Vol. 11, No. 3 Printed in U.S.A.

INnCTION AND IMMUNrrY, Mar. 1975, p. 429-435 Copyright 0 1975 American Society for Microbiology

Escherichia coli Enterotoxin: Stimulation of Adenylate Cyclase in Broken-Cell Preparations FRIEDRICH DORNER*

AND

PETER MAYER

Sandoz Forschungsinstitut Wien, A 1235 Vienna, Austria Received for publication 31 October 1974

The enterotoxin from cell-free filtrates of the enteropathogenic Escherichia coli strain P-263 was found to stimulate adenylate cyclase activity in broken-cell preparations from myocardial tissue. Particulate and detergent-solubilized fractions from cat heart were incubated with enterotoxin and assayed for adenylate cyclase activity. Adenylate cyclase activity was stimulated by enterotoxin; the extent of stimulation was proportional to the concentration of enterotoxin. The data demonstrate that stimulation of enterotoxin-sensitive adenylate cyclase in this system provides a sensitive in vitro assay, either as an accurate measure of enterotoxin concentration or as an assay for antitoxic titers in sera. A parallel comparison showed that stimulation of fluid production in rabbit intestinal loops by enterotoxin was less sensitive.

Enteropathogenicity of Escherichia coli strains in pigs has been shown to be related to the production of two forms of enterotoxin, described as heat labile and heat stable (8, 15, 17, 21-23). Both enterotoxins were found to be controlled by transmissible plasmids, and it has been suggested that they are probably two forms of essentially the same toxin (22). Various models developed for the study of the pathophysiological effects of Vibrio cholerae enterotoxin have also been used to characterize the biological activities of the E. coli enterotoxin itself (2-5, 10, 16, 17, 19, 20). It seems evident that one must first prepare pure enterotoxin in order to fully evaluate its role in disease and to study the antitoxic immunity. The limitations of the assay procedures have so far prevented the purification and characterization of E. coli enterotoxins. A simple reliable assay would be useful in order to facilitate purification of enterotoxin. Enterotoxin activity can be demonstrated by the ligated intestinal loop procedure or by feeding preparations containing enterotoxin to piglets (11, 22, 24). These techniques are only semiquantitative and time consuming and allow considerable latitude of error. In addition, a comparatively large amount of enterotoxin is necessary to produce fluid accumulation. These features make the assay unsuitable for evaluating fractionation and purification procedures. It has been shown that enterotoxin stimulates adenylate cyclase activity in the jejunal mucosa of rabbits (5) when added in vivo. Several reports show no stimulation when added to broken-cell preparations. This study was under429

taken to investigate the effect of E. coli enterotoxin on the activity of adenylate cyclase in broken-cell preparations from myocardial tissue. In this report we describe our observations concerning the usefulness of the in vitro test in identifying the heat-labile enterotoxin of E. coli strain P-263 and we show the correlation of increased adenylate cyclase activity with diarrheagenic activity. Our aim was -to develop a test that is easy to perform, highly reproducible, superior in sensitivity to the current methods, and economical, since multiple assays can be done with the same adenylate cyclase preparation in stock along with minimal amounts of enterotoxin. In addition, the cyclic 3',5'-adenosine monophosphate (AMP) assay can be used to measure serum antitoxin titers. We hope that application of this assay routinely in purification studies will prove valuable for isolation and characterization of pure E. coli enterotoxin. MATERIALS AND METHODS Adenylyl imidodiphosphate (AMP-PNP) and the radioisotope dilution test system with cyclic 3',5'AMP binding protein were obtained as a test kit from Boehringer (Mannheim, Germany). Phosphatidylserine and Crotalus adamanteus venom, grade II, were purchased from Sigma Chemical Co. (St. Louis, Mo.). Theophylline, glucagon, and L-thyroxine were obtained from Serva (Heidelberg, Germany). [8JH ,adenosine-3',5'-cyclic phosphate ammonium salt was obtained from the Radiochemical Centre (Amersham, England). Trypticase soy broth medium was obtained as lyophilized powder from BBL (Cockeysville, Md.). Dowex 50X2 (Bio-Rad AG 50 W-X2, 200

430

DORNER AND MAYER

to 400 mesh) and Dowex 2X8 (Bio-Rad, AG 2-X8, 100 to 200 mesh, chloride form) were obtained from

