237

Biochimica et Biophysica Acta, 421 ( 1 9 7 6 ) 2 3 7 - - 2 4 5 © Elsevier Scientific P u b l i s h i n g C o m p a n y , . A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 27816

PROPERTIES OF CHOLERA TOXIN- AND NaF-STIMULATED ADENYLATE CYCLASE FROM MOUSE THYMOCYTES

V A N C E J. P E T R E L L A a n d T E R R Y V. Z E N S E R

United States A r m y Medical Research Institute o f Infectious Diseases, Frederick, Md. 21701 (U.S.A.) ( R e c e i v e d J u n e 3rd, 1 9 7 5 ) ( R e v i s e d m a n u s c r i p t received O c t o b e r 7 t h , 1 9 7 5 )

Summary Kinetic parameters of mouse t h y m o c y t e adenylate cyclase activity were determined. NaF and cholera toxin stimulated adenylate cyclase. Stimulation by either agent did not change the pH or Mg2÷ optima relative to control (unstimulated cyclase). The Km value for ATP of adenylate cyclase stimulated by NaF was significantly reduced from control. By contrast, cholera toxin treatment did not change the Km relative to control. Adenylate cyclase, when stimulated by NaF, had an optimum for Mn 2÷ alone, or Mn 2÷ in combination with Mg:÷, at least twice that of control. In contrast, cyclase activity prepared from cells treated with cholera toxin remained unchanged with regard to these divalent cations when compared to control. Addition of NaF to adenylate cyclase prepared from cells treated with cholera toxin resulted in a significant reduction (30%) in activity suggesting that both NaF and cholera toxin were acting on the same cyclase. NaF inhibition of cholera toxin-stimulated activity was shown to be a direct interaction of fluoride on the stimulated cyclase enzyme. This inhibition appeared to be immediate and independent of pH, Mg2÷ or ATP concentrations. Although NaF inhibition was lost when Mn 2÷ was present in the reaction mixture, the activity expressed by addition of NaF to cyclase prepared from cholera toxin-treated cells was much less than by addition of NaF to control. As observed with cholera toxin stimulation alone, activity expressed by the inhibited enzyme {cholera toxin treated + NaF) exhibited a Km for ATP and an optimum for Mn ~÷ alone or in combination with Mg2÷ similar to control.

In c o n d u c t i n g the research d e s c r i b e d in this r e p o r t , the investigators a d h e r e d to the "Guide for t h e Care and Use of Laboratory Animals," as p r o m u l g a t e d by the C o m m i t t e e on Revision o f the Guide for Laboratory Animal Facilities and Care o f the I n s t i t u t e o f L a b o r a t o r y Animal Resources, N a t i o n a l R e s e a r c h C o u n c i l . T h e facilities are fully a c c r e d i t e d b y the A m e r i c a n A s s o c i a t i o n o f A c c r e d i t a t i o n o f L a b o r a t o r y A n i m a l Care.

238 These data suggest that cholera toxin stimulation of mouse t h y m o c y t e adenylate cyclase is kinetically different from that of NaF. Furthermore, NaF appeared to inhibit the cyclase prepared from cells treated with cholera toxin in a non-competitive manner with respect to ATP substrate, that is, at some site other than the catalytic site. From analogous observations of NaF inhibition of hormonal stimulation in fat cells, it could be postulated that cholera toxin may effect the "coupling" site.

