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Home , Cyclic nucleotide, Deoxyadenosine, Deoxyribonucleoside, Polynucleotide, Ribonucleoside, Ribonucleotide

Proc. Nat. Acad. Sci. USA Vol. 71, No. 4, pp. 1299-1303, April 1974

Regulation of a Plant 5'(3')- Phosphohydrolase by Cyclic and , , and Cytokinin Ribosides (higher plants/5'-nucleotidase/phytohormones) G. M. POLYA Biochemistry Department, La Trobe University, Bundoora, Melbourne, Victoria 3083, Australia Communicated by Rutherford Robertson, December 10, 1973

ABSTRACT A highly specific 5'(3')-ribonucleotide biochemical evidence as yet for - or translation- phosphohydrolase has been purified extensively from wheat modifying cAMP-binding or cAMP-activated seedling leaves. While catalyzing the of a wide variety of phosphomonoesters, the has Km values in higher plants. A protein has been obtained for 5'-monophosphate and adenosine 3'-mono- from soya-bean callus but this is not activated by cAMP in the micromolar range and appears highly (30). A 5'(3')-ribonucleotide phosphohydrolase from wheat specific for 5'- and 3'- monophosphates seedlings has been extensively purified in this laboratory and as substrates at pH 5.0. The cyclic nucleotides adenosine 3': 5'-cyclic monophosphate, 3': 5'-cyclic mono- shown to be competitively inhibited by cAMP (14). This phosphate, and 8-bromoadenosine 3':5'-cyclic monophos- communication demonstrates the regulation of this specific phate, as well as 8-bromoadenosine 5'-monophosphate, 5'-nucleotidase by other nucleotides, including cyclic nucleo- are powerful competitive inhibitors of the enzyme; the tides, that have been shown to be active as plant-growth apparent Ki values for these nucleotides are 3.4 MM, promoters. This enzyme is also shown to be regulated by 1.6 Mm, 26 MM, and 9.1 MM, respectively. The enzyme is inhibited noncompetitively by a variety of pyrimidine and , including cytokinin ribosides. purine (including cytokinin) ribosides and 2'-deoxyribo- sides. Since the cyclic competitive inhibitors MATERIALS AND METHODS are also active growth promoters of higher plants, and, furthermore, since elevation of cyclic AMP levels appears Purification of the 5'-Nucleotidase. The 5'-nucleotidase was to be a consequence of some phytohormone treatments purified as described (14), but with the addition of two further that promote plant growth, it is suggested that negative control of this 5'(3')-ribonucleotide phosphohydrolase purification steps. The 5'-nucleotidase, prepared as in ref. 14, may be a significant component of growth regulation was eluted near the void volume from a Sephadex G-75 through the maximizing of the levels of nucleotide pre- column (2 X 64 cm) equilibrated and eluted with buffer A cursors for RNA and DNA synthesis. containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1 M KCl, and 0.03% 2-mercaptoethanol. There is considerable interest in the The active fractions currently possibility that from this step were concentrated by precipitation at 90% adenosine 3': 5'-cyclic monophosphate (cAMP) may be ammonium sulfate saturation and a DEAE- involved in metabolic and growth regulation in higher plant washed through in cellulose (Cellex-D, 0.67 mequiv/g) column (5 X 10 cm) in cells a fashion analogous to its role in bacterial (1-3) and buffer B mM Tris*HCl 1 mM animal (2, 3) systems. Direct evidence for the occurrence of [10 (pH 8.0), EDTA, and 0.03% 2-mercaptoethanol]. The 5'-nucleotidase is not retained cAMP in higher plants and algae has been obtained by a DEAE-cellulose and is ahead of other that variety of procedures (4-9). In addition, cAMP phospho- by eluted proteins diesterase has been detected in are slightly retarded by DEAE-cellulose under these con- higher plants (10-14), including ditions (14). The overall