Vol. 25, No. 2INFECTION AND IMMUNITY, Aug. 1979, p. 586-5960019-9567/79/08-0586/11$02.00/0

Purification and Chemical Charcterization of the Heat-LabileEnterotoxin Produced by Enterotoxigenic Escherichia coli

STEVEN L. KUNKEL AND DONALD C. ROBERTSON*Department ofMicrobiology, University of Kansas, Lawrence, Kansas 66045

Received for publication 21 May 1979

Heat-labile enterotoxin (LT) produced by a human strain of enterotoxigenicEscherichia coli (286C2) was purified to homogeneity from pH extracts offermentor-grown cells by ultrafiltration, (NH4)2SO4 fractionation, hydrophobicchromatography on norleucine-Sepharose 4B, hydroxylapatite chromatography,and Bio-Gel P-150 filtration. Purified LT preparations exhibited biological activitycomparable to that of cholera toxin in four bioassays specific for the two entero-toxins (Y-1 adrenal tumor cells, Chinese hamster ovary cells, pigeon erythrocytelysates, and skin permeability test). The overall yield of LT protein was 20%,which represented a 500-fold purification over pH extracts. A native molecularweight of 73,000 was determined by gel electrophoresis. The toxin dissociatedupon treatment with sodium dodecyl sulfate, pH 7.0, into two components withmolecular weights of44,000 and 30,000. Purified LT preparations were remarkablystable over a wide range of storage conditions, temperatures, and pH's. Thebiological activity was increased by incubation with trypsin and completelydestroyed by pronase and proteinase K, whereas deoxyribonuclease I, ribonucle-ase, and phospholipase D had no effect. The amino acid composition of purifiedLT was quite different from that of cholera toxin. Neither carbohydrate norlipopolysaccharide was present in purified preparations. The purification schemeappeared applicable to LT produced by other human and porcine enterotoxigenicstrains, but reflected the amount of LT produced by each strain. These data showthat LT and cholera toxin share many common chemical and physical properties,but must be purified by different techniques.

Enterotoxigenic (ENT') strains of Esche-richia coli have been implicated as the etiolog-ical agent of diarrheal disease in children (45,47) and adults (48), common traveler's diarrhea(41, 46), and colibacillosis of neonatal animals(25, 30, 51). Clinical isolates of ENT' E. colifrom both humans and neonatal animals pro-duce one or two kinds of enterotoxins: either alow-molecular-weight nonantigenic heat-stablemolecule or a high-molecular-weight antigenicprotein (LT) or both. The mechanism of actionof LT is similar to that of cholera toxin, sinceboth enterotoxins activate membrane-boundadenylate cyclase with a subsequent increase inintracellular cyclic adenosine 3',5'-monophos-phate in epithelial cells of the small intestine(17, 47). The heat-stable enterotoxin does notstimulate adenylate cyclase (47) but, instead,appears to increase levels of cyclic guanosine3',5'-monophosphate in intestinal tissue throughactivation of guanylate cyclase (16). The heat-stable enterotoxin produced by a porcine strainof ENT' E. coli has been purified and chemi-cally characterized (1). There were no unique

chemical properties, except the presence of sixhalf-cystine residues which may be linkedthrough disulfide bonds, to explain the heatstability.Although several reports have appeared on

the purification of LT (8, 10, 15, 18, 31, 35, 40,49, 53, 56), there is no general agreement as tothe molecular weight and chemical nature oftoxin as it is released from whole cells. Molecularweights ranging from 23 x 103 to over 106 havebeen determined, but, more importantly, thepreparations noted above were 103- to 106-foldless active than cholera toxin in bioassays spe-cific for the two enterotoxins.The purification of LT produced by a human

strain of ENT' E. coli (286C2) with biologicalactivity almost identical to that of cholera toxinis described in this report. Techniques have beendeveloped to increase yields of cell-associatedLT (22, 32) and to maintain the protein in itsholotoxin form, where biological activity inwhole-cell assays would be facilitated by a bind-ing component. The purification scheme wasalso applied to the isolation of LT from porcine

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PURIFICATION AND PROPERTIES OF LT 587

strains, but the yields were about 10-fold lower.It appears that E. coli LT and cholera toxinshare many chemical and physical properties.