Introduction The action of Vibrio cholerae in the small intestine is the result of an enterotoxin, cholera toxin, elaborated by this organism [1]. The massive fluid and electrolyte losses which occur in clinical cholera infection are the result of cholera toxin stimulation of intestinal adenylate cyclase [2]. In addition to its effect on intestinal cells, cholera toxin has also been shown to increase adenylate cyclase activity in a number of tissues [3--6] including t h y m o c y t e s [7,8]. Cholera toxin stimulation of adenylate cyclase, in contrast to hormones and fluoride, requires preincubation of toxin with intact tissue [2,6] and occurs only after a considerable time lag [6,8]. However, the mechanism by which cholera toxin, once bound to its receptor, stimulates adenylate cyclase has not been well established. By contrast, kinetic analysis has distinguished hormonal from NaF stimulation of adenylate cyclase [9,10]. To elucidate the molecular mechanism by which cholera toxin acts, kinetic properties of mouse t h y m o c y t e adenylate cyclase under control and cholera toxin-stimulated conditions with or without NaF have been investigated. Materials and Methods Purified cholera toxin (Lot 1071) was prepared for the National Institutes of Allergy and Infectious Diseases by Dr. R.A. Finkelstein, Dallas, Texas, as previously described [11], and provided by Dr. Carl Miller, National Institutes of Health. [a-32 p] ATP (8--10 Ci/mmol), cyclic [G-3 H] AMP (24 Ci/mmol) and Riafluor were obtained from New England Nuclear Corp., Boston, Mass. ATP, cyclic AMP, creatine phosphate, creatine phosphokinase and neutral type WN-3 alumina were purchased from Sigma Chemical Co., St. Louis, Mo. Male, C57BL/6 mice, 3--4 weeks old were supplied by R.B. Jackson Laboratories, Bar Harbor, Me. Preparations of thymocytes. Mice were killed by cervical spine dislocation; t h y m o c y t e s were prepared by a mechanical procedure previously described [12]. T h y m o c y t e s (3.2 • 10~--3.7 • 107 cells/ml) were suspended in Hank's balanced salt solution and 30 ml preincubated at 37°C for 30 min in the presence or absence of a maximally stimulating concentration [8] of cholera toxin (500 ng/ml). Preparation and assay o f adenylate cyclase. After preincubation, cells were pelleted by centrifugation and piazma membranes were obtained as previously described [13]. This membrane preparation was suspended in 0.75 ml 3 mM Tris • HCI, pH 7.5, 2 mM MgSO4, 0.5 mM EDTA, and 1.0 mM dithiothreitol.

239 This resulted in a 4-fold enrichment of adenylate cyclase activity with a 60% yield of activity when compared to t h e whole homogenate. The incubation mixture for assaying adenylate cyclase activity was 0.075 ml in volume and contained the following: 2.0 mM [ a - 3 2 p ] ATP (14--130 cpm/ pmol), 1.3 mM cyclic AMP, 5.0 mM MgSO4, 20 mM caffeine, 20 mM creatine phosphate, 70 units/ml creatine phosphokinase, 0.4 mg/ml bovine serum albumin. The reaction, which was initiated by addition of 40--80 pg of protein, was linear during the 10-min incubation at 30°C. Adenylate cyclase reactions were stopped by addition of 0.02 ml of 100 mM EDTA containing cyclic [ 3H] AMP (20 000 cpm) to monitor recovery, and heated to coagulate proteins. The cyclic AMP formed was isolated from neutral alumina columns as described by White and Zenser [14]. Adenylate cyclase activity is expressed as pmol of cyclic AMP formed/min per mg protein. Protein concentrations were estimated according to Lowry et al. [ 15], using bovine serum albumin as a standard. Data for Lineweaver-Burk plots were obtained by varying the concentration of ATP between 0.1 and 2.0 mM. Incubation time was shortened to 3 min to insure reaction linearity at the lower substrate concentrations. Adequate substrate availability was assessed by studying ATP breakdown in the following manner. After a 3-min incubation, aliquots of the adenylate cyclase incubation mixture were cochromatographed with carrier ATP, ADP and AMP on polyethyleneimine-impregnated cellulose thin-layer sheets. Chromatographs were developed with 1 M LiC1 and 20 mM K2HPO4, pH 4.0. The R F values for ATP, ADP and AMP were 0.1, 0.3 and 0.5, respectively. Using the assay outlined above, < 2 0 % of the ATP (0.2 raM) was metabolized. In all experiments, the mean of at least triplicate determinations for each condition is presented. Results

