Application of a Fluorometric Assay for Characterization of the Catalytic Competency of a Domain III Fragment of Pseudomonas aeruginosa Exotoxin A

Analytical Biochemistry 292, 26 –33 (2001) doi:10.1006/abio.2001.5052, available online at http://www.idealibrary.com on

Application of a Fluorometric Assay for Characterization of the Catalytic Competency of a Domain III Fragment of Pseudomonas aeruginosa Exotoxin A 1 S. Armstrong and A. R. Merrill 2 Guelph-Waterloo Centre For Graduate Work in Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada N1G-2W1

Received September 25, 2000; published online April 2, 2001

Pseudomonas aeruginosa exotoxin A (ETA) is a member of the family of bacterial ADP-ribosylating toxins that use NAD ⴙ as the ADP-ribose donor. The reaction catalyzed by ETA involves the nucleophilic attack of the diphthamide residue on the anomeric carbon of the nicotinamide ribose forming a new glycosidic bond. A fluorometric assay involving the use of etheno␤-nicotinamide adenine dinucleotide (⑀-NAD ⴙ), an analog of NAD ⴙ, has been found to provide a rapid, reliable, and sensitive procedure for assessing the kinetic parameters of this class of enzymes including ETA and its C-terminal fragment, PE24. Furthermore, application of this new assay facilitated the determination of the kinetic parameters for the protein substrate of ETA, elongation factor, which has previously been difficult to characterize. These findings provide new insights into catalytic mechanism of dipthamide-specific ribosyltransferases. In addition, this assay should also prove valuable for the study of NADases or NAD ⴙglycohydrolase enzymes (B. Weng, W. C. Thompson, H. J. Kim, R. L. Levine, and J. Moss, 1999, J. Biol. Chem. 274, 31797–31803; Y. S. Cho, M. K. Han, O. S. Kwark, M. S. Phoe, Y. S. Cha, N. H. An, and U. H. Kim, 1998, Comp. Physiol. B: Biochem. Mol. Biol. 120, 175–181) and the poly-ADP-ribosyltransferases (A. A. Pieper, A. Verma, J. Zhang, S. H. Snyder, 1999, Trends Pharmacol. Sci. 20, 171–181; M. K. Jacobson and E. L. Jacobson, 1999, Trends Biochem. Sci. 24, 415– 417). © 2001 Academic Press

Key Words: fluorescence; exotoxin A; mono-ADP-ribosyltransferase; kinetics.

1 Supported by the Canadian Institutes of Health Research and Canadian Cystic Fibrosis Research Foundation. 2 To whom correspondence should be addressed. Fax: (519) 7661499. E-mail: [email protected].

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Pseudomonas aeruginosa (PA) 3 is a ubiquitous, gram-negative, opportunistic pathogen that is commonly found in soil, water, sewage and even in hospital environments (1). This bacterium is a leading cause of infections in AIDS, burn, cystic fibrosis, postoperative patients, and in other various immune-compromised hosts. PA synthesizes a number of extracellular toxic products believed to be involved in the pathogenesis of these infections. The most toxic factor secreted by PA is the 66-kDa protein, exotoxin A (ETA). ETA belongs to a larger family of enzymes that catalyze the transfer of ADP-ribosyl moiety from NAD ⫹ to acceptors (2– 4). More specifically, ETA is a member of the family of enzymes known as mono(ADP-ribosyl) transferases (5–7) and is an NAD ⫹-diphthamide ADP-ribosyl transferase (EC 2.4.2.36). The enzyme domain of ETA catalyzes the transfer of ADP-ribose from NAD ⫹ to the diphthamide residue in eukaryotic translation factor protein, eEF-2. A catalytic mechanism based on the X-ray structure (8, 9) and our own recent work has been proposed for this reaction (10 –12). Although elucidation of the three-dimensional structure (8, 9) and other approaches based on site-directed mutagenesis (13) and photo-affinity labeling (14) have 3 Abbreviations used: PA, Pseudomonas aeruginosa; PE24, Pseudomonas aeruginosa exotoxin A-24 kDa C-terminal fragment; PE40, Pseudomonas aeruginosa exotoxin A 40-kDa C-terminal fragment; ADPRT, ADP-ribosyl transferase; ⑀-AMP, etheno-adenosine monophosphate; ⑀-NAD ⫹, etheno-␤-nicotinamide adenine dinucleotide (oxidized form); eEF-2, eukaryotic elongation factor 2; ETA, Pseudomonas aeruginosa exotoxin A; IPTG, isopropyl-␤-D-thiogalactopyranoside; K d, substrate-binding (dissociation) constant; K M, Michaelis constant; NAD ⫹, ␤-nicotinamide adenine dinucleotide (oxidized form); PARP, poly-ADP-ribosyltransferase; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Tris, tris(hydroxymethyl)aminomethane; WT, wild-type; Caps, 3, cyclohexylamino-1-propanesulfonic acid.