Bio-Rad (Richmond, Calif.) Diethylaminoethyl-cellulose (DE-52) was obtained from H. Reeve Angel & Co. (London, England). Fluothane was obtained from Imperial Chemical Ltd. (Wilmslow, England). Lubrol-PX was a gift from ICI Austria Inc. All other chemicals and the animals were obtained from local commercial sources. Adenylate cyclase assay. (i) Tissue preparation. Preparation of a partially purified membrane fraction containing myocardial adenylate cyclase of the cat left ventricular muscle was performed essentially according to Neer (18). After anesthesia with pentobarbital, 25 to 35 mg/kg intraperitoneally, the heart was quickly excised and chilled in a sucrose buffer of the following composition: 0.25 M sucrose, 0.2 M tris (hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 7.4), 10 mM MgCl2, and 1 mM ethylenediaminetetraacetic acid. All of the following steps were performed at 4 C. After the left ventricle was dissected free of endocardium and epicardium, approximately 300 to 350 mg of muscle was minced, and the tissue was homogenized in 10 volumes (wt/ vol) of sucrose buffer with 10 strokes of a loosely fitting all-glass Dounce homogenizer. The homogenate was filtered through nylon gauze and centrifuged at 1,500 x g for 10 min. The supernatant was discarded and the pellet was washed with sucrose buffer, resuspended, and recentrifuged. The second 1,500 x g pellet was taken up in about one-fourth of the original volume of sucrose buffer, and the total volume was measured. An amount of 2.00 M sucrose buffer was added such that the final concentration of sucrose was 1.40 M. The mixture was homogenized once more with one or two strokes to make an evenly divided suspension, which was put into centrifuge tubes. A small volume of 0.25 M sucrose buffer was layered over the top and the tubes were spun in an ultracentrifuge at 95,000 x g for 3 h. The whitish material banding at the interface of the two sucrose solutions was collected, diluted with 0.25 M sucrose buffer such that each milliliter contained between 0.8 and 3 mg of protein as determined by the method of Lowry et al. (14), divided into aliquots, and stored at -70 C. The adenylate cyclase activity remained stable for at least 1 month. Preparations of solubilized adenylate cyclase were derived from the same hearts as described by Levey (12). Briefly, this involves solubilization of tissue with a buffered Lubrol-PX solution, removal of the 105,000 x g pellet, and diethylaminoethyl-cellulose chromatography, which removes the detergent. (ii) Incubation method. The method used to assay the adenylate cyclase activity was a modification of the radioisotope dilution test with cyclic 3',5'-AMP binding protein described by Gilman (6). Instead of adenosine triphosphate, AMP-PNP was used as the substrate and was purified before use by passage over a column of Dowex 50X2 in the H+ form. The reaction medium consisted of 50 mM trishydrochloride buffer at pH 7.4, 5 mM MgCl2, 10 mM theophylline, 0.1% bovine serum albumin, and 2 mM AMP-PNP. Including enzyme and test materials or solvent controls whenever appropriate, the final vol-

INFECT. IMMUN.

