P. Ingram, J. Food Sci. 34, 637 (1969). 76. D. Y. Wang, BJ 83, 633 (1962). 77. S. Johanason and I. B. Toljedal, Endocrinology 82, 173 (1968). 78. M. J. Hardonk and 6. Koudstaal. Hitochemie 15, 290 (1968). 79. M. J. Hardonk and H. G. A. de Boer, Histochemie 12,!29 (1968). 80. A. W. Murray and B. Friedrichs, BJ 111, 83 (1989).

15.

NUCLEOTIDE PHOSPHOMONOESTERASEE

349

a,

(Ki 0.4 and 4.8 respectively). The enzyme is strongly inhibited by Znz+.5’-Nucleotidase in these cells has also been examined by Paterson and Hori (81) who found the enzyme located primarily in nuclei. Nuclei prepared from a 6-mercaptopurine-resistant subline were markedly deficient in enzymic activity. L. 5’-NUCLEOTIDASE

FROM POTATOES

The presence of an enzyme with 5’-nucleotidase activity in extracts of potato were referred to by Heppel (1).The enzyme has been purified 200-fold by Klein (8B) and studied kinetically (83). All major 5’nucleotides are hydrolyzed a t similar rates. The preparation also hydrolyzed 3’-nucleotides a t a substantial rate (2040% that of 5’-AMP). However, kinetic data (83)suggested that the purified preparation was perhaps a mixture of specific 5’- and 3’-nucleotidases.

M. COMPARISON OF

THE

ENZYMES

It seems clear that the 5‘-nucleotidases are a somewhat heterogeneous group of enzymes with many differences and yet having certain properties in common. It must be emphasized that most of the enzyme preparations do not represent pure proteins, and there can be many variations in experimental procedures which might account for some of the differences. Table I presents a summary of some of the similarities and contrasting features of the enzyme from several sources. It can be seen that the relative rates of hydrolysis of the major 5’-nucleotides differ in a seemingly random manner. The E . coli and S. sonnei enzymes differ from those of other sources in that they possess nucleoside diphosphosugar hydrolase activity and also hydrolyze ATP. The B. subtilis enzyme is different from that of the two above organisms in that it does not hydrolyze ATP and converts UDPG to nucleoside and sugar phosphate (16) whereas the E . coli and S. sonnei enzymes degrade this compound to The yeast enzyme is unique base, sugar, and inorganic phosphate (9,13). in that it possesses nucleotide pyrophosphatase activity, converting NAD to NMN, adenosine and inorganic phosphate. The liver lysosomal enzyme appears to have yet a different substrate profile, hydrolyzing both nucleoside 2‘- and 3’-phosphates in addition to 5‘-phosphates. 81. A. R. P. Paterson and A. Hori, Can. J . Biochem. Physwl. 41, 1339 (1963). 82. W. Klein, 2. Physwl. Chem. 307, 247 (1957). 83. W. Klein, 2. Physwl. Chem. 307, 254 (1957).

W

cn

0

TABLE I SUMMARY OF PROPERTIES OF VARIOUS 5!-NUCLEOTIDASES

Enzyme murce

E. wli

Cellular localisstion

Apparent Other eubstrah

K,

5'-AMP (mM)

PH optimum

Activators

Deoxyribo0.03(0.12 6.0for5'Cd+.Mn'+, Ca*+ nucleotidea for ATP) AMP; 6.8 ATP. UTP, for ATP GTP. UDPglucose E . sonnei Surface A > U > G > C > I Deoxyribo0.012 (0.13 5.8 for 5'Cd+. Mn*+ periphic nucleotidea for ATP) AMP: 7-8 for UDPG ATP, ADP. (0.11 for UDP-glucoee UDPG) 8 UDPG. CDPG, 0.0018 for B. uubtilis A >G >U >C ADPG UDPG 5.57 C d + , Ni'+ A>>G = I > U > C NAD. NADH, 0.2 Yesst FAD, ATP S.wiformis

Snake venom B. droz

Surface periplaamic

Relative rate of hydrolyaia of 5'ribonucleotideaa A > G > C > U > I

A > C > I > G > U

Deoxyribonucleotidea NMN

9

W+.Mg'+

and Ni'+ reveme EDTA inhibition

Inhibitor8

MW

EDTA citrate 52,000

D%grS?% of purity

Ref.

