Effect of Self-association on Activity of an ADP-ribosyltransferase from Turkey Erythrocytes

Transcription

1 Vol. 256, No. 22. Issue of November 25. pp , 1981 Printed in U.S. A. Effect of Self-association on Activity of an ADP-ribosyltransferase from Turkey Erythrocytes CONVERSION OF INACTIVE OLIGOMERS TO ACTIVE PROTOMERS BY CHAOTROPIC SALTS* Joel Moss+, Sally J. Stanley.+, and James C. Osborne, Jr.8 (Received for publication, March 3, 1981) From the *Laboratory of Cellular Metabolism and the 8 Molecular Disease Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland The activity of a highly purified guanidine-specific ADP-ribosyltransferase, assayed at low concentrations of acceptor, was increased >lo-fold by certain inorganic salts; chaotropic salts were most active with SCN- > Br- > Cl- > F- > PO1 G; the order of effectiveness followed the Hofmeister series. NaCl was maximally effective at 200 mu; at this concentration, NaCl decreased the K,,, for NAD. In NaCl, the Lineweaver- Burk plot for the ADP-ribose acceptor, a&nine methyl ester, was converted from one consistent with substrate activation with limiting slopes corresponding to K,,, values of 3.8 and 60 mu into one displaying Michaelis-Menten kinetics with a K,,, of 1.3 mu. With ovalbumin as the ADP-ribose acceptor, low concentrations of NaCl increased the release of Ica&onyZ- 4C]nicotinamide from [cu&~~~z- ~C]NAD and the incorporation of [acze&ne-u- 4C]ADP-ribose from [adenine-u-14c]- NAD into protein; at higher concentrations of NaCl, [ca&onyz- 4C]nicotinamide release, but not [a&mine-u- %]ADP-ribose incorporation, was increased. In contrast, with agmatine as ADP-ribose acceptor, NaCl increased ADP-ribosylation in parallel with [cc&onyz- Clnicotinamide release. In the absence or presence of 200 mu NaCl, the ratio of [adenine-u- 4C]ADP-ribose coupled to agmatine to [carbonyz- 4C]nicotinamide released using [adenine-u-14c]nad and [cu&o~~yz- ~C]- NAD, respectively, as substrates was The ADP-ribosyltransferase was converted by NaCl from a rapidly sedimenting oligomeric form(s) to slowly sedimenting protomeric species. In the absence of NaCl, the transferase eluted in the void volume of a Sephadex G-200 column; in the presence of NaCI, the enzyme possessed a K., similar to that of chymotrypsinogen. These data are consistent with the highly purified erythrocyte transferase existing as relatively inactive, high molecular weight oligomers which are converted by chaotropic salts to high activity protomeric species. ADP-ribosyiation of proteins is important in the regulation of many metabolic pathways (1). Choleragen, Escherichia coli heat-labile enterotoxin, diphtheria toxin, and Pseudomonas Exotoxin A exert their effects on cells by catalyzing the NAD-dependent (mono)adp-ribosylation of specific cellular proteins (2-7); infection of E. coli with T-4 phage leads to ADP-ribosylation of RNA polyrnerase (8-10). Poly(ADP-ribosylation) may be involved in the regulation of DNA repair, chromatin structure, or DNA replication (1). Although * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (mono)adp-ribosylation has been observed in animal tissues, its biological role is uncertain. The specificity of ADP-ribosylation is, in part, determined by the amino acid which serves as the acceptor. Choleragen and E. coli heat-labile enterotoxin catalyze the ADP-ribosylation of what appears to be an arginine in a 42,000-dalton particulate protein from several types of cells (2-5, 11, 12); diphtheria toxin and Pseudomonas Exotoxin A modify a hi&dine derivative in elongation factor 2 (13, 14). The acceptor for poly(adp-ribose) synthetase appears to be the carboxy1 moiety of a glutamate residue or a terminal lysine (15-18). The ability to ADP-ribosylate arginine is not restricted to viral transferases and bacterial toxins (10-12). An avian erythrocyte transferase has been purified which catalyzes the ADPribosylation of arginine with a turnover number, when measured with high concentrations of acceptor, of 10,000 mol. mir-.mol- (19). We have investigated factors which may regulate the enzymatic activity of the erythrocyte (mono) ADP-ribosyltransferase. In the studies reported here, we demonstrate that dissociation of enzyme oligomers to protomers with SCN-, Cl-, or PO4 G is associated with apparent increases in its affinities for both NAD and arginine and a >lofold increase in catalytic activity. EXPERIMENTAL PROCEDURES Assays-NAD glycohydrolase and ADP-ribosyltransferase activities were determined as described previously (II, 20). The standard assay contained 50 mm potassium phosphate (ph 7.0), 32.4 pm [carbonyl- C]NAD (-40,000 cpm), ovalbumin.