ume of incubation was 0.1 ml. The usual enzyme concentration was 0.15 mg of protein per assay. Whenever NaF was included in the assay, its concentration was 10 mM. Crystalline glucagon was dissolved in 0.01 N HCl, and the concentration used was 10-6 M. L-Thyroxine was dissolved in 0.01 N NaOH to a final concentration of 5 x 10-6 M. Incubation was initiated by the addition of enzyme and was performed at 37 C for 30 min unless otherwise noted. Determinations were routinely done in triplicate, and for each set of incubations a reaction blank without enzyme was used. The reaction was terminated by adding 500 ul of 7.5% trichloroacetic acid and keeping the mixture at 4 C for 10 min. The precipitate was removed by centrifugation at 1,500 x g for 15 min, and the supernatant material was added to 5 ml of ethyl ether saturated with water; the mixture was thoroughly shaken and, after separation of the layers by centrifugation, the upper ether phase was removed and discarded. The ether extraction was repeated two additional times. The aqueous solutions were kept in a 60 to 70 C water bath and concentrated to dryness in a stream of air. By adding a known amount of tritiated cyclic AMP tracer to one sample, the appropriate correction for extraction recovery (usually 90 to 98%) was made for each set of experiments. The residue was dissolved in 1.0 ml of 0.2 M acetate buffer (pH 4.0), and aliquots were used for the cyclic AMP assay according to the procedure provided by the manufacturer. Bacterial strain. E. coli strain P-263, serotype 08:K87, K88a, b:H19, a porcine enteropathogen, was obtained from H. W. Moon, National Animal Disease Laboratory, Ames, Iowa. Enterotoxin preparation. An 18-liter volume of Trypticase soy broth medium was inoculated with 200 ml of an 18-h Trypticase soy broth culture of strain P-263. Growth was for 18 h under high aeration in a 20-liter fermentor (Klein-Submersanlage "Marburg", E. Schutt, Jr., G6ttingen, Germany). The cells were removed by centrifugation at 1,500 x g for 45 min, and the supernatant fluid was filtered through 0.45ym membrane filter. The filtrate was checked for sterility on blood agar plates. The heat-labile toxin was isolated and semipurified from the supernatant fluid of the broth cultures by gel filtration and ion exchange chromatography. The final preparation was purified approximately 500 times. A detailed description of the purification procedure will be published in a subsequent* paper. Lyophilized samples of such preparations were stored at -70 C. Rabbit ligated intestinal loop assay. Enterotoxin activity of the toxin preparations was assayed in rabbit intestinal loops by the method of Burrows and Musteikis (2), which depends upon the partial gross reactions produced by marginal toxin concentrations and allows the interpolation of a 50% dose or unit of toxin. New Zealand white rabbits averaging 1.0 to 2.0 kg were used. The animals were starved for 48 h before use. They were anesthestized by inhalation of Fluothane, and the abdomen was opened. The small bowel was flushed with 20 ml of Tyrode solution, and ligated segments, usually six averaging 10 to 12 cm long, were prepared by using a single tie between segments. Each

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431

E. COLI ENTEROTOXIN

segment received a 2.0-ml intralumenal injection consisting of 0.2 mg of heat-labile P-263 enterotoxin (positive control) or Tyrode solution (negative control). The remaining four loops were used for test materials. All materials were tested in duplicate in each rabbit and in random sequence, using a minimum of six rabbits per study. After injection of the loops, the abdomen was closed. The animals were autopsied at 18 h; a volume (in milliliters)-to-length (in centimeters) ratio was calculated for each loop, and the toxin unit, i.e., that amount required to give a 50% reaction, was obtained by interpolation. Results were considered only if the positive and negative controls gave the appropriate responses. Preparation of antitoxin. The procedure for emulsifying the antigen was the following: 2 volumes of a 0.5% solution of heat-labile E. coli strain P-263 enterotoxin in 0.5% sodium bicarbonate was mixed with 2 volumes of a Willstatter aluminium hydroxide suspension (dry weight, 15 mg/ml) and left in the refrigerator for at least 1 h. The suspension of adsorbed antigen (4 volumes) was emulsified with Freund complete adjuvant (6 volumes) by dropwise additions under vigorous stirring. Goats were injected in multiple subcutaneous sites near the axillary and pelvic lymph nodes with 10 ml of the emulsified antigen. After 6 weeks, each goat received another 10 ml of the antigen and monthly booster doses thereafter. All animals were bled 10 to 12 days after each booster. A pool derived from the same goats before immunization served as control serum. Before starting an immunization procedure, serum samples were tested for the presence of antibodies versus antigen to be injected.

Toxin neutralization studies. Antitoxin was ti-

venom, the reaction mixture was applied to a column of Dowex 2 x 8 and the labeled adenosine was then separated from other nucleotides by elution with 0.04 M Tris-hydrochloride buffer, pH 7.5. Aliquots of the Tris fractions were counted in a liquid scintillation spectrometer. RESULTS

Heat-labile E. coli enterotoxin was found to stimulate the activity of myocardial adenylate cyclase preparations. The amount of cyclic 3',5'-AMP synthesized by the particulate myocardial fraction was increased in the presence of toxin throughout the experiment up to 30 min, although the reaction rate gradually decreased (Fig. 1). The kinetic curve of heat-inactivated enterotoxin (not shown) coincided with that of the control. The effect of enterotoxin on the accumulation of cyclic 3',5'-AMP became clearly evident by less than 5 min. This rapid effect suggests that the site of toxin action is either directly on adenylate cyclase or on a closely related step. After incubation for 60 min or longer, the amount of cyclic 3',5'-AMP accumulated was less than at 30 min, probably because of destruction of the nucleotide by cyclic 3',5'-AMP phosphodiesterase. For simplifying the assay, AMP-PNP instead of an adenosine triphosphate-generating system was used. To ascertain the responsiveness of the adenylate cyclase preparation to stimulation, the hormones glucagon and thyroxin were included in every set of experiments (Table 1). The amount of cyclic 3',5'-AMP accumulated in the presence of toxin was proportional to the concentration of the myocardial adenylate cyclase preparations (Fig. 2) over a large range. Enterotoxin without the particulate myocardial adenylate cyclase preparation did not promote the formation of cyclic 3',5'-AMP in the test