0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

FLUOROMETRIC ASSAY FOR EXOTOXIN A

provided valuable insights into the nature of amino acid residues participating in the catalytic mechanism, a detailed analysis of the ADPRT reaction still remains obscure. A major contributing factor to this situation is the reliance on the currently employed assay procedure, which involves the use of radioactively labeled substrate. This approach requires multiple experiments for the determination of the initial rate of the enzyme catalyzed process and hence demands access to adequate amounts of highly purified preparation of eEF-2, a condition which, often, is not easily achievable. Two factors appear to be responsible for the limited accessibility to eEF-2 and these are: (i) its expression in an eukaryotic system is under stringent control which retards its overexpression; and (ii) the formation of diphthamide functionality, its ADP-ribosylation site, involves a series of posttranslational events, a feature that cannot be duplicated in prokaryotes. These considerations necessitated the development of an alternate assay procedure. In this connection, the feasibility of using ⑀-NAD ⫹, a fluorescent analogue of NAD ⫹, which has been successfully employed in following the ADP-ribosylation of agmatine (15), to monitor the reaction catalyzed by ETA and its C-terminal fragment, PE24, is explored. This report demonstrates the reliability, reproducibility and other advantages afforded by the fluorometric assay for the evaluation of the kinetic parameters of the catalytic fragment of ETA. Furthermore, this assay should also be highly useful for measuring the NADase activity of the NAD ⫹-glycohydrolase family of enzymes. MATERIALS AND METHODS

The Centriprep concentrators, 10- and 50-kDa MW cutoff, were supplied by Amicon Inc. (MA). The following materials were supplied by Sigma (St. Louis, MO): IPTG, ampicillin, ⑀-AMP, and ⑀-NAD ⫹. Chelating agarose and Fast Flow Q-Sepharose resins were purchased from Bio Rad (Amersham-Pharmacia Biotech, Baie d’Urfe, QU). Wheat germ was purchased from New-life Mills (Hanover, ON). Overexpression and purification of PE24. The Cterminal fragment of ETA (PE24) was overexpressed in Escherichia coli strain BB101 (␭DE3) cells and purified using the protocol of Beattie and Merrill (10) with slight modifications. Two microliters of plasmid pPE24H, which contains a CAT repeat that codes for a poly His sequence at the C-terminus of the protein, was used to transform BB101 (␭DE3) cells. The transformation mixture was plated onto two 2X YT medium plates containing ampicillin 100 ␮g/mL and left to grow at 37°C overnight. Each plate was scraped, and the cells were placed into 50 mL of 2X YT broth containing ampicillin 100 ␮g/mL. The culture was grown at 37°C to a high cell density (⬃1 h) and an aliquot (10