MATERIALS AND METHODSBacterial strains. The ENT' E. coli strains were

kindly supplied by Harley Moon, National AnimalDisease Center, Ames, Iowa, and R. Bradley Sack,Johns Hopkins University School of Medicine, Balti-more, Md.; although several strains were screened inearly stages of these experiments, most of the purifi-cation work was done with strain 286C2, a humanstrain, and strain 263, a porcine strain.

Preparation of media. The M-9 minimal saltsmedium containing 10 mM N-tris(hydroxy-methyl)methyl glycine (Tricine), 0.5% glucose, andthree amino acids (methionine, lysine, and either as-partic acid or glutamic acid) was prepared as describedpreviously (22). The medium for growth in the fer-mentor was prepared by autoclaving 27 liters of dis-tilled water plus MgCl2 and FeCl2 in the fermentorvessel. The amino acids were dissolved in 3 liters ofdistilled water, adjusted to pH 7.5 with 5 N NaOH,and sterilized separately by autoclaving. One liter of20% glucose and 10-fold-concentrated basal salts, pH7.5, were each autoclaved separately. Before inocula-tion, the amino acids, basal salts, and glucose wereadded to the fermentor containing the sterile waterand trace salts.Growth conditions. Starter cultures were grown

aerobically with shaking (300 rpm) in 250-ml Erlen-meyer flasks containing 50 ml of Trypticase soy brothor defined medium. After 8 h of incubation at 370C,the starter culture was used to inoculate four Fernbachflasks, each containing 1 liter of defined medium. Thevolume of the starter culture was sufficient to adjustthe initial absorbance at 620 nm to 0.05. After 8 h ofshaking (300 rpm) at 37°C, the fourth Fernbach flaskswere used to inoculate 36 liters of defined medium inan F-50 New Brunswick fermentor. The initial absorb-ance at 620 nm varied from 0.2 to 0.3. Growth in thefermentor vessel was for 5 h at 37°C with vigorousaeration (2.0 CFM at STP) and stirring (200 rpm).The initial pH of the medium was 7.5, which decreasedafter 5 h of growth to about 6.8. Cells were collectedwith a Du Pont Sorvall RC-5 refrigerated centrifugeand an SZ-14 continuous-flow rotor at 18,000 rpm.About 250 g of cells was collected per 40 liters ofdefined growth medium.Toxin assays. Several biological assay systems

were used to examine preparations for LT activity.The Y-1 adrenal tumor cell assay of Donta et al. (9)was used to scan column fractions and to determinethe biological activity of purified LT preparations. Theadrenal cells were maintained on F-10 medium(GIBCO Laboratories, Grand Island, N.Y.) supple-mented with 15% horse serum, 2.5% fetal calf serum,and 50,ug of gentamicin per ml. Assays for enterotoxinactivity were performed in 24-well cluster dishes (Co-star, Cambridge, Mass.). Each well contained 1 ml ofmedium and was seeded with 105 cells. After 2 days ofgrowth at 37°C in a humidified atmosphere of 95% airand 5% C02, 0.05 ml of sample was added to each well.Toxin activity was determined by estimating the

amount of rounding at 18 h and in some experimentsby extracting steroids. Under these assay conditions,1 ng of either purified LT or cholera toxin inducedgreater than 90% rounding, and 5 to 10 pg induced 20to 30% rounding. The pigeon erythrocyte lysate (PEL)assay of Gill and King (21) was also used extensivelyto complement the tissue culture assay just described.Samples for the PEL assay were preincubated in 0.15%sodium dodecyl sulfate (SDS)-0.02 M sodium phos-phate, pH 7.0, for 15 min before the assay (22). TheChinese hamster ovary (CHO) cell assay was per-formed as described by Guerrant et al. (24), and theskin permeability test was performed by the methodof Craig (5).