trated by a conventional neutralization technique. Serial dilutions of inactivated and thoroughly dialyzed serum were incubated for 1 h at 37 C with a standard amount of toxin and the residual toxin titrated either in the loop or in the adenylate cyclase assay system. Control mixtures consisted of appropriate dilutions of control serum, enterotoxin without serum, boiled enterotoxin, and Tyrode solution, respectively. The amount of antiserum required to neutralize 1 unit of toxin was determined as described under Results and was considered to be 1 unit of system. antitoxin (10). Antitoxin titer, then, could be exThe enterotoxin-mediated increase in cyclic pressed as antitoxin units per milliliter of serum. 3',5'-AMP accumulation could be due either to

Phosphodiesterase assay. Cyclic nucleotide phos-

phodiesterase activity was assayed as described (1) by conversion of [3H]cyclic 3',5'-AMP to labeled 5'AMP, which in turn is subsequently converted to [3HJadenosine by C. adamanteus phosphatase. In these experiments each reaction mixture contained 40 mM Tris-hydrochloride, pH 7.5, 2.0 mM MgCl2, and 1.0 uM [3H]cyclic 3',5'-AMP. Including enzyme and solvent controls when appropriate or either active or heat-inactivated enterotoxin, the final volume of the incubation was 0.15 ml and the usual enzyme concentration was approximately 0.06 mg of protein. Incubations were performed for 10 min at 37 C and were terminated by immersing the tubes in a boiling water bath for 1 min, after which excess C. adamanteus venom (0.1 mg) was added and the mixture was incubated for 10 min at 30 C. After incubation with

an increase in the conversion of AMP-PNP to cyclic 3',5'-AMP by adenylate cyclase or to a decrease in destruction of cyclic 3',5'-AMP by the phosphodiesterases. To distinguish between these two mechanisms, cyclic 3',5'-AMP hydrolysis was tested in the presence and absence of enterotoxin. No effect of enterotoxin on cyclic 3',5'-AMP hydrolysis was found (Table 2). We therefore concluded that the observed increase in cyclic 3',5'-AMP concentration was in fact due to an activation of adenylate cyclase. In comparison, the semipurified E. coli enterotoxin preparation was used to investigate the response in the ligated intestinal loop test as a function of concentration. Both assay systems

432

DORNER AND MAYER 840 C. 780 E 720 ' 660 600 540-

I NFECT . I MMUN .

E.coli P 263 enterotoxin control

-

31480 :-300 ,E 240

a.

/

10

20

30

40

50

60

70

minUteS Of incikation

80

90

00

110

FIG. 1. Time course of the effect of heat-labile E. coli P-263 enterotoxin on the accumulation on cyclic

3',5-AMP in myocardial adenylate cyclase preparations. Each reaction mixture contained 150 ysg of particulate adenylate cyclase and, where indicated, 5 mtg of toxin. TABLE 1. Effect of hormones and heat-labile E. coli enterotoxin on cardiac adenylate cyclase

Experimental conditions

Cyclic 3'.5'-AMP accumulated (pmol/30 min per mg of protein)5

Tissue control .................... ACa ......................... AC + glucagon (10-6 M) .......... AC + thyroxin (5 x 10-6 M) ....... AC + E. coli enterotoxin

2

E. coli P 263 enterotoxin control

--

--

E

100 u=0 810 -

96 ± 10

x80 70

60u.5

50

u'

40

30 30

0

E

0 ..