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mL) was transferred to each of two culture flasks containing 500 mL of super L-broth supplemented with MgSO 4 0.4% and glucose 0.5%. These cultures were grown to an OD 650 value between 0.5 and 0.7. Subsequently, the cells were grown for an additional 90 min at 37°C following induction with isopropyl ␤-D-galactopyranoside (1 mM). The cells were collected by centrifugation (10,000g) for 10 min and the cell pellet was suspended in 100 ml of 20 mM Tris, 10 mM EDTA, pH 7.9, containing sucrose (20%). The periplasmic fraction was isolated as previously described by Rasper and Merrill (16), and the extract was loaded onto a QSepharose Fast-Flow anion-exchange column (10 ml), previously equilibrated in buffer A (20 mM Tris 䡠 HCl, pH 7.8) containing NaCl (50 mM). The column was washed with 20-bed volumes of buffer A to ensure removal of EDTA from the extract and eluted with buffer A containing NaCl (300 mM). The eluent was then passed through a 1.0-mL chelate-agarose affinity column (Pharmacia) charged with 50 mM NiSO 4. The column was washed initially with 5 ml buffer B (20 mM Tris 䡠 HCl, 500 mM NaCl, pH 7.9) containing 5 mM imidazole and subsequently with 10 mL of buffer B at a higher imidazole concentration (60 mM). The PE24 protein, possessing a poly His sequence, was eluted from the column with 8 mL of buffer B containing imidazole 250 mM. All fractions (1 mL each) were analyzed by SDS-PAGE and those exhibiting a major band at M r 28 kDa were pooled and dialyzed overnight in buffer A containing 1 mM EDTA. The dialyzed sample was concentrated to 1.0 mL using an Amicon Centriprep concentrator (10 kDa MWCO) dispensed into small volume aliquots, quick frozen in a dry ice-methanol bath, and stored at ⫺70°C. Purification of eEF-2. Wheat germ served as the source of this protein and the procedure employed was similar to that previously described (19) with slight modifications. Crude extract from wheat germ (obtained from Hanover Mills; 450 g) was adjusted to pH 5.0 and the precipitate formed was collected and dissolved in 2 L of Tris 50 mM, pH 8.1, containing MgOAc (5 mM), CaCl 2 (4 mM), KCl (100 mM), and ␤-mercaptoethanol (0.7%). The precipitate formed upon addition of ammonium sulfate (35% saturation) was collected and redissolved in 1.5 L of buffer C (KCl 50 mM, glycerol 5%, pH 7.0) containing potassium phosphate (25 mM) and applied to 8 ⫻ 15-mL columns of hydroxyapatite equilibrated with buffer C. The chromatographic mixture was washed with buffer C containing potassium phosphate (60 mM) prior to elution with buffer C containing potassium phosphate (300 mM). The protein fraction recovered was subjected to further chromatography on Fast Flow Q-Sepharose and the protein preparation was concentrated to (10 –20 mg/ mL) and stored at ⫺70°C.