purification was about 50-fold, and activities inhibited by methyl (10, 11) which are further purification was not feasible because of the small final known to inhibit mammalian phospho- yield of protein diesterase (2, 3). However, the specificities-of these phospho- (about 6 mg/kg fresh weight). diesterases remain to be resolved. Enzyme Assays. Phosphomonoesterase and phosphodiester- The possibility that cAMP may act as a "second messenger" ase activities were determined at 300 in a medium containing in higher plant cells by mediating the effect of phytohormones 100 mM acetate [Na+, (pH 5.0)], 4 mM MgCI2, and the has been strengthened by experiments indicating increased appropriate substrate. Phosphate release was determined by turnover of cAMP in plant tissue in response to application of the method of Allen (31), and p-nitrophenol generated from indole-3-acetic acid (8, 9) and gibberellic acid (GA) (7). p-nitrophenylphosphate or bis-p-nitrophenylphosphate was Elevation of cAMP levels in Avena coleoptiles occurs as a measured from the extinction at 400 nm in alkali (32). cAMP consequence of indole-3-acetic acid treatment (4). In phyto- activity was assayed radiochemically -replacement experiments, cAMP has been shown to essentially as described (14), except that the reaction products apparently mimic responses to indole-3-acetic acid (8, 15-17) were resolved by chromatography on polyethyleneimine thin- and GA (18-29) in appropriate bioassays. layer plates developed in a solvent system containing 5% While there is already considerable evidence suggesting a borate and 0.5 M ammonium acetate (pH 7.5). The poly- growth-regulatory role for cAMP in higher plants, there is no ethyleneimine thin-layer plates were prepared according to the procedure of Randerath and Randerath (33). The rates of Abbreviations: cAMP, adenosine 3': 5'-cyclic monophosphate; phosphate ester hydrolysis were linear functions of protein GA, gibberellic acid. concentration under the conditions used. The assay conditions 1299 Downloaded by guest on September 23, 2021 1300 Biochemistry: Polya Proc. Nat. Acad. Sci. USA 71 (1974) TABLE 1. Substrate specificity of the 5'(3')-ribonucleotide tometric parameters. Both 8-bromonucleotides were chro- phosphohydrolase matographically homogeneous and resolved from the parent nucleotides in three chromatographic systems, namely, Relative rates ascending chromatography on polyethyleneimine thin Substrate* of layers hydrolysist eluted with 5% borate-0.5 M ammonium acetate (pH 7.5) Adenosine 5'-monophosphate 1.00 (for 8-Br cAMP) or 0.75 M LiCl-1.0 M acetic acid (for 8-Br Adenosine 3'-monophosphate 0.40 5'-AMP) and descending paper chromatography on Whatman Adenosine 2'-monophosphate 0.005 3M paper eluted with 95% ethanol-i M ammonium acetate 2'- 5'-monophosphate 0.92 (pH 7.5) (7:3, v/v) or 1-butanol-acetic acid-water (5:2:3, 5'-monophosphate 1.17 v/v/v). Both 8-bromonucleotides chromatographed 5'-monophosphate 1.14 essen- 5'-monophosphate 1.06 tially as reported for the latter system (35). 5'-monophosphate 1. 19 Chemicals. All nucleotides and nucleosides were purchased 5'-monophosphate 1.25 from Sigma Chemical Co. and other chemicals were of Guanosine reagent 5'-monophosphate 1.25 grade. [8-8H]cAMP was purchased from the Radiochemical 8-Bromoadenosine 5'-monophosphate 0. 