20 10

0.1

produced a near-linear dose response curve in the range used, although the cyclic 3',5'-AMP assay was clearly more sensitive. For example, 10 ug of enterotoxin had no detectable loop activity, whereas there was a nearly threefold increase in cyclic 3',5'-AMP accumulation over the basal value in the cyclic AMP test system. Here and in all other instances in which it was applied, the slopes and upper asymptotes of the dose response curves of both test systems remained substantially identical, and the values of the toxin units were highly reproducible. The effects of E. coli enterotoxin on solubilized myocardial adenylate cyclase were also investigated. As shown by Levey (12), the solubilized enzyme is refractory to the effect of added hormones known to stimulate the par-

0

u m

203 27 337 ± 23 345 ± 18

421 ± 16 (5 Ag/assay) ................... AC + E. coli enterotoxin (65 C, 30 min; 5 gg/assay) ..... .... 202 ± 21 a AC, Particulate myocardial adenylate cyclase. b Each value represents the mean + standard error of six samples.

140 130 120

0.2

0.3

0.4

0.5

0Q6

mg protein FIG. 2. Accumulation of cyclic 3',5'-AMP as a function of the concentration of particulate adenylate cyclase preparations. Each reaction mixture was incubated for 30 min and contained 5 lAg of toxin where indicated.

ticulate adenylate cyclase. The responsiveness to hormone activation can be restored by addition of phospholipids such as phosphatidylserine (13). The toxin concentration response curve in the presence of the phospholipid was almost identical to that for the toxin activation of particulate myocardial adenylate cyclase (Table 3). For the assay of neutralizing antibody, more than 1 unit of toxin is required to allow titration

E. COLI ENTEROTOXIN

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433

of residual activity. The amounts of toxin used incubated for 1 h at 37 C with constant agitahere were 2 units per ml for the loop assay and 3 tion, and titrated for residual activity. A neuunits per ml for the adenylate cyclase assay. tralization coefficient (NC) was calculated for The toxin solution was mixed with equal vol- each dilution by dividing the mean volumeumes of serial dilutions of inactivated serum, length ratio (X) by 2.66, the calculated upper asymptote of the dose response curve (Fig. 3), and subtracting this value from 1: NC = 1 TABLE 2. Lack of effect of enterotoxin on cyclic (X/2.66). Assuming that this kind of calculation 3',5'-AMP hydrolysis is applicable to the adenylate cyclase test system, the corresponding values are y (the mean Cyclic 3,5-AMP Experimental conditions hydrolyzed (pmol/10 min)a picomoles of cyclic 3',5'-AMP accumulated under the conditions of the test system), and +

....... 4.22 Control ..... Toxin (5 ag/assay) ............ 4.10 a

i

1.5 1.3

Values expressed as in Table 1.

TABLE 3. Effect of stimulators on solubilized cardiac adenylate cyclase Cyclic 3',5'-AMP accumulated (pmol/30 min per mg of protein )a

Experimental conditions

Solubilized AC + 6 Ag of phos-

Solubilized AC

pholipid/assay

Control Glucagon (10 M) NaF (10- 2 M) E. coli enterotoxin (Ag/assay):

280

4

7

532

+

16

431 ± 13 650 ± 10 767 28

1 2 5 8 10

ileaL loop system I.9x l0 mlAb/UAg Ab 526 U/mL o-o

482 732 926 1,077 ± 1,133 ±

27 18 21 28 36

a AC, Particulate myocardial adenylate cyclase. Values are expressed as in Table 1.

; 1.0 .20.8

'o 0.6o* 0.4

727/2.6 x 10-3 1.3xIOF3mLAb/UAg

-

Ab= 769

/

e c 0.2

//

0.5

U/ ml

adenylate cyclase system

4 810 15 30 antiserum ml x I103 FIG. 4. Interpolation of the 50%o point in the titration of antitoxin by log-log plot of neutralization coefficient versus serum concentration.

1

2

0

Z 950 *' 900

2.66 2.5

-' 800

2.0 o

., 700

3D

600

1.5 X

Eu

to

I 500

1.0 0

-I -c'-

400

.9

0.5

UZ 300

1 200 .5.

2

5

10

20 30 50

100

200

500

,ug enterotoxin / assay FIG. 3. Titration of heat-labile E. coli P-263 enterotoxin in the in vitro myocardial adenylate cyclase system and in the rabbit intestinal loop system. 200 pmol of cyclic 3',5'-AMP accumulated/mg of protein per 30 min represents the value of the basal activity of the particulate adenylate cyclase preparation under the conditions of the test.

434

DORNER AND MAYER

I NFECT . I MMUN .