Effects of NaF concentration, time, and pH on mouse thymocyte adenylate cyclase activity The effect of NaF on mouse t h y m o c y t e adenylate cyclase isolated from cells preincubated for 30 min at 37°C in the presence or absence of cholera toxin is shown in Fig. 1. Cholera toxin treatment dramatically increased cyclase activity 5--6-fold. NaF elicited a concentration-dependent increase in control activity with 10 mM being optimum and higher concentrations inhibitory. By contrast, NaF caused a concentration-dependent decrease in cholera toxin-treated cyclase activity. A consistent 30% inhibition (P < 0.005) was observed with 10 mM NaF. Since NaC1 (10 or 20 mM) neither increased basal nor inhibited cholera toxin-stimulated activities, both effects of NaF were attributed to fluoride. To further determine whether this inhibitory effect of fluoride was due to direct interaction with the activated enzyme or with cholera toxin, the following experiment was performed. Cholera toxin was mixed with or without 10 mM NaF for 15 min at 30°C. These mixtures were then preincubated with intact t h y m o c y t e s at 37°C for 30 min with the final concentration of cholera toxin and NaF being 6" 10 -9 and 3.3 • 10 -6 M, respectively. Then cyclase activity was determined. Pretreatment of cholera toxin with NaF prior to addition to intact t h y m o c y t e s did not inhibit the cholera toxin response. However,

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when the activity of this preparation was determined in the presence of 10 mM NaF the 30% inhibition was observed. The activity of control (unstimulated) and cholera toxin-stimulated cyclase was linear for 15 min. NaF stimulation of control or inhibition of cyclase prepared from cells treated with cholera toxin appeared immediate and remained constant during this period. The preincubation time-course of cholera toxin stimulation was defined for the t h y m o c y t e system to determine what effect, if any, it had on NaF inhibition (Fig. 2). During the first 10 min of preincubation, activity expressed by cholera toxin with NaF was usually five times that of cholera toxin without NaF, which was similar to control. After 10 min, cholera toxin activity gradually increased above control resulting in a

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lowered ratio of cholera toxin with NaF to cholera toxin w i t h o u t NaF activity. By 20 min and continuing to 60 min, the ratio dropped below 1.0 and remained between 0.7 and 0.6 (approx. 30% inhibition). Maximal stimulation by cholera toxin was seen after 20 min. Although control activity was rather insensitive to changes in pH, the stimulated activities exhibited pH optima between 7.5 and 8.0 (Fig. 3). NaF stimulation of control and inhibition of cyclase prepared from cells treated with cholera toxin was independent of pH. Effects o f various concentrations o f Mg 2÷ and A T P on mouse t h y m o c y t e adenylate cyclase activity Control and stimulated cyclase activities rose with increasing Mg2÷ concentrations reaching a maximum at 5 mM and remaining constant to 20 mM. A similar Mg2÷ concentration dependency was shown by Sharp et al. [16]. NaF inhibition of cholera toxin-stimulated cyclase activity was apparent at concentrations of Mg2÷/> 1.25 mM. As shown in the Michaelis-Menten plot (Fig. 4 inset), enzyme activities were maximal near 1.0 mM ATP. No substrate inhibition was noticed from 0.1 to 2.0 mM ATP. NaF inhibition of cholera toxin-stimulated cyclase activity was not dependent on the ATP concentration. Km values for ATP exhibited by control and cholera toxin-stimulated enzyme, with or w i t h o u t NaF, were similar (approx. 0.46 mM, Fig. 4). However, when control enzyme was stimulated with NaF, a significant (P < 0.025) decrease in Km for ATP was noted (0.28 mM). Effects o f Mn 2+, Ca 2÷ and Co 2÷ on mouse t h y m o c y t e adenylate cyclase activity Cholera toxin-stimulated cyclase activity with or without addition of NaF along with control cyclase activity displayed a Mn 2÷ optimum of 2.5 mM (Fig. 5). By contrast, the o p t i m u m for control with NaF was 5 mM. Mn 2÷ alone (Fig. 5) or in combination with Mg2÷ {Fig. 6) resulted in the loss of NaF inhibi-