(0.3 mg), and 75 rnm arginine methyl ester in a total volume of 0.3 ml. Under these conditions, transferase activity is maximal and independent of added salt. The effects of salt were determined at low concentrations of acceptor (56 mu). After 30 min at 30 C, two O.l-ml samples were run over AG l- X2 columns (0.5 x 4 cm) to isolate [carbonyl-%]nicotinamide for radioassays (20). One unit of transferase corresponds to 1 pmol of GDP-ribose transferred to an acceptor/min. Protein was determined by the method of Lowry et al. (21). The ADP-ribosyltransferase was purified from the soluble fraction of turkey erythrocytes by successive chromatography on phenyl-sepharose, carboxymethylcellulose, NAD-agarose, and concanavalin A agarose (19). The purified enzyme exhibited one major protein band on sodium dodecyl sulfate-polyacrylamide gels (19). Materials-Turkey erythrocytes were purchased from Pel-Freez; potassium and sodium phosphate, propylene glycol, sodium fluoride, and sodium thiocyanate from Fisher; sodium chloride and sodium bromide from Baker; ovalbumin, agmatine, arginine, arginine methyl ester, guanidinopropionate, guanidine, trypsin inhibitor, and chymotrypsinogen from Sigma; catalase from Boehringer-Mannheim; Tris from BRL, Sephadex G-200 from Pharmacia; AG 1-X2 from Bio-Rad; [carbonyl-winad (specific activity 53 mci/mmol) and [adenine-u- CJNAD (specific activity 265 mci/mmol) from Amersham. RESULTS The activity of the ADP-ribosyltransferase, assayed at low 11452

2 Activation of ADP-ribosyltransferase by Chaotropic Salts acceptor concentrations, was enhanced by several inorganic salts (Table I, Fig. 1). Chaotropic salts were most effective with SCN- > Br- > C1- > F- > PO:-; the activity of the salt corresponded to its position in the Hofmeister series for the salting out of englobulins. The nature of the monovalent cation (Na, K, or Li) had little influence on the effectiveness of a given anion (Table I). Activation did not result from stabilization alone, since under the assay conditions at 30 "C the reaction rate was linear both in the absence and presence of NaCl (Fig. 2). In the absence of NaCl, double reciprocal plots gave limiting slopes corresponding to apparent Michaelis constants of 3.8 and 50 mm, respectively, for arginine methyl ester (22). The standard V versus substrate plot was consistent with the presence of two enzyme species (Fig. 3A). In the presence of NaCI, the double reciprocal plot was linear with an apparent K, of 1.3 mm (Figs. 3B and 4). Other guanidino derivatives also stimulated [carb~nyl-'~c]nicotinamide release (Fig. 4); classical Michaelis-Menten kinetics was observed in the presence ofnac1. The substrates possessing positively charged groups near the guanidino moiety showed significantly more activity: arginine methyl ester > agmatine 2 arginine > guanidinopropionate 2 guanidine; creatine was inactive (Fig. 4, data not shown). In the presence of NaC1, the TABLE I Effect of salt on the activity of the erythrocyte ADPribosyltransferase Assays containing 50 mm potassium phosphate (ph 7.0), 3 nm arginine methyl ester, ovalbumin (1 mg/ml), 100 PM [carb~nyj-'~c] NAD, and the indicated addition were initiated with transferase (0.79 ng) and incubated for 30 min at 30 "C. Means of values from duplicate assays are reported. Addition (100 m ~ ) Nicotinamide released A. None NaF NaCl NaBr B. LiCl NaCl KC1 C. Buffer" KC1 KSCN a Potassium phosphate, ph 7.0 (100 mm). pmol. min". mg" TIME (min) FIG. 2. Effect of NaCl on the rate of [mrbonyl-"c]nicotinamide release from [~arbonyl-'~c]nad catalyzed by the erythrocyte transferase. Assays (total volume, 1.8 ml) containing 50 mm potassium phosphate (ph 