Purification of LT. Cells collected from 40 litersof defined growth medium were suspended in 2 litersof cold 0.12 M tris(hydroxymethyl)aminomethane(Tris)-chloride, pH 6.5, and stirred for 45 min at roomtemperature. The cells were collected and suspendedin 1.6 liters of 0.12 M Tris-chloride, pH 8.5. The pH-adjusted cells were incubated at 370C in Fernbachflasks with gentle shaking until the temperature of theculture reached 34WC (about 1 h). The pH was thenadjusted again to 8.5 and incubated for an additional30 min. The cell suspension was centrifuged at 10,000x g, and the supernatant was concentrated sixfold byultrafiltration through PM-10 Diaflo membranes(Amicon Corp., Lexington, Mass.).

(i) (NH4)2SO4 fractionation. Solid (NH4)2SO4 wasadded with stirring at 4VC to the ultrafiltration reten-tate to 90% saturation. After centrifugation, the pelletwas extracted with 30% (NH4)2SO4 to yield a proteinconcentration of about 15 mg/ml. Any particulatematter was removed by centrifugation, and the super-natant was applied to a column of norleucine cova-lently coupled to Sepharose 4B (hydrophobic chro-matography).

(ii) Hydrophobic chromatography. A modifica-tion of the procedure of Cuatrecasas (7) was used toconjugate norleucine to the activated resin. Large-scale preparation of norleucine-Sepharose was basedon the following pilot experiment. Five grams of cyan-ogen bromide in 20 ml of distilled water was added to20 ml of packed Sepharose 4B. The pH of the suspen-sion was maintained at 11 by the slow addition of 15to 16 ml of 5 N NaOH. Chipped ice was added tomaintain the temperature below 30°C. After 9 to 11min, the resin suspension was poured through a sin-tered-glass filter and washed with 1 liter of ice-cold 0.1M NaHCO3, pH 9.0. The activated Sepharose wasadded to a solution containing 98 mg (750 mmol) ofnorleucine dissolved in 20 ml of 0.1 M NaHCO3, pH9.0. The suspension was gently stirred for 16 h andwashed extensively with distilled water. A column (2.5by 18 cm) of norleucine-Sepharose was equilibratedwith 1.25 M (NH4)2SO4-20 mM Tricine, pH 8.0 (runbuffer). The supernatant from the 30% (NH4)2SO4back extraction was loaded onto the column andwashed with run buffer until the optical density re-turned to base line. A linear decreasing salt gradient(800 ml) from 1.25 M (NH4)2SO4-20 mM Tricine, pH8.0, to 20mM Tricine, pH 8.0, containing 50% ethyleneglycol was used for elution. Fractions (20-ml) werecollected at a flow rate of 60 ml/h. Fractions contain-ing LT activity were pooled, dialyzed to remove eth-

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588 KUNKEL AND ROBERTSON

ylene glycol, concentrated by ultrafiltration with aPM-10 Diaflo membrane, and dialyzed against 100volumes of 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5.

(iii) Hydroxylapatite chromatography. A col-umn (1.5 by 20 cm) of hydroxylapatite was equilibratedwith 10 mM HEPES, pH 7.5, as the run buffer. Thepartially purified LT recovered from the norleucinecolumn was loaded onto the hydroxylapatite columnand eluted with an increasing potassium phosphategradient (400 ml) from 10 mM HEPES to 10 mMHEPES-400 mM potasium phosphate, pH 7.5. Frac-tions (10-ml) were collected at a flow rate of 30 nil/h.