950 pM cyclic 3',5'-AMP (the upper asymptote validity of the results obtained. The present of the dose response curve in this system). NC study suggests that the adenylate cyclase syshas a range of 0 to 1, with 0 representing no tem may indeed be a valid assay. neutralization and 1 complete neutralization. The data suggest several possibilities of both When log NC was plotted against log milliliters practical and theoretical importance. From the of antiserum, the points were best fitted by a practical point of view it has been demonstrated slightly curved line, but the departure from that the myocardial adenylate cyclase preparalinearity appeared to be sufficiently small over tion is a sensitive indicator for the presence of the range NC = 0.4 to 0.8 that half neutraliza- biologically active E. coli enterotoxin. This tion, NC = 0.5, could be reasonably well inter- finding provides an in vitro assay requiring only polated from a straight line. several hours. The myocardial adenylate cyIn the titration illustrated in Fig. 4, 2 units of clase preparation from one cat suffices for the toxin was used in the ileal loop assay, and NC = assay of 400 samples. The test system can be 0.5 indicates the neutralization of 1 unit of arranged either as an accurate means for meatoxin. From the interpolation of the 50% point, a suring toxin or as a test for antitoxic titers in value of 526 units per ml as the antitoxin titer sera. From the theoretical point of view, this was determined. For the adenylate cvclase sys- responsive system permits a variety of studies tem 3 units of toxin were used; in this experi- on the mechanism of action of enterotoxin that ment, NC = 0.5 indicates the neutralization of 2 would not be so readily accomplished in whole units, and 769 units per ml was found to be the tissue or the intact animal. antitoxin titer. ADDENDUM DISCUSSION During the preparation of this manuscript, two The detection of enterotoxigenic activity of E. other simple and reliable in vitro techniques for the coli has relied almost exclusively on the use of assay of the toxins of both V. cholerae and E. coli, intestinal loop systems. Recently, it has become i.e., the Y1 adrenal cell assay of Donta et al. (S. T. H. W. Moon, and S. C. Whipp. 1974. Science clear that the action of enterotoxin is mediated Donta, 183:334-335) and the alteration of Chinese hamster by increase of intracellular concentrations of ovary cell morphology of Guerrant et al. (R. L. Guercyclic 3',5'-AMP secondary to stimulation of rant, L. L. Brunton, T. C. Schnaitman, L. I. Rebhun, adenylate cyclase and that the fluid accumula- and A. G. Gilman. 1974. Infect. Immun. 10:320-327) tion produced by E. coli enterotoxin might be were published. associated with increased adenylate cyclase acLITERATURE CITED tivity (5, 7, 9). Attempts in many laboratories to reproducibly demonstrate a direct stimulatory 1. Beavo, J. A., J. G. Hardman, and E. W. Sutherland. 1970. Hydrolysis of cyclic guanosine and adenosine effect of enterotoxin on isolated cell membranes 3',5'-monophosphate by rat and bovine tissues. J. Biol. or on other broken-cell preparations have so far Chem. 245:5649-5655. failed. The effects of the toxin on the activity of 2. Burrows, W., and G. M. Musteikis. 1966. Cholera infecthis enzyme were demonstrable only when the tion and toxin in the rabbit ileal loop. J. Infect. Dis. 116:183-190. intact tissue was incubated with the toxin at 3. De, S. N., and D. N. Chatterjee. 1953. An experimental 37 C for more than 1 h (5, 9), since in these test study of the mechanism of action of Vibrio cholerae on systems one of the most peculiar characteristics the intestinal mucous membrane. J. Pathol. Biol. of the previously described action of enterotoxin 66:559-562. 4. Etkin, S.. and S. L. Gorbach. 1971. Studies on enterois the consistent lag phase that exists between toxin from Escherichia coli associated with acute the time of addition of the toxin and the time of diarrhea in man. J. Lab. Clin. Med. 78:81-87. onset of the effect of enterotoxin measured. The 5. Evans, D. J., Jr., L. C. Cheni, G. T. Curlin, and D. G. lack of success in applying one of these systems Evans. 1972. Stimulation of adenyl cyclase by Escherichia coli enterotoxin. Nature (London) New Biol. to a comparatively large number of samples and 236:137-138. making these assays adequately quantitative 6. Gilman, A. G. 1970. A protein binding assay tor adenosine for our purification studies on the heat-labile 3':5'-cyclic monophosphate. Proc. Nat. Acad. Sci. enterotoxin of E. coli strain P-263 caused us to U.S.A. 67:305-312. 7. Guerrant, R. L., N. F. Pierce. U. Gangtily, W. B. try the cardiac adenylate cyclase system preGreenough III, and C. K. Wallace. 1972. Mechanism of sented. action of Sn E. coli enterotoxin. J. Clin. Invest. The myocardial adenylate cyclase system 51 :39a-40a. 8. Gyles, C. L., and D. A. Barnum. 1969. A heat-labile offers a number of advantages over the current enterotoxin from strains of Escherichia coli enteropathsystems in use, especially over the intestinal ogenic for pigs. J. Infect. Dis. 120:419-426. loop system, in terms of expense, convenience, 9. Kantor, H. S., P. Tao. and S. L. Gorbach. 1974. Stimulaspace requirements, and availability. The prition of intestinal adenyl cyclase by Escherichia coli enterotoxin: comparison of strains from an infant and mary consideration, however, should be the