7.0), 3 mm arginine methyl ester, ovalbumin (1 mg/ml), 100 FM [~arbonyl-'~cjnad (41,600 cpm), and either no NaCl (0) or 0.3 M NaCl (A) were initiated with transferase (4.73 rg). At the indicated times, two 0.14 samples were removed for isolation of [~arbonyl-'~c]nicotinamide. A 1 I I I I 1 I CONC (mm) /C,(rnM) FIG. 3. Effect of NaCl and arginine methyl ester on the ac- CONC (mm) tivity of the erythrocyte ADP-ribosyltransferase. Assays (0.3 ml, total volume) containing 50 mm potassium phosphate (ph 7.0), Ftc. 1. Effect of NaCl and NaSCN on the activity of the ovalbumin (1 mg/ml), 100 p~ [~arbonyz-'~c]nad(41,600 cpm), and erythrocyte ADP-ribosyltransferase. Assays were carried out as the indicated concentrations of arginine methyl ester with (0) or described intable I with the indicated concentrations of NaSCN (8) without (0) 200 mm NaCl were initiated with transferase (0.41 ng) or NaCl (0). and incubated for 30 min at 30 "C, as described previously.

3 11454 Activation ADP-ribosyltransferase of by Chaotropic Salts TABLE I1 Effect of NaCI on the ADP-ribosylation of agmatine Assays, in a total volume of 0.3 ml, containing 20 mm potassium phosphate (ph 7.0), ovalbumin (1 mg/ml), 2 mm agmatine, and either 100 p [adenine-u-"c'jnad (78,300 cpm) or 100,UM [carbonyl-"cj NAD (37,300 cpm) were initiated with transferase (5.4 ng in the absence of NaCl and 0.54 ng in the presence of NaCI) and incubated for 30 min at 30 "C. Means of values from duplicate assays are reported. adenine-u- carbonyl- Addition LIADP-ri- bose trans- klvicotin- arnlde re- Ratio (A,BI ferred (A) leased (B) pmol.min".mg" 0.95 None NaCl(200 mm) o 1 IC (mm) FIG. 4. Effect of substrate on the activity of the erythrocyte ADP-ribosyltransferase. Assays containing 50 mm potassium phos- phate (ph 7.0), 200 mm NaCI, ovalbumin (1 mg/ml), [carbonyl- ~ ~ '%]NAD (36,300 cpm), and the indicated concentrations of arginine methyl ester (O), agmatine sulfate (O), arginine (A), or guanidine (A) were initiated with transferase (0.41 ng) and incubated at 30 "C for 30 min. Two 0.1-ml samples of each were used for isolation of [~arbonyz-~~c]nicotinamide. r I I I, I I + NaCl 0 4 d/"= - t RADIUS (cm) I 100 FIG. 6. EfTect of NaCl on the rate of sedimentation of the ADP-ribosyltransferase. A, reaction mixture (4 ml) containing Na CI (mm) transferase (1.08 ng), trypsin inhibitor (5 mg), or chymotrypsinogen FIG. 5. Effect of NaCI on the release of [~admnyl-'~c]nicotin- (5 mg) and 25% propylene glycol, 50 mm potassium phosphate (ph amide from [cuh~nyl-'~c]nad and on transfer of [adenine-u- 7.0), 100 PM NAD, 3 mm arginine methyl ester, and 200 mm NaCl was "CIADP-ribose from [adenine-u-"c]nad to ovalbumin cata- centrifuged at 326,000 X g for 66 h at 0 "C. Samples (0.2 ml) were lyzed by the erythrocyte ADP-ribosyltransferase. Assays (total removed from the top of the tube and assayed as described under volume, 0.3 ml) containing 20 mm potassium phosphate (ph 7.0), "Experimental Procedures." B, two samples of transferase (0.16 ng) ovalbumin (1 mg), [~arbonyl-'~c]nad(36,200 cpm) or [adenine-u- containing 10 mm Tris(Cl-) (ph 7.0), 25% propylene glycol, 30 PM "CINAD (41,200 cpm), and the indicated concentration of NaCl were NAD, 5 mm arginine methyl ester, catalase (1 mg/ml), one with and initiated with transferase (6.3 ng) and incubated for 30 min at 30 "C. one without 200 mm NaC1; 3.4 ml of each was layered in a centrifuge From duplicate assays containing [carbonylr4c]nad, two 0.1-ml tube on 0.6 ml of 100% propylene glycol and tubes were centrifuged samples were removed for isolation of [~arbonyl-~~c]nicotinnamide. at 326,000 X g for 3 h. Fractions (0.2 ml) were collected from the top To assays Containing [~denine-u-'~c]nad, 0.5 ml of 10% trichloroacetic acid was added followed by 0.5 mg of ovalbumin (in 0.1 ml). After 30 min at 0-4 "C, samples were filtered to collect precipitated protein for radioassay (23) RADIUS (crn) of the tube and 10-pl samples of each were assayed for transferase activity, Recovery was 77% in the absence of NaCl and 94% in its presence. Only the sedimentation pattern in the absence of salt is shown; no sedimentaion was observed in its presence.