17 Adenosine 5'-diphosphate 0.081 Centre, Amersham, U.K. Nucleotide concentrations were Adenosine 5'-triphosphate 0.017 determined spectrophotometrically (35). p-Nitrophenylphosphate 1.24 RESULTS 5'-phosphate 0.098 fl-Glycerophosphate 0.008 Substrate Specificity of the 5'-Nucleotidse. It was previously Adenosine 3': 5'-cyclic monophosphate 0.001 observed that 5'-nucleotidase, ribose 5'-, and a Bis(p-nitrophenyl)phosphate 0.017 low level of cAMP phosphodiesterase activity purified with the nucleotidase and that, in gel filtration of the highly puri- * All substrates were at 1 mM concentration. fied enzyme, peaks of phosphomonoesterase activity deter- t Initial rates of hydrolysis relative to that with 5'-AMP as mined with 5'-AMP, 5'-IMP, 3'-AMP, ribose 5'-phosphate, substrate (1.00) were determined from time-courses of ester hy- and p-nitrophenylphosphate as sub- drolysis at pH 5.0 (300) as described in jg-glycerophosphate, Methods. strates exactly coincided (14). One peak of phosphomono- were chosen so that less than 10% of the substrate was hy- esterase was obtained from gel filtration, and only one phos- drolyzed and phosphate ester hydrolysis was a linear function phomonoesterase band was detected after starch or polyacryl- of time. All assay values were corrected by use of appropriate amide gel electrophoresis of the highly purified 5'-nucleotid- zero time controls. ase. Table 1 shows the relative initial rates of hydrolysis of a Synthesis of 8-Bromopurine Nucleotides. 8-Bromo 3':5'- variety of monophosphates, phosphomonoesters, cAMP and 8-bromo 5'-AMP were synthesized by bromination and phosphodiesters catalyzed by the 5'-nucleotidase. 5'- of 3': 5'-cAMP and 5'-AMP, respectively, by the procedure Nucleoside monophosphates are clearly the most effective of Muneyama et al. (34). The products were purified by substrates and are hydrolyzed at comparable rates in the con- passage through Dowex 50 (H+) (eluted with H20), and the ditions used. 2'-Deoxy 5'-nucleoside monophosphates (2'- solutions containing the purified nucleotides were lyophilized. deoxy 5'-AMP and 2'-deoxy 5'-TMP) are hydrolyzed at much The purified products of the bromination of 3': 5'-cAMP and the same rate as 5'-AMP. A notable exception is 8-bromo 5'-AMP were chromatographically homogeneous and identical 5'-AMP; the rate of hydrolysis of this substrate is less than to 8-bromo 3:5'-cyclic AMP and 8-bromo 5'-AMP, respec- one-fifth of that for 5'-AMP. 3'-AMP is less effective than tively, in terms of chromatographic and spectrophotometric 5'-AMP as a substrate, and 2'-AMP much less so. While p- parameters. For 8-bromo 3': 5'-cyclic AMP, Xmaxfl+ = 263 TABLE 3. Apparent Ki values of the 5'(S')-ribonucleotide nm and XmaxOH = 265 nm, as reported previously for this phosphohydrolase for nucleoside noncompetitive inhibitors compound (35). For 8-bromo 5'-AMP, Xm xH+ = 263 nm, XminH+ = 231 nm, Xmax°0 = 265 nm, Xmin°0 = 232 nm, Nucleoside Ki* (M) A2ws/A260 (pH 1) = 0.51, and A280/A260 (pH 11) = 0.47, essentially as described before for this compound (35). Both Kinetin riboside 1.2 X 10-4 8-bromonucleotides differ from the nonbromi- 2'-Deoxyadenosine 1.9 X 10-4 significantly Adenosine 2.8 X 10-4 nated parent nucleotides with respect to these spectropho- Inosine 6.6 X 10-4 TABLE 2. Apparent Ki values of the 5'(S')-ribonucleotide 3'-Deoxyadenosine 1.2 X 10-3 phosphohydrolase for nucleotide competitive inhibitors Uridine 2. 1 X 10-3 Zeatin riboside 2.3 X 10-3 Nucleotide Ki* (/AM) (2'-d)-Thymidine 3.0 X 10-3 Guanosine 3.3 X 10-3 3':5'-cyclic AMPt 3.4 N-6-Benzyladenosine 5.2 X 10-3 8-Bromo-3': 5'-cyclic AMP 26 Cytidine 7.1 X 10-3 8-Bromo-5'-AMP 9.1 2',3'-Isopropylideneguanosine 1.2 X 10-2 3':5'-cyclic GMP 1.6 2',3'-Isopropylideneadenosine 2.4 X 10-2 N-6-Dimethyladenosine 8.0 X 10-2 * The estimates are derived from plots of 1/V against 1/S in the presence and absence of inhibitor in experiments using ribose * The apparent Ki values were derived from plots of the recipro- 5'-phosphate and p-nitrophenylphosphate as suhbstrates. cal of the rate of 5'-AMP hydrolysis as a function of inhibitor t Data from ref. 14. concentration. Downloaded by guest on September 23, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Regulation of 5'-Nucleotidase 1301