5000-fold ( 9 )

urea

EDTA citrate 44,000-

3000-fold

(13)

p

(16)

P

(17$1 )

1OOO-fold

(83)

53 ,000 137,000

Zn*+, Cu'+. EDTA nucleosidea. particularly adenoeine En'+, EDTA

A

>U

=G

>I

Lyemornea

Rat liver

I =G >U >A I00.OOO X u supernatant U = A > C > G > I " P b membrane" U >C =G >A

Calf intestinal mucw Bovine pituitary Sheep brain

Rat heart

Membrane bound

F i h skeletal muscle Ascitea Nuclear tumor cells membrane a

6.5-7 7 . 8 and 9 . 2

C > A > I > G

Rat liver

Rat liver

0.13

A > C = U > C

Snake venom N. naja olra BuU seminal P b

2'-, 3'-Mononucleotides. 5'-deo4nucleotidea CDeoxyribonucleotidea

3.7-5.5

6.8

6.3

Mn'+. Mg'+

NP+, Znz+

Cd+.Ni'+

Znt+. Cu'+ nucleonidea nucleotidea Zn*+. NP+

Cd+,Mn*+.

P Nucle&dea

P P

6.04.5

Cd+. MIL¶+, ME¶+

EDTA nucleosidea

G > U = C > I >A

0.06

9

ME'+ r e v e m EDTA

EDTA Zn'+ nucleonidea

A = I

0.007

7.3

C > U = A > I >G

0.018

9

U =I

>C >G

U > A > C > G > I

5'-Deoxyrib* nucleotidea

8 0.067

P

ME'+

0.012

inhibition None required

Mnt+, ME'+

Hihly P

and Niz+ do n d activate

0.05

5'-Deoryribcnuoleotiden

1O.OOO

237 ,OOO

ATP. UTP. Cot+. En:+ ATP (Ri 1.8 r M ) EDTA. ZnZ+ ATP, Zn'+

P P

D P

120,OOO

Nuclec&de-5'-monophospha~are represented only by the baee letter. Degree of purity designated nu p representa partial purification.

P

352

Q. I. DRUMMOND AND M. YAMAMOTO

The most notable similarities relate to activation and inactivation by metal ions and other materials. I n most instances (Table I) each is activated by one or more of the cations Co2+,Mn2+,NiZ+,Mg2+,or Ca2+ of which the former three are usually most effective. Here again, however, there are differences. Thus, the B. atrox enzyme is not activated by ions, but they serve to reverse EDTA inhibition (23). The rat liver lysosomal enzyme is also not activated by divalent metals (31) ; the sheep brain enzyme does not seem to require divalent cation and in fact it is inhibited by Co2+ (67). In most cases EDTA has been reported to be an effective inhibitor; Znz+and Cu2+are frequently inhibitory. Whereas ATP is a substrate for the bacterial enzyme, it and other nucleosidetriphosphates are powerful inhibitors of the enzyme from several mammalian sources (a7, 67,68). The enzymes differ markedly in molecular weight, varying from 10,OOO for the snake venom (Naja atra) enzyme (27) to 237,000 for the enzyme from bovine pituitary (64). It appears certain that there is more than one 5‘-nucleotidase present in most mammalian tissues. This is best established for liver. In other cases it has not been possible to determine the exact intracellular origin because of the nonselective extraction procedures used. However, those enzymes isolated from acetone powder preparations of chicken liver and rat liver appear to have properties essentially identical to the enzyme present in 100,000 x g supernatant fraction of rat liver and therefore may be cytoplasmic in origin. This could also be the case for the intestinal mucosa enzyme. Very little can be said about the physiological function of the enzyme except that it is obviously involved in normal cellular catabolism of nucleosidemonophosphates. Its surface localization in microorganisms must have metabolic relevance; its presence in membrane structures in mammalian tissues also points to specialized functions. Perhaps, even the nucleoside product has physiological functions yet to be discovered.

II. 3’-Nucleotidase

In contrast to 5‘-nucleotidases, enzymes which hydrolyze nucleoside3‘-phosphates have attracted comparatively little attention. Enzymes possessing some specificity for 3’-nucleotides seem to occur predominantly in the plant kingdom and several have been only partially purified and characterized.

15.

NUCLEOTIDE PHOSPHOMONOESTERASES

353

A. RYE GRASS3’-NUCLEOTIDASE An enzyme purified from rye grass capable of specifically hydrolyzing 3‘-nucleotides has been available for some years (84). The enzyme has also been purified (50-fold) from germinating rye seedlings (85) and seems quite specific for 3’-nucleotides. 3’-Deoxymononucleotides are not attacked (86) nor is arabinonucleoside 3’, 5’-diphosphate (87) . Enzymic activity increases 10-fold during germination (85).