4 Activation of ADP-ribosyltransferase by Chaotropic Salts FRACTION FIG. 7. Effect of NaCl on the elution of the ADP-ribosyltransferase from Sephadex G-200. Columns of Sephadex G-200 (1.2 X 46 cm) were equilibrated at 4 C with 0.2 M glycine buffer, ph 7.0, containing 25% propylene glycol with (A) or without (B) 1 M NaCl and after application of transferase (1.7 ).rg in 1 ml) were eluted with the same buffer. Fractions of 0.65 ml (A) or 0.92 ml (B) were collected and samples assayed for transferase activity under the optimal conditions given under Experimental Procedures. In A, blue dextran was eluted at 16 ml, chymotrypsinogen at 45 ml, and r3h]atp at 55 ml. In B, blue dextran was eluted at 23 ml, chymotrypsinogen at 45 ml, and [SH]ATP at 60 ml. K,,, for [carbonyl- %]NAD was reduced slightly (data not shown). Ovalbumin added to the assay stabilized the ADP-ribosyltransferase and acted as an ADP-ribose acceptor. In the presence of ovalbumin, 50 to 150 mm NaCl increased [adenine-u- %]ADP-ribosylation of ovalbumin and release of [carbonyl- *C]nicotinamide from [carbonyz- 4C]NAD in parallel. Further increase in NaCl concentration to 250 mm enhanced nicotinamide release but did not further increase ADP-ribosylation, i.e. the ratio of [adenilze-u- 4CJADP-ribose incorporated to [carbonyl- 4C Jnicotinamide released declined (Fig. 5). With agmatine as acceptor, the ratio of [adenine-u- C]ADP-ribose incorporated to [carbonyz- 4C]nicotinamide released (-0.9) was unaffected by increasing NaCl; the rates of both processes were increased -lo-fold by 200 mm NaCl (Table II). In the presence of NaCl and propylene glycol, a large fraction of the ADP-ribosyltransferase exhibited a sedimentation coefficient similar to that observed for chymotrypsinogen (Fig. 6A). On Sephadex G-200 in the presence of NaCl and propylene glycol, the enzyme had a KBy similar to that of chymotrypsinogen (Fig. 7A). As noted previously, the transferase shows a molecular weight of 28,300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (19). In the absence of NaCl, the transferase (Fig. 6B) showed a sedimentation pattern consistent with the presence of multiple associated species and was eluted in the void volume of a Sephadex G- 200 column (Fig. 7B). Assays run under conditions identical with those used to examine the sedimentation and gel permeation properties of the transferase showed that NaCl increased the activity of the transferase 7 lo-fold (data not shown). DISCUSSION The data presented here are consistent with the conclusion that the erythrocyte ADP-ribosyltransferase can exist in a relatively inactive, rapidly sedimenting form which elutes in the void volume of a G-200 gel permeation column, and a more active, slowly sedimenting species with a Kay similar to chymotrypsinogen. The amount of the latter form is increased in the presence of salt. Assays performed under conditions identical with those used for ultracentrifugation and gel permeation chromatography demonstrated that, in salt, the transferase was over lo-fold more active. In the presence of salt, the apparent K,,, for arginine methyl ester was reduced dramatically; interpretation of the nonlinear double reciprocal plots for arginine methyl ester in the absence of salt is complicated by the fact that increasing the substrate concentration increases the salt in the assay and thus would tend to activate the enzyme. Increasing the concentration of salt in the assay converted the nonlinear Lineweaver-Burk plot to one consistent with classical Michaelis-Menten