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Spectroscopic measurements. All fluorescence measurements were performed using a PTI Alphascan spectrophotometer (Photon Technology International, South Brunswick, NJ) equipped with a water-jacketed sample chamber set to 25°C. Excitation and emission slitwidths were 1 and 2 nm, respectively, unless otherwise stated, and the excitation wavelength used for the assay was 305 nm. The emission monochromator was set to 0 nm, and a 309-nm cutoff filter (Oriel Corporation, Stratford, CT) was placed on the sample chamber side of the emission monochromator to maximize signal detection. ADPRT assay. A typical assay, in a final volume of 70 ␮l, consisted of ⑀-NAD ⫹ (desired concentration), eEF-2 (20 ␮M), and buffer (20 mM Tris 䡠 HCl, pH 7.8) in an ultramicrocuvette of pathlength 3 mm (Helma Inc., Concord, ON) placed in the sample chamber of the fluorometer. Following temperature equilibration for 10 min, the reaction was initiated by the introduction of PE24 (5 nM). The reaction was monitored by recording the increase in fluorescence intensity with time (excitation 305 nm, emission 309-cutoff filter). These experiments were performed over a wide range (0 –700 ␮M) of ⑀-NAD ⫹ using appropriate aliquots of a 57 mM stock solution of this compound in the above buffer. In experiments where eEF-2 concentration was varied, the protein was used over the range of 2–25 ␮M. In all experiments the KCl concentration was kept at 50 mM concentration. ⑀-AMP standard curve and assay calibration. A stock solution of 44.7 mM ⑀-AMP in distilled water was 265 prepared (⑀ M ⫽ 10,000 M ⫺1 cm ⫺1) along with a series of standards, containing 0 –7 ␮M ⑀-AMP in buffer (20 mM Tris, pH 7.8). The fluorescence emission of each standard was measured and a standard curve constructed, which was used to calibrate the enzyme-catalyzed reaction rate. Kinetic analysis. Determination of K M and V max for PE24 involved measurement of initial rates at 10 different concentrations of substrates. Over a range of 2–25 ␮M in the case of eEF-2 and 50 –700 ␮M for ⑀-NAD ⫹. When the concentration of ⑀-NAD ⫹ was varied, eEF-2 was kept constant (20 ␮M) and conversely when eEF-2 concentrations were varied, ⑀-NAD ⫹ concentration was kept at 500 ␮M. These conditions fulfilled the requirement of maintaining the concentration of the second substrate at saturation level. The activity at each substrate concentration was determined in triplicate and all the experiments were performed at least three times. An activity profile for the effect of pH on the enzyme activity was obtained by varying the pH between 2 and 11. The buffer media used with the pH range shown in parentheses were sodium acetate (30 mM, pH 2– 4), Bis-Tris (30 mM, pH 4.5–7.0), Tris-HCl (30 mM, pH

7.4 – 8.5), and Caps (30 mM, pH 9 –11). All the measurements were taken at 25°C and spontaneous hydrolysis of the ⑀-NAD ⫹ substrate at each pH was not sufficient to interfere with the ADPRT reaction. The effect of temperature on activity of PE24 was also examined. The ADPRT reaction was measured through a temperature range from 0- to 40°C in 5°C increments using a digital thermistor with a fine-wire thermocouple that bathed in a reference cuvette filled with water in the multicell holder of the fluorometer. The reaction mixture was incubated at each temperature for 10 min to equilibrate the sample before the toxin was added to the solution. Furthermore, the ADPRT activity of PE24 was measured at various KCl concentrations ranging from 50 to 800 mM (V max conditions). The concentrations of both ⑀-NAD ⫹ and eEF-2 substrates were kept constant at 500 and 20 ␮M, respectively. All measurements were taken at 25°C. Quenching of intrinsic protein fluorescence. The NAD ⫹-dependent quenching of intrinsic protein fluorescence was monitored as a function of the NAD ⫹ concentration as previously described by Beattie and Merrill (10). The effect of KCl concentration on the binding of NAD ⫹ was also examined by varying KCl in buffer in a 50 – 600 mM range. RESULTS

The ADPRT assay. The assay procedure is based on the increase in fluorescence intensity (quantum yield) accompanying the removal of the nicotinamide moiety from ⑀-NAD ⫹ (15). A calibration curve was constructed by monitoring fluorescence intensity as a function of concentration of ⑀-AMP, which served as a standard for the ADP-ribosylated product. The calibration curve (Fig. 1) is in excellent agreement with that demonstrated in a previous report (15). The relatively high degree of purity of eEF-2 and PE24 attained in this study is illustrated in Fig. 2. Limited access to highly purified EF-2 has served as an impediment for achieving a meaningful kinetic analysis of diphthamide-specific ADP-ribosyl transferases. The current fluorometric assay by virtue of its ability to provide a continuous means for data collection (20 pts per second, 6000 data points collected for a typical kinetic trace and adapted for a ultramicrocuvette (0.3 ⫻ 0.3 mm)) minimized the need for large amounts of eEF-2 or PE24. A plot of velocity against substrate concentration for both substrates fits a rectangular hyperbola function, suggesting that the enzyme follows simple MichaelisMenten kinetics (17). A Hanes-Woolf linear transformation of the data shows that the fit to the MichaelisMenten kinetic model is good (Fig. 3). The kinetic parameters for ⑀-NAD ⫹ and eEF-2 substrates are