(iv) Gel filtration. A Bio-Gel P-150 column (1.5 by85 cm) was equilibrated with 10 mM Tricine, pH 8.0,plus 50 mM NaCl as the run buffer. Fractions (2-ml)were collected at a flow rate of 6 ml/h.Determination of LT recovery by single radial

immunodiffusion. The single radial immunodiffu-sion technique developed by Mancini et al. (37) wasused to determine LT recovery at each purificationstep. The antiserum against purified LT was kindlyprepared by P. H. Gilligan of this laboratory as follows.New Zealand white rabbits, 3 to 5 months of age, wereinjected intradermally at multiple sites on a shavedportion of the back and in one hind footpad with 100Ag of purified LT in Freund complete adjuvant. Theanimals were bled from the central ear artery atweekly intervals 3 weeks after immunization. The titerof humoral antibody against 286C2 LT remained highfor as long as 15 weeks. The antiserum raised topurified 286C2 LT exhibited a single precipitin bandwhen reacted with crude pH extracts and partiallypurified preparations.Molecular weight determination. The molecular

weight of native LT was determined by comparing theslopes obtained in Ferguson plots with those obtainedusing proteins of known molecular weights (26). Thesubunit molecular weights were determined using slabgels prepared as described by Ames (2) with the gelcomposition of Lugtenberg et al. (36) and in glasstubes (0.6 by 10 cm) by the procedure of Weber andOsborn (58), except that the phosphate concentrationwas reduced to 0.1 M.

Sedimentation coefficient. Analytical ultracen-trifugation studies were performed with a Spincomodel E analytical ultracentrifuge equipped withphase plate schlieren optics. The sedimentation coef-ficient for purified LT was determined by the proce-dure of Chervenka (4).

Sulfhydryl group determination. Determinationof protein sulfhydryl groups was performed with Ell-man reagent (13) by the procedure of Mohler et al.(39).Amino acid analysis. Before hydrolysis, samples

were extensively dialyzed against distilled water, trans-ferred to hydrolysis ampoules, and lyophilized. Theampoules were degassed and hydrolyzed at 110°C for24 to 72 h. After hydrolysis, excess HC1 was removed,and the residue was dissolved in citrate buffer, pH 2.2.The samples were applied to a Beckman 120C aminoacid analyzer. The observed threonine and serine val-ues were divided by 0.95 and 0.90, respectively, tocorrect for destruction during hydrolysis (52). Half-cystine residues were determined as cystine or as S-

carboxymethylcysteine (6). Tryptophan was deter-mined by the method of Edelhoch (11).Amino-terminal residue. The procedure used for

the identification of the amino-terminal residue(s) wasa minor modification of that described by Gray (23).One milligram of dialyzed LT was lyophilized andsuspended in 0.5 ml of 0.5 M NaHCO3, and 0.5 ml ofdansyl chloride (2.5 mg/ml) was added, followed byincubation at 37°C for 4 h. The hydrolysate was ex-tracted twice with ethyl acetate and suspended inacetone-glacial acetic acid (3:2, vol/vol) for applicationto polyamide sheets as described by Woods and Wang(59). Solvents used for thin-layer chromatographywere water-90% formic acid (200:3, vol/vol) in the firstdimension and n-heptane-n-butanol-glacial aceticacid (3:3:1, vol/vol/vol) in the second dimension.

Stability studies. (i) Enzyme treatment of LT.Several hydrolytic enzymes (pronase, trypsin, protein-ase K, deoxyribonuclease, ribonuclease, and phospho-lipase D) were incubated with purified LT at LT/enzyme ratios (wt/wt) of 1:50, 1:10, 1:1, and 10:1. Theprocedure of Efling et al. (12) was used for proteindegradation by proteinase K. Protease activity wasterminated after 60 min by adding either phenylmeth-ylsulfonyl fluoride or soybean trypsin inhibitor.

(ii) pH treatment of LT. Hydrochloric acid (1 N)or sodium hydroxide (1 N) was added with a micro-syringe to 1-mi samples of LT (22 pg/ml). The pH wasdetermined by spotting on litmus paper until the fol-lowing values were reached: 1, 3, 5, 7.2, 10, and 12. Thesamples were then incubated at 37°C for 60 min andadjusted to pH 7.2 before assay.