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tion of cholera toxin-stimulated cyclase activity. In the presence of an optimum a m o u n t of Mg2÷, Mn 2÷, up to 1.25 or 5 mM, increased enzyme activity. In contrast to Mg2÷, high levels of Mn 2÷ were inhibitory (Figs. 5 and 6). Neither Ca 2÷ nor Co 2÷ could satisfactorily substitute for Mg2÷ or Mn 2÷. Discussion Fluoride exhibited the expected biphasic concentration curve for cyclase with a magnesium-fluoride complex probably responsible for stimulation and F- itself inhibiting at high concentrations [17,18]. In contrast, cholera toxinstimulated cyclase activity was inhibited by this magnesium-fluoride complex. This inhibition suggested that fluoride and cholera toxin were acting on the same cyclase, but by different mechanisms. Both the stimulatory and inhibitory effects of fluoride appeared immediate. By contrast, the cholera toxin stimulation of cyclase was delayed and exhibited a time course similar to that reported when mouse t h y m o c y t e cyclic AMP content was measured [8]. Although similar inhibitory effects of NaF on cholera toxin-stimulated cyclase activity have recently been reported [19--21], no detailed kinetic analysis was conducted. In our study, NaF inhibition was dependent on the length of time cholera toxin was preincubated with t h y m o c y t e s prior to cellular disruption and assay of cyclase activity. Inhibition was seen only after cholera toxin maximally stimulated the cyclase. To investigate the possibility that fluoride inhibition of Cholera toxin-stimulated cyclase activity might have been due to either removal of magnesium or formation of a fluoride-magnesium-ATP complex involving the catalytic site, cyclase activity was assessed at various ratios of

244 magnesium to ATP. At all concentrations tested, fluoride inhibited cholera toxin-stimulated cyclase activity. This inhibition was also independent of pH and was shown to be the result of direct interaction of fluoride (magnesiumfluoride complex) with the cholera toxin-stimulated enzyme. These data suggested that fluoride inhibition of cholera toxin-stimulated cyclase activity occurred at a site distinct from th.e catalytic site. NaF stimulation of adenylate cyclase was different from cholera toxin in several ways. NaF stimulation reduced the Km value for ATP to half that of control while cholera toxin, as previously shown in rabbit ileum by Sharp et al. [16], had no effect on Kin. Furthermore, addition of NaF to the cholera toxin-stimulated cyclase resulted in non-competitive inhibition with respect to ATP as described by Plowman [22]. When Mn 2÷ was substituted for Mg2÷ or added in combination with Mg ~÷, the kinetic responses of the control and cholera toxin-stimulated cyclase were consistently similar but different from the control with NaF-stimulated cyclase. Because the precise nature of the adenylate cyclase system is not known, speculation as to the mode of the stimulatory or inhibitory processes just described is difficult. However, fluoride inhibition of hormonal stimulation has been observed in fat cells [18], and an analogous situation may be represented in our study. In isolated fat cells, it was shown that fluoride inhibition was not due to inhibition of hormonal interaction with their receptor nor, as also shown here, to inhibition of substrate utilization at the catalytic site. To explain this inhibitory action of fluoride in fat cells, it was suggested that fluoride "uncouples" hormonal activation subsequent to hormone-receptor interaction. Coupling of the hormonal receptor to the catalytic component requires the involvement of lipids and GTP [23--26]. By contrast, lipids and GTP are not required for fluoride stimulation and, in some instances, even cause inhibition [23--25]. Therefore, since the cellular receptor for cholera toxin is a glycolipid [27] and cholera toxin has been shown to increase the sensitivity of adenylate cyclase for hormones ("coupling") [19,20] as does GTP [25,26], it is possible that cholera toxin activated the cyclase in a manner which irreversibly altered this coupled state to one more sensitive to hormonal, than fluoride stimulation. A positive relationship between cholera toxin and hormonal stimulation has been demonstrated by the fact that both are similarly affected by fluoride [18], Mn 2÷ [10] and pyrophosphate [10,16]. In summary, kinetic properties of the immunologically important mouse t h y m o c y t e adenylate cyclase have been described. Cholera toxin and NaF appeared to stimulate the same cyclase. However, differences in the Mn 2÷ optima and Km for ATP of the NaF- and cholera toxin-stimulated cyclase were observed. Furthermore, since the activity expressed by the cholera toxin-stimulated cyclase with NaF with regard to Mn 2÷ optimum and Km was similar to that of the cholera toxin-stimulated enzyme, it is postulated that the configuration induced by cholera toxin stimulation was not changed to a fluoride form, but rather inhibited non-competitively by fluoride. NaF inhibition of cholera toxin stimulation was found to be dependent on the extent of cholera toxin activation and Mg 2÷. This kinetic evidence suggests that cholera toxin stimulates mouse t h y m o c y t e adenylate cyclase in a manner different from NaF perhaps via the "coupling" site.