40 vF (A) + KINETIN 0-33mM RIBOSIDE I - 30 V 0220mM

OOmM o d . - :r If O-5m tI

I 1I aa I -1*O -0-5 0 O-5 10 1-5 (e-A M P)- (m M)-

1-04mM (8) + ADENOSINE 15

1~~~~~~01m

I I .1 I I I I I. I -3 -2 -1 0 1 2 3 (5'-AMP)-l (M)-1 Fig. 1. Noncompetitive inhibition of the 5'-nucleotidase by kinetin riboside and adenosine. The reciprocals of the rates of 5'-AMP hydrolysis (1/v) are in arbitrary units. The levels of added kinetin riboside are shown in (A). The mean levels of adenosine present were determined by correction from the adenosine produced by hydrolysis of 5'-AMP and are shown in (B).

nitrophenylphosphate is hydrolyzed at comparable rates to for 8-Br cAMP is an order of magnitude greater. 8-Br 5'- 5'-AMP, ribose 5'-phosphate and 5'-ADP are less than one- AMP is more effective than 8-Br cAMP as a competitive tenth as effective. ,B-Glycerophosphate and 5'-ATP are, inhibitor of the 5'-nucleotidase. respectively, less than 1/100 and 1/50 as effective as sub- Noncompetitive Inhibition the strates in these conditions as 5'-AMP. 3':5'-cAMP phos- of 5'-Nucleotidase by Nucleo- phodiesterase purifies with the 5'-nucleotidase; the 3': 5'- sides. In the course of analyzing the structural requirements for nucleotide inhibition of was cAMP phosphodiesterase rates are 1/1000 of the 5'-AMPase cyclic the 5'-nucleotidase, it found rates under the same conditions. Phosphodiesterase activity that purine nucleosides (9-B-D-ribofuranosylpurines) with bis-p-nitrophenylphosphate is less than 1/50 that of 5'- are effective noncompetitive inhibitors of the enzyme. Fig. 1 AMPase. shows that N-6 furfurylaminopurine riboside (kinetin ribo- side) and adenosine act as hyperbolic noncompetitive in- Competitive Inhibition of the 5'-Nucleotidase by Cyclic hibitors of the 5'-nucleotidase; the maximum rate of hydroly- Nucleotides. cAMP was previously shown to compete with sis is lowered and the Km for 5'-AMP is elevated in the pres- 5'-AMP, p-nitrophenylphosphate, and ribose 5'-phosphate ence of these nucleosides. Table 3 shows the relative effective- as substrates for the 5'-nucleotidase; the Ki of the 5'-nucleo- ness of a wide variety of nucleosides as noncompetitive tidase for cAMP was 3.4 AM (14). It was, therefore, of interest inhibitors of the 5'-nucleotidase. The most effective non- to seen whether other nucleotides, including cyclic nucleo- competitive inhibitors are kinetin riboside, 2'-deoxyadeno- tides, that can elicit plant growth-promoting effects could sine, adenosine, and inosine; the 5'-nucleotidase had sub- inhibit the 5'-nucleotidase with comparable specificity. 3':5'- millimolar Ki values for these compounds. Kinetin (1.4 mM) Cyclic GMP (cGMP), 8-bromo 3': 5'-cyclic AMP (8-Br did not inhibit significantly under these conditions. At very cAMP) and 8-bromo 5'-AMP (8-Br 5'-AMP) all inhibit the high 5'-AMP concentrations (about 10 mM), the inhibition 5'-nucleotidase competitively. The apparent Ki values of the by adenosine can be partly overcome, suggesting weak 5'-nucleotidase for these nucleotides are presented in Table 2. binding of 5'-AMP at the noncompetitive site. Ribose and 2'- While the Ki for cGMP is lower than that for cAMP, the Ki do not inhibit the 5'-nucleotidase. Downloaded by guest on September 23, 2021 1302 Biochemistry: Polya Proc. Nat. Acad. Sci. USA 71 (1974)