(iii) Temperature treatment of LT. Purified LTwas stored at a concentration of 2.2 mg/ml at -70,-20, and 4°C for 2 months and assayed for biologicalactivity. Samples (1-mi) of purified LT (22 ,ug/mi)were heated at 50, 60, 70, 80, and 90°C for 30 min. Thesamples were cooled to room temperature and assayedfor LT activity.

Ganglioside treatment of LT. The procedure ofVan Heyningen et al. (55) was used to examine theeffects of mixed gangliosides (Sigma type III) on thebiological activity of purified LT. Mixed gangliosideswere prepared in 0.1 M phosphate buffer, pH 7.2,containing 0.2% gelatin and added to LT at LT/gan-glioside (wt/wt) ratios of 1:100, 1:50, 1:25, 1:10, 1:1, and100:1. The solutions were then incubated at 370C for30 min and assayed for LT activity.

Protein and carbohydrate determination. Pro-tein was determined by the absorbance at 280 and 260nm (57). Total carbohydrate was determined by thephenol-sulfuric technique, with glucose as the stan-dard, and qualitative detection of lipopolysaccharidewas by the colorimetric assay for 2-keto-3-deoxyocton-ate (29). Lipopolysaccharide of Salmonella typhimu-rium was a positive control.Reagents used. All medium components and re-

agents employed throughout this study were pur-chased from Sigma Chemical Co., St. Louis, Mo.,unless otherwise indicated. The electrophoresis re-agents and Bio-Gel P-150 (100- to 200-mesh) werepurchased from Bio-Rad Laboratories, Richmond,Calif. Thin-layer chromatography plates were pur-chased from Brinkmann Instruments Inc., Westbury,N.Y. Purified cholera toxin was kindly provided by J.

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PURIFICATION AND PROPERTIES OF LT 589

Peterson, University of Texas, Galveston.

RESULTSPurification of LT. The procedures devel-

oped for the purification of E. coli LT are sum-marized in Fig. 1. Yields of cell-associated LTwere increased by growing cells in a definedmedium (22) and extracting fermentor-growncells under conditions which rapidly released asignificant amount of LT (32). The pH extractfrom 80 liters of cells was concentrated approx-imately sixfold with an Amicon TC5E ultrafil-tration unit with PM-10 Diaflo membranes andfractionated by the addition of (NH4)2SO4 to90% saturation. After stirring for 1 to 2 h at 40C,the precipitate was removed by centrifugationat 10,000 X g and extracted with 30% (NH4)2SO4.The 30% (NH4)2SO4 extract was applied to acolumn of norleucine covalently coupled toSepharose 4B (hydrophobic chromatography).The LT activity, quantitatively retained by thenorleucine column, was eluted free from contam-inating lipopolysaccharide, shown by the ab-sence of 2-keto-3-deoxyoctulosonic acid (KDO)with a linear decreasing (NH4)2SO4 gradient anda linear increasing gradient of 50% ethylene gly-col (Fig. 2). It should be noted that LT was oneof the most hydrophobic proteins in the pHextract and that a 15- to 20-fold purification wasroutinely observed. The LT activity in pH ex-tracts of a porcine strain, 263, eluted at a slightlyhigher concentration of (NH4)2SO4, but LT ac-tivity in preparations from most strains elutedbetween concentrations of 0.5 and 0.25 M(NH4)2S04 (Fig. 2). Addition of ethylene glycolwas necessary to prevent spreading of toxin ac-tivity. The LT activity produced by some strainsof ENT' E. coli, which precipitated below 40%(NH4)2SO4, did not bind to the hydrophobic