kinetics with a low K,,, for ADP-ribose acceptor (Fig. 3). Similar effects were observed with other substrates; those with a positive charge near the acceptor guanidino moiety were better ADP- ribose acceptors. The effectiveness of salts followed the Hofmeister series for the salting out of englobulins with SCN- > Br- > Cl- 7 F- > POd3- (24, 25); as expected, the effectiveness of a salt appeared to be related to the anionic rather than cationic species. Ionic strength was not the primary determinant of transferase activation; the most active salts were those which are effective in disrupting macromolecular structure, i.e. are chaotropic. Salt appears to increase the activity of related enzymes. The ADP-ribosyltransferase activity of choleragen is increased by salt (20). Although it did not enhance the synthesis of long chain poly(adp-ribose), NaCl increased the NAD glycohydrolase activity of poly(adp-ribose) synthetase (26). There is no evidence, however, that salt affects the associative properties of these enzymes or that the effect was related to an increase in ionic strength alone. It is evident, however, that the catalytic activity of the erythrocyte ADP-ribosyltransferase (with subsaturating concentrations of ADP-ribose donor and acceptor) is clearly greater under conditions in which it is dissociated. Since the self-association is clearly reversible n (Transferase) + (Transferase), and has a dramatic effect on activity, this transition, perhaps influenced by as yet uniden- tified effecters, may have a role in the physiological regulation of enzyme activity. REFERENCES 1. Hayaishi, 0.. and Ueda, K. (1977) Anna. Reu. Biochem. 46, Cassel, D., and Pfeuffer, T. (1978) Proc. N&Z. Acad. Sci. U. S. A. 75, Gill, D. M., and Meren, R. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, Johnson, G. L., Kaslow, H. R., and Bourne, H. R. (1978) J. Biol. Chem. 263, Gill, D. M., and Richardson, S. H. (1980) J. Infect. Dis. 141, Honjo, T., Nishizuka, Y., Hayaishi, 0.. and Kato, I. (1968) J. Biol. Chem. 243, Iglewski, B. H., and Kabat, D. (1975) Proc. Natl. Acad. Sci. U. S. A. 72,22&G Goff, C. G. (1974) J. Biol. Chem. 249, Rohrer, H., Zillig, W., and Mailhammer, R. (1975) Eur. J. Biothem. 60, Zillig, W., Mailhammer, R., Skorko, R., and Rohrer, H. (1977) Curr. Top. Cell. Regal. 12, Moss, J., and Vaughan, M. (1977) J. Biol. Chem. 252, Moss. J.. and Richardson. S. H. (1978) J. Clin. Invest MOSS, J., and Stanley, S. J., unpublished data.

5 11456 Activation ADP-ribosyltransferase of 13. Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1980) J. Biol. Chem. 255, Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1980) J. Bid. Chem. 255, Riquelme, P. T., Burzio, L. O., and Koide, S. S. (1979) J. Biol. Chem. 254, Burzio, L. O., Riquelme, P. T., and Koide, S. S. (1979) J. Biol. Chem. 254, Ogata, N., Ueda, K., and Hayaishi, 0. (1980) J. Biol. Chem. 255, Ogata, N., Ueda, K., Kagamiyama, H., and Hayaishi, 0. (1980) J. Biol. Chem. 255, Moss, J., Stanley, S. J., and Watkins, P. A. (1980) J. Biol. Chem. 255, by Chaotropic Salts 20. Moss, J., ManganieIlo, V. C., and Vaughan, M. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, Moss, J., Stanley, S. J., and Oppenheimer, N. J. (1979) J. Biol. Chem. 254, Moss, J., and Vaughan, M. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, Von Hippel, P. H., and Hamabata, A. (1973) J. Mechanochem. Cell Motility 2, Edelhoch, H., and Osborne, J. C., Jr. (1976) Adu. Protein Chem. 30, Ueda, K., Okayama, H., Fukushima, M., and Hayaishi, 0. (1975) J. Biochem. (Tokyo) 77,lP