245

Acknowledgements We acknowledge the expert technical assistance of Ernest Fischer, Brian Sanders, and Nancy Reamy and we thank Mrs. Phebe Summers for her aid in the preparation of this manuscript. References 1 De, S.N. (1959) Nature 183, 1533--1534 2 Kimberg, D.V., Field, M., Johnson, J., Henderson, A. and Gershon, E. (1971) J. Clin. Invest. 50, 1218--1230 3 Gorman, R.E. and Bitensky, M.W. (1972) Nature 235, 439--440 4 Beckman, B., Flores, J., Witkum, P.A. and Sharp, G.W.G. (1974) J . . Clin. Invest. 53, 1202--1205 5 Wolff, J°, Temple, R. and Cook, G.H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2741--2744 6 Bourne, H.R., Lehrer, R.I., Llchtenstein, L.M., Welssmann, G. and Zurier, R. (1973) J. CHn. Invest. 52, 698--708 7 Boyle, J.M. and Gardner, J.D. (1974) J. Clln. Invest. 53, 1149--1158 8 Zenser, T.V. and Metzger, J.F. (1974) Infect. Immun. I 0 , 503--509 9 Birnbaumer, L., Poh], S.L. and Rodbell, M. (1969) J. Biol. Chem. 244, 3468--3476 10 Birnbaumer, L., Pohl, S.L. and Rodbell, M. (1971) J. Biol. Chem. 246, 1857--1860 11 Finkelstein, R.A. and LoSpalluto, J.J. (1970) J. Infect. Dis. 121, $63--$72 12 Adler, W.H., Taklguchi, T., Marsh, B. and Smith, R.T. (1970) J. Exp. Med. 131, 1049--1078 13 Zenser, T.V. (1975) Biochim. Blophys. Acta~ 404, 202--213 14 White, A.A. and Zenser, T.V. (1971) Anal. Biochem. 4 1 , 3 7 2 - - 3 9 6 15 Lowry, O.H., Rosebrough, N~., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 16 Sharp, G.W.G., Hynle, S., Ebel, H., Parkinson, D.K. and Witkum, P. (1973) Blochim. Biophys. Acta 309, 339--348 17 Dru mmond, G.I. and Duncan, L. (1970) J. Biol. Chem. 245, 9 7 6 ~ 9 8 3 18 Harwood, J.P. and Rodbell, M. (1973) J. Biol. Chem. 248, 4 9 0 1 - - 4 9 0 4 19 Bennett, V., O'Keefe, E. and Cuatrecasas, P. (1975) Proc. Natl. Acad. Sci. U.S. 72, 33--37 20 Field, M. (1974) Proc. Natl. Aead. Sci. U.S. 71, 3299--33 03 21 Bennett, V. and Cuatrecasas, P. (1975) J. Membrane Biol. 22, 1--28 22 Plowman, K.M. (1972) Enzyme Kinetics, p. 57, McGraw-Hill Book Co., New York 23 Pohi, S.L., Krans, H.M.J., Kozyreff, V., Bh'nbaumer, L. and Rodbell, M. (1971) J. Biol. Chem. 246, 4447--4454 24 Rubalcava, B. and Rodbell, M. (1973) J. Biol. Chem. 248, 3831--3837 25 Rodbell, M., Birnbaumer, L., Pohi, S.L. and Krans, H.M.J. (1971) J. Biol. Chem. 246, 1877--1882 26 Rodbell, M. (1972) Glucagon (Lefebvre, P~. and Unger, R.H., eds.), p. 68, Pergamon Press, Oxford 27 King, C.A. and van Heyningen, W.E. ( 1 9 7 3 ) J . Infect. Dis. 1 2 7 , 6 3 9 - - 6 4 7