E.D

z

FRACTION

column. Since these preparations were high inKDO color, it seemed likely that LT was tightlycompleted with outer membrane components.The amount of soluble LT protein back ex-tracted by 30% (NH4)2SO4 from either a 0 to 90%(NH4)2SO4 cut or a 40 to 90% (NH4)2SO4 cutvaried widely from strain to strain and deter-mined whether sufficient amounts could be pu-rified for chemical characterization. The finalyield ofLT was not dependent on the amount ofprotein applied to the hydrophobic column sinceup to 5 was loaded with little effect on overallrecovery.The hydrophobic column fractions with LT

activity were pooled, dialyzed, and concentrated.Before hydroxylapatite chromatography, thepartially purified preparation was dialyzedagainst 50 to 100 volumes of 10mM HEPES, pH7.5. The LT activity eluted from hydroxylapatiteas a symmetrical peak at 150 mM phosphate,

PH EXTRACT(CELL-ASSOCIATED LT FROM 80 LITERSOF CELLS)

ULTRAFILTRATION USING PM-10 DIAFLOMEMBRANES

(NH4)2S04 (90Z SATURATION)

HYDROPHOBIC CHROMATOGRAPHY ON NORLEUCINE-SEPHAROSE

HYDROXYLAPAT I TE CHROMATOGRAPHY

4GEL FILTRATION ON BIO-GEL P-150

FIG. 1. Purification scheme for E. coli LT.

z

Z

) zt;

FRACTION

FIG. 2. Fractionation of 286C2 LT by hydrophobic chromatography, using norleucine-Sepharose 4B. Thesolid line reflects absorbance at 280 nm, and the broken line represents Y- 1 adrenal cell activity detected at4 h with 25 I&d ofa 1:1K00 dilution ofeach fraction. The slashes indicate those fractions with maximum roundingactivity but not further diluted. The gradient was started at the point indicated by the arrow.

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590 KUNKEL AND ROBERTSON

pH 7.5 (Fig. 3), using a linear 0 to 400 mMphosphate gradient. Fractions containing LT ac-tivity were pooled, concentrated to about 2 mlwith an Amicon stirred cell, and applied to aBio-Gel P-150 column (1.5 by 85 cm). Althoughsome rounding activity appeared in the voidvolume ofthe column, the biological activity wasassociated primarily with the second major pro-tein peak (Fig. 4). Peak fractions were pooled,with about 10% of the LT activity on either sideof the peak not included. Finally, the pooled gelfiltration fractions were concentrated and storedat 2 to 4 mg/ml.The complexity and relative purity of LT at

each stage of the purification scheme are shownin Fig. 5. Note that LT was the major proteineluted from the norleucine-Sepharose columnand the predominant band eluted in the secondpeak from hydroxylapatite. A final gel filtrationstep was necessary to yield a preparation esti-mated to be 95 to 98% pure. The gels wereoverloaded with 150 ,ug of protein to detect tracesof impurities.The recovery of LT from strain 286C2 at each

stage of the purification is shown in Table 1.The minimal LT activity that did not adsorb tothe hydrophobic column is probably associatedwith lipopolysaccharide since most of the KDOcolor eluted in the first peak. Since the predom-inant immune response in rabbits is directedagainst the binding component of the toxin (P.H. Gilligan and D. C. Robertson, unpublishedobservations), the radial immunodiffusion assaydetected both the B component and holotoxinin pH extracts. Therefore, the yield of biologi-cally active holotoxin is likely higher than thatshown in Table 1.Molecular weight. The native molecular

weight of LT was determined by the method ofHedrick and Smith (26). The mobility of LT andseveral protein standards in 6, 8, 10, and 12%

,00

190

70

a60 Z

0

50C

30 £

FRACTION

FIG. 3. Fractionation of 286C2 LT by hydroxylap-atite chromatography. The lines were as described inthe legend to Fig. 2.

90

so

70 CD

60 z

20

so "

0 us

-j04 10 IS2 5 3 S 4FRCIO

FIG. 4. Gel filtration of 286C2 LT on Bio-Gel P-150. The lines were as described in the legend to Fig.2.

*... ..