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FLUOROMETRIC ASSAY FOR EXOTOXIN A

FIG. 1. Standard curve of ⑀-AMP as measured by fluorescence intensity. ⑀-AMP concentration was determined by absorbance spec265 troscopy ⑀ M ⫽ 10,000 M ⫺1 cm ⫺1 and series of dilutions were prepared from a stock solution in buffer A.

shown in Table 1. The k cat/K M values indicate that PE24 catalytic fragment is a moderately efficient enzyme with k cat/K M values near 10 8 M ⫺1 min ⫺1. Effect of pH upon ADP-ribosylation reaction catalyzed by PE24. Although previous studies have determined that the catalytic fragment PE24 is functionally analogous to the parent protein, the complete characterization of its kinetic parameters remained to be addressed. The pH dependence of ADP-ribosylation activity for PE24 showed a typical bell-shaped curve, with maximal activity around pH 7.8 (data not shown). The effect of pH upon the measured kinetics parameters, k cat and k cat/K M, of PE24 is shown in Figs. 4A and B, respectively. The toxin is most stable at neutral pH

FIG. 3. (A) Michaelis-Menten plot of toxin ADPRT activity as a function of eEF-2 concentration. Measurements were obtained at 500 ␮M ⑀-NAD ⫹ and 20 nM toxin at 25°C. (B) Dependence of velocity on ⑀-NAD ⫹ substrate concentration according to the Michaelis-Menten relationship. Measurements were obtained at 20 ␮M eEF-2 and 5 nM toxin at 25°C.

and remains in solution when incubated in buffer at pH values between 5 and 11. Although both substrates NAD ⫹ and eEF-2 are stable over the wide pH range studied (10) eEF-2 was observed to precipitate out of TABLE 1

Kinetic Parameters for Toxin ADPRT Activity Substrate

FIG. 2. Polyacrylamide gel electrophoresis with SDS. Lane 1, standards; lane 2, 10 ␮g of PE24; lane 3, 12 ␮g of eEF-2.

Parameter

NAD ⫹

eEF-2

K M (␮M) V max (pmol min ⫺1) k cat (min ⫺1) k cat/K M (M ⫺1 min ⫺1)

275 ⫾ 52 234 ⫾ 30 675 ⫾ 85 2.5 䡠 10 6

8.0 ⫾ 1.8 258 ⫾ 24 734 ⫾ 67 92.8 䡠 10 6

Note. The kinetic parameters were determined as described under Materials and Methods. The values represent the mean ⫾ SD from two to six independent experiments with each experiment consisting of three separate samples.

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ARMSTRONG AND MERRILL

FIG. 4. pH activity profile of PE24. ADPRT activity was measured using the fluorescence-based approach. The pH was varied in the enzymatic reactions by using a series of buffers: pH 2–5, 30 mM NaOAc; pH 6 –7, 30 mM Bis-Tris; pH 7–9, Tris 䡠 HCl; pH 10 –12, 30 mM Caps. The reaction temperature was 25°C and 20 nM toxin was used in the assay. The pH profile of (A) log k cat and (B) log k cat/K M [NAD⫹] for ADP ribosylation of eEF-2 by PE24. Insets: (A) log k cat versus pH and (B) log k cat/K M[NAD] versus pH.

solution at a pH value close to its isoelectric point of 5.0 –5.3. The V max was maximal at pH 7.8, which is in close agreement with that observed in the case of the intact ETA using radiolabeled NAD ⫹ substrate (10). The maximum value for the enzyme turnover number, or k cat, was observed at pH 7.0. At pH values less than

4.0 and greater than 10.0, the enzymatic activity was negligible (Fig. 4A). The K M for NAD ⫹ was strongly pH dependent as indicated by the shift to larger values with increasing pH. A plot of k cat/K M as a function of pH (Fig. 4B) exhibited a relatively sharp profile centered at pH 7.8.