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an adult with diarrhea. J. Infect. Dis. 129:1-9. 10. Kasai, G. J., and W. Burrows. 1966. The titration of cholera toxin and antitoxin in the rabbit ileal loop. J. Infect. Dis. 116:606-614. 11. Kohler, E. M. 1968. Enterotoxic activity of filtrates of Escherichia coli in young pigs. Am. J. Vet. Res. 29:2263-2274. 12. Levey, G. S. 1970. Solubilization of myocardial adenyl cyclase. Biochem. Biophys. Res. Commun. 38:86-92. 13. Levey, G. S. 1971. Restoration of glucagon responsiveness of solubilized myocardial adenyl cyclase by phosphatidyl serine. Biochem. Biophys. Res. Commun. 43:108-113. 14. Lowry, 0. H., N. J. Rosebrough. A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 15. Moon, H. W., D. K. Sorensen, and J. H. Sautter. 1966. Escherichia coli infection of the ligated intestinal loop of the newborn pig. Am. J. Vet. Res. 27:1317-1325. 16. Moon, H. W., S. C. Whipp, and A. L. Baetz. 1971. Comparative effects of enterotoxin from Escherichia coli and Vibrio cholerae on rabbit and swine small intestine. Lab. Invest. 25:133-140. 17. Moon, H. W., S. C. Whipp, G. W. Engstrom, and A. L. Baetz. 1970. Response of the rabbit ileal loop to cell-free products from Escherichia coli enteropatho-

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genic for swine. J. Infect. Dis. 21:182-187. 18. Neer, E. J. 1973. The Vasopressin-sensitive adenylate cyclase of the rat renal medulla. J. Biol. Chem. 248:4775-4781. 19. Pierce, N. F., and C. K. Wallace. 1972. Stimulation of jejunal secretion by a crude Escherichia coli enterotoxin. Gastroenterology 63:439-448. 20. Sack, R. B., S. L. Gorbach, J. G. Banwell, B. Jacobs, B. D. Chatterjee, and R. C. Mitra. 1971. Enterotoxigenic Escherichia coli isolated from patients with severe cholera-like disease. J. Infect. Dis. 123:378-385. 21. Smith, H. W., and C. L. Gyles. 1970. The effect of cell-free fluids prepared from cultures of human and animal enteropathogenic strains of Escherichia coli on ligated intestinal segments of rabbits and pigs. J. Med. Microbiol. 3:403-409. 22. Smith, H. W., and C. L. Gyles. 1970. The relationship between two apparently different enterotoxins produced by enteropathogenic strains of Escherichia coli of porcine origin. J. Med. Microbiol. 3:387-401. 23. Smith, H. W., and S. Halls. 1967. Studies on Escherichia coli enterotoxin. J. Pathol. Bacteriol. 93:531-543. 24. Stevens, J. B., C. L. Gyles, and D. A. Barnum. 1972. Production of diarrhoea in pigs in response to Escherichia coli enterotoxin. Am. J. Vet. Res. 33:2511-2526.

Escherichia coli enterotoxin: stimulation of adenylate cyclase in broken-cell preparations.

Vol. 11, No. 3 Printed in U.S.A. INnCTION AND IMMUNrrY, Mar. 1975, p. 429-435 Copyright 0 1975 American Society for Microbiology Escherichia coli En...
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