FIG. 5. Polyacrylamide gels of LT fractions: (A)(NH4)2S04 precipitate ofpH extract; (B) pooled frac-tions after hydrophobic chromatography; (C) pooledfractions eluted from hydroxylapatite; (D) final prep-aration after Bio-Gel P-iSO0 gel filtration.

acrylamide gels was determined, and a slope wascalculated by plotting the log mobility versusthe percent acrylamide concentration (Fig. 6).The molecular weight of 73,000 was derived fromthe relationship between the slope and molecu-lar weight of protein standards. Sedimentationvelocity experiments run at a protein concentra-

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PURIFICATION AND PROPERTIES OF LT 591

TABLE 1. Summary of LTpurification from EN7'E. coli strain 286C2

Total LT pro-Step protein teina Recov- Fold pu-

(mg) (mg) ery ( rifiCation

pH-released LT (ultra- 7,800 81 100filtration with PM-10 Diaflo mem-branes)

90% (NH4)2SO4 frac- 3,168 61 75 2.5tionation

Hydrophobic chroma- 196 31 37 40tography

Hydroxylapatite chro- 30 23 28 260matography

Bio-Gel P-150 filtra- 17 17 21 459tiona Determined as LT antigen by Mancini assays.

- 2.6"L 24z 22020

(D 1.8wi 1.6LL 1.40 1.2hia. 1.00-i

35 55 7.5 95 11.54 13.5MOLECULAR WEIGHTX 10

acidic pH treatment suggests that the A frag-ment is more stable than the holotoxin and, also,that heating at 650C for 30 min may not besufficient to remove all LT activity from crudepreparations. In addition, storage of LT at -70and -20OC for months did not affect activity.Since these preparations consisted of purifiedextracellular LT released by pH adjustment,there was no need for activation with proteases,although the biological activity was enhancedby trypsin digestion. Pronase and proteinase Kcompletely destroyed the biological activity ofpurified LT. In contrast to protease treatment,there was no effect on LT activity by deoxyri-bonuclease I, ribonuclease, and phospholipaseD. Purified preparations contained neither car-bohydrate nor lipopolysaccharide, as shown bya negative test for the presence of KDO. Non-dissociated LT did not possess surface sulfhydrylgroups, as no reaction occurred with dithiobis-nitrobenzoic acid (DNTB-Ellman reagent). Fur-thermore, DNTB did not react with purified LTin the presence of 8 M urea.

Identification of amino-terminal resi-

FIG. 6. Determination of molecular weight of286C2 LT by disc gel electrophoresis.

tion of 4 mg/ml (50,000 rpm, 20°C) did notreveal any polydispersity in purified LT prepa-rations. The s2ow value was found to be 5.65. Noattempt was made to calculate a molecularweight from these limited data.The purity of LT after Bio-Gel P-150 gel

filtration was further assessed by SDS-gel elec-trophoresis. Upon incubation in neutral SDS atroom temperature, two stained bands corre-sponding to molecular weights of 44,000 and30,000 were observed (Fig. 7). The species witha molecular weight of 30,000 had activity in thePEL assay and likely corresponds to the samefragment synthesized by a porcine ENT' strain,1362 (32). The 44,000-molecular-weight compo-nent dissociated upon heating in SDS to a spe-cies which corresponded to a molecular weightof 11,000. This subunit is probably the bindingcomponent of LT. LT purified from a porcinestrain of ENT' E. coli (strain 263) had a similarsubunit structure (Fig. 7).

Stability and chemical characteristics ofLT. As shown in Table 2, LT retained a portionof its biological activity at extremes of both pHand temperature. The biological activity of LTobserved in the PEL assay after heating and

^,,§.. S

*: w}

:. :? <;:....; .;.

vs. .:

.:...;,: ..

.: .....

.: .... ::

... ..

.: .: ..:

...... ..:.

....: ..;

:.: .s .;': :::

f:S .,;.... ^.....

:,

z

:X :: ....... :0. ..

.; :..: :': .