FLUOROMETRIC ASSAY FOR EXOTOXIN A

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some variability in their sensitivity to KCl concentrations, the general trend was similar. The concentration of KCl at which the ADPRT activity of PE24 was 50% of its original value was between 150 and 180 mM. Ionic strength effect on NAD ⫹ binding of PE24. The titration of PE24 with NAD ⫹ exhibited behavior similar to that previously observed in our laboratory for this C-terminal catalytic fragment (20), for PE40, and for whole toxin (10). The intrinsic fluorescence of the WT was quenched in a dose-dependent manner to less than 20% of the original fluorescence (data not shown). The effect of ionic strength on the Trp fluorescence quenching of PE24 was examined by varying the concentration of KCl in the range of 50 – 600 mM. The effect of KCl concentration on the dissociation constant for NAD ⫹ binding is shown in Fig. 7. Although the 3-fold decrease in K d at high KCl concentrations is not very significant, the general trend is interesting. This finding indicates that the electrostatic interactions do not play a dominant role between PE24 and NAD ⫹ but that the source of salt sensitivity of ADPRT activity is likely the protein-protein interaction between PE24 and eEF-2. DISCUSSION

FIG. 5. Effect of temperature on PE24 activity. Samples containing saturating amounts of eEF-2 and various concentrations of ⑀-NAD ⫹ in a range from 50 to 500 ␮M in 20 mM Tris buffer, pH 7.8, were prepared at room temperature. The ADP-ribosylation activity was measured at various temperatures following a 10-min incubation at the each specified temperature.

The effect of temperature. The applicability of the fluorometric method to study the ADP-ribosylation catalyzed by PE24 was examined. As shown in Fig. 5A, the method permitted evaluation of kinetic parameters over a range of temperature (2– 40°C) and the data obtained aided in the estimation of the activation energy, E a (39 ⫾ 0.5 kJ/mol) for the PE24-catalyzed reaction (Fig. 5B) Effect of ionic strength. Previous studies have indicated that the eEF-2 protein (substrate) is not stable in low ionic strength buffers (18). Furthermore, other investigators have indicated that the activity of exotoxin A seems to be affected by the presence of salt in the buffer (19). In order to further investigate this matter, a study of the effect of increasing KCl concentrations on the activity of PE24 was conducted and a general trend of decreasing activity at higher salt concentrations was observed (Fig. 6). At a concentration of 800 mM KCl there is almost total loss of activity of PE24. Although the different preparations of eEF-2 showed

The goal of this kinetic study was to gather insight into the kinetic mechanism, illustrating the validity of the fluorescence-based assay and to characterize the kinetic parameters of the toxin-catalyzed reaction for both substrates. The results presented here are the first report of such parameters for the eEF-2 substrate of ETA. The results recorded in this study demonstrated the suitability of the fluorometric assay for monitoring the ADP-ribosylation reaction catalyzed by PE24. The kinetic parameters obtained using this

FIG. 6. Dependence of PE24 ADPRT activity on total KCl concentration. The activity of PE24 was measured at 200 ␮M ⑀-NAD ⫹, 20 ␮M eEF-2, 20 nM toxin in various KCl concentrations (50 – 800 mM) in 20 mM Tris-HCl buffer, pH 7.9, at 25°C. The activities were normalized to a value of 1.0 for the ADPRT activity at 50 mM KCl.

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FIG. 7. The effect of KCl concentrations on the binding of NAD ⫹ to PE24. Various concentrations of KCl (50 – 600 mM) were included in the assay buffer (20 mM Tris, pH 7.9) and the intrinsic fluorescence quenching of PE24 by NAD ⫹ was measured as described under Materials and Methods.