B. MUNGBEAN3’-NUCLEOTIDASE Walters and Loring (88) have purified a 3’-nucleotidase about 50-fold from mung bean sprouts (Phaseolus aureus Roxb.). The enzyme hydrolyzes 3’-AMP, 3’-GMP, 3’-CMP in decreasing order and also hydrolyzes the 3I-phosphate group of coenzyme A. (89), but it has no significant activity for 2’- or 5’-ribonucleotides. For 3’-GMP, 3’-AMP, 3’-UMP, and 3’-CMP, K , values are 0.67, 1.1, 7.7, and 15 mM, respectively. The enzyme preparation also contained acid stable ribonuclease activity (89). Both 3’-nucleotidase and acid ribonuclease were inactivated reversibly at pH 5.0 and by dialysis and this inactivation could be prevented by Zn2+.The two activities were similarly inactivated by heat at pH 5 and 7.5. Such data indicate that the two are metalloproteinsprobably zinc metalloproteins. These similarities and other kinetic data provide evidence that the 3’-nucleotidase and ribonuclease activities reside in the same protein.

c. 3’-NUCLEOTIDASE FROM

WHEAT

SEEDLENGS

An enzyme similar to the 3‘-nucleotidase of mung bean has been isolated from germinating wheat seedlings and purified 800-fold (90). The preparation possessed DNase, RNase, and 3’-nucleotidase activities. These three activities were similar in p H optima, requirements for Znz+ and sulfhydryl compounds, stability to storage, temperature inactivation L. Shuster and N. 0. Kaplan, “Methods in Enzymology,” Vol. 2, p. 551, 1955. L. Shuster, JBC 229, 289 (1967). L. Cunningham, JACS 80, 2546 (1958). G. R. Barker and G. Lund, BBA 55, 987 (1962). T. L. Walters and H. S. Loring, JBC 241, 2870 (1966). H. S. Loring, J. E. McLennan, and T. L. Walters, JBC 241, 2876 (1966). 90. D. M. Hanson and F. L. Fairley, JBC 244, 2440 (1969).

84. 85. 86. 87. 88. 89.

354

G. I. DRUMMOND AND M. YAMAMOTO

and reactivation and inhibition by metal ions and EDTA. The three activities also co-chromatographed on DEAE-cellulose and phosphocellulose and migrated identically on gel filtration. The three activities thus seem to reside in a single protein. The activity in wheat seedlings increases over 80-fold during germination (91). D. 3’-NUCLEOTIDASE

FROM

MICROORGANISMS

Numerous microorganisms possess a cyclic 2’, 3’-ribonucleoside phosphate diesterase which has 3‘-nucleotidase activity. This “double headed” enzyme has been vigorously studied and is described in the chapter on nucleoside cyclic phosphate diesterases (Chapter 16 by Drummond and Yamamoto, this volume). Suffice it to say that the ability of microorganisms producing this enzyme to catabolize 3’-nucleotides is well established. An enzyme from Bacillus subtilis, which hydrolyzes a variety of nucleoside 3’-phosphates, has been briefly described (92). The enzyme was found to be present in culture filtrates after removing the cells by centrifugation. Whether or not this enzyme is identical to the nucleoside cyclic phosphate diesterase in this organism is unclear. Because of its specificity for 3‘-nucleotides it has been proposed (93) as a specific method for preparing 2’-nucleotides. Becker and Hurwitz (94) have found that after infection of E . coli B with T-even bacteriophages a novel 3‘-deoxynucleotidase activity appears. They purified the enzyme 2000-fold. In addition to its attack on 3’-deoxymononucleotides, the enzyme selectively removes the 3’-phosphoryl groups from DNA. It does not attack 3‘-ribonucleotides, 3’phosphoryl groups of RNA, or 5’-phosphate esters. Like bacterial 5‘nucleotidases, this enzyme is markedly activated by Mg2+and Co2+and is inhibited by EDTA. The enzyme appears to be a phage-induced enzyme; the activity rises early after injection with T-even phages and formation of the enzyme is blocked with chloramphenicol.

91. L. Shuster and R. H. Gifford, Arch. Biochem. Biophys. 96, 534 (1962). 92. S. Igarashi and A. Kakinuma, Agr. Biol. Chem. ( T o k y o ) 26, 218 (1962). 93. A. Kakinuma and S. Igarashi, Agr. Biol. Chem. ( T o k y o ) 28, 131 (1964). 94. A. Becker and J. Hurwitz, JBC 242, 936 (1967).