DISCUSSION phodiesterases (2, 3), suggesting by analogy that these effects The wheat seedling 5'(3')-nucleotidase is the only enzyme are mediated by increased cAMP levels. from higher plants as yet demonstrated to be regulated by The effects of cyclic nucleotides discussed above provide cyclic nucleotides. As previously discussed (14), the enzyme is some support for the hypothesis that cyclic nucleotides, and in best described as a 5'(3')-ribonucleotide phosphohydrolase in particular cAMP, may mediate the actions of certain phyto- view of its substrate specificities. While the enzyme catalyzes , and the hypothesis is strengthened by observations the hydrolysis of a wide variety of phosphate esters, the Km of increased turnover of cAMP in plant tissues treated with values for 5'-AMP and 3'-AMP at pH 5.0 (1.4 ,uM and 6.1 GA (7) or indole-3-acetic acid (8, 9) and transient elevation of jIM, respectively) are very low, while the Km values for sub- cAMP levels by indole-3-acetic acid (4). There is no biochemi- strates such as p-nitrophenylphosphate, ribose 5'-phosphate, cal evidence for specific mechanisms of regulation of macro- and 0-glycerophosphate are in the millimolar range (14). The molecular in higher plants by cyclic nucleotides. rates of hydrolysis of all ribonucleoside 5'-monophosphates However, the evidence presented in this paper renders feasible and 2'- 5'-monophosphates are compara- the possibility that one consequence of phytohormone treat- ble, with the exception of 8-bromo 5'-AMP which is a rela- ments that enhance cAMP levels may be the inhibition of a tively poor substrate for the enzyme. Other phosphate esters specific 5'-nucleotidase of higher plants. The kinetic param- tested are poor substrates compared with 5'-AMP at 1 mM eters reported here and elsewhere (14) indicate that the 5'(3')- concentration, except for p-nitrophenylphosphate (Table 1). nucleotidase is highly specific for 5'-nucleotides, and the Km However the Km for the latter is about 300 times greater for 5'-AMP (1.4 uM) is of the same order of magnitude as than the Km for 5'-AMP (14). The very low Km values of the physiological 5'-AMP levels in cells (38). The Ki enzyme for nucleotides argues for a physiologically significant for cAMP (3.4 MM) is of the same order of magnitude as role for the enzyme. Physiological levels of 5'-AMP in animal cAMP levels in animal (2, 3) and plant cells (4-6). Thus, the and plant cells are of the same order of magnitude as the Km kinetic data indicate the feasibility of physiological control of of the enzyme for 5'-AMP (1.4 /AM). nucleoside monophosphate levels by way of the effect of cAMP The inhibition of 5'(3')-nucleotidase by cAMP and related on the 5'-nucleotidase. Such regulation of the 5'-nucleotidase compounds could be involved in the mechanism by which could then be a significant component of phytohormone- these compounds influence plant growth. However, other induced plant growth responses through the maximizing of specific enzyme reactions may also be regulated by these com- the levels of nucleotide precursors for syn- pounds in higher plants, and it is relevant to note the diversity thesis. Evidence for proposals similar to this in implicating of responses evoked by cyclic nucleotides in higher plant control of highly specific 5'-nucleotidases in such nonspecific systems. cGMP (Ki = 1.6 MM) applied at 1 mM has been regulation of polynucleotide synthesis and growth has shown to mimic GA3 in a-amylase induction and enhancement been obtained for animal (38, 39) and bacterial (40) systems. of reducing production in barley aleurone (28) and to A particularly relevant system is that of the - have GA-like effects in flowering induction in perennial plants oscillations of partially hepatectomized rats and the associated (28). 8-Bromo cAMP (Ki = 26,M) and 8-bromo 5'-AMP out-of-phase oscillations of levels of a specific 5'-nucleotidase (Ki = 9.1 ,uM) applied at 0.1 mM, both mimic the effect of in these cells (39). kinetin (0.5 mg/liter) in promoting growth (by cell division) While cyclic nucleotides competitively inhibit the 5'(3')- of excised tobacco pith parenchyma tissue (36). nucleotidase, certain purine ribosides exert a noncompetitive cAMP (Ki = 3.4 ,uM) applied at millimolar levels to barley inhibition. There is clearly no correlation between the cell- aleurone mimics the effect of GA3 on induction of a-amylase division-promoting (cytokinin) activity of the tested purine (18, 19, 28), secretion of soluble sugar (28), induction of ribosides (see 41, 42) and their ability to inhibit the 5'(3')- phosphatase (20-22), and induction of protease (20). At these nucleotidase. This casts doubt on the regulatory significance levels, cAMP also stimulates the effect of GA but not of of the observed noncompetitive inhibitions. Nevertheless, the kinetin on lettuce seed germination (25). At somewhat lower noncompetitive inhibitions may be of significance in experi- levels (10-5-10-4 M) cAMP can enhance GA3-induced a- mental situations involving application of high concentrations amylase production by barley aleurone (18, 23). However, it (>10-4 M) of cytokinins or cytokinin ribosides to higher plant must be noted that application of 1 mM cAMP can increase cells, although assessment of the data in a correlative sense is GA3 levels in barley aleurone (28) and further that the "a- obviously complicated by parameters such as cytokinin trans- amylase" measured in the above studies may be a combination port, compartmentation, and . Inhibition of the of glucan hydrolases other than a-amylase (37). cAMP at 5'(3')-nucleotidase by purine ribosides would be expected to 10-6-10-4 M mimics the effect of GA in stimulating lettuce elevate nucleotide levels and thus influence biosynthetic seedling hypocotyl elongation (27) and enhancing auxin- processes. It is relevant to note that application of 10-4 10-1 induced growth of Avena coleoptiles (29). At 10-5-10-4 M, M 6-benzylaminopurine causes a marked elevation of ATP cAMP can enhance auxin-induced cell expansion of Jerusalem levels in axillary buds of tobacco (43) and a major increase in artichoke tuber slices (24, 26) and can overcome the effect of 5'-AMP and ATP levels in pea roots (44). 2-chloroethyl trimethylammonium chloride (an inhibitor of The findings reported in this paper demonstrate a specific GA biosynthesis) in this system (26). cAMP can also have site of action of cAMP, cGMP, 8-bromo cAMP, and 8-bromo auxin-like effects in delaying abscission of debladed Coleus 5'-AMP in a biochemical system from higher plants. It is possi- petioles (15) and in promoting elongation of excised apical ble that inhibition of this specific 5'(3')-nucleotidase by segments of etiolated wheat coleoptiles (17). These experi- cAMP may be a significant consequence of certain phyto- ments with cAMP have been complemented in some cases hormone applications. with comparable effects by methylxanthines such as theo- This work was supported by Grant D72/15265 from the phylline, aminophylline, and (18, 29). These com- Australian Research Grants Committee. Acknowledgment is also pounds are known to inhibit mammalian cyclic AMP phos- due to Dr. D. S. Letham for helpful comments and discussion. Downloaded by guest on September 23, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Regulation of 5'-Nucleotidase 1303

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