method are very similar to those recorded using a traditional radioactively labeled substrate assay in our laboratory. Current assays for ADP-ribosylation are limited because of their discontinuous nature, requiring the laborious process of individually processing numerous samples and resulting in the assembly of only a partial reaction progress curve. Previously, the use of the traditional radioactive assays for ADP-ribosylation made it difficult to study the inhibition mechanisms of specific inhibitors for ETA-catalyzed ADPribosylation reactions. However, the fluorometric procedure has the advantage of being more rapid and sensitive and boasts the capability of continuous acquisition of data. As emphasized earlier, its main advantage pertains to obviating the need for large amounts of eEF-2, the obligatory and precious cosubstrate. Furthermore, the spectrofluorometric ADP-ribosylation assay described herein is amenable to automation in assay procedures involving the use of a fluorescence microplate reader. This makes it feasible as a possible rapid screening assay for the detection of enzyme inhibitor candidates. The use of fluorescent NAD ⫹ derivatives has proven to be advantageous in other systems as well since it provides a more direct method for kinetic studies. Basso and co-workers (21) have used ⑀-NAD ⫹ to study the kinetics of binding for glutamate dehydrogenase. They were able to probe the nucleotide binary and ternary complex formation using ⑀-NAD ⫹. The high sensitivity of fluorescent derivatives of NAD ⫹ in measuring ADP-ribosylation reactions has also been shown by Davis and co-workers (22). They used ⑀-NAD ⫹ to detect poly-ADP-ribosyltransferase (PARP) activity in an immunohistochemical assay in which a specific antibody to ethenoadenosine was used to detect the production of modified poly(ADP-ribose) in cell extracts.

Other investigators, including our own laboratory, have observed that the ADPRT activity of the diphthamide-specific ribosyltransferases is sensitive to the ionic strength of the reaction mixture (14, 18, 19). Furthermore, at moderate salt concentrations total loss of ADPRT activity for the enzyme is observed. In the present study, it was seen that at concentrations above 500 mM KCl there was almost complete inhibition of activity as indicated in Fig. 6. In order to further pursue the mechanism behind these observations, the effect of KCl concentration on NAD ⫹-binding capacity of PE24 was also explored. As shown in Fig. 7, the NAD ⫹-toxin interaction is relatively insensitive to high salt in the binding reaction solution. Thus, the high salt sensitivity of the toxin’s ADPRT activity suggests that a major component of the toxin’s interaction with eEF-2 must involve an electrostatic effect. The ID 50 value for KCl was determined to be in the range of 150 –180 mM, indicating that protein-protein interactions between toxin and eEF-2 are largely electrostatic in nature. This is in agreement with previously published data (18), which illustrated that the binding of toxin to immobilized eEF-2 showed high sensitivity to salt concentrations and no binding was observed at relatively high NaCl concentrations (500 mM). The resourcefulness of the method is indicated by its applicability over a wide range of pH, temperature, and ionic strength values. The kinetic data in this report show that PE24, the C-terminal fragment of ETA, is an efficient catalyst like its parent protein in promoting the ADP-ribosylation reaction. Interestingly, the catalytic function of PE24 at 37°C remains relatively unimpaired, indicating the preservation of its active conformation under conditions of assay. Interpretation of the other aspects of the data in terms of an overall kinetic mechanism of PE24 will be discussed elsewhere, since the primary aim of this report is to emphasize the salient aspects of the analytical procedure. In summary, this assay is much more amenable to an automated procedure, which could provide a high throughput approach to study the enzyme kinetics and catalytic mechanism of diphthamide-specific ADPRT enzymes such as ETA and diphtheria toxins as well as NADase and glycohydrolase enzymes. In this report, the improved approach for the acquisition of the kinetic properties of the toxin ADPRT activity provided additional insight into the catalytic mechanism and properties of this toxin-enzyme. Further investigations in our laboratory are being conducted to elucidate the order of substrate binding and product release mechanism for mono-ADPRT enzymes utilizing this new assay. ACKNOWLEDGMENTS We thank Dr. T. Viswanatha for review of the manuscript and helpful comments. We are grateful to Gerry Prentice for the devel-

FLUOROMETRIC ASSAY FOR EXOTOXIN A opment of the eEF-2 purification procedure and for technical assistance throughout the duration of this project. Furthermore, we thank Yolanda Weir and Monica Tory for their contributions during the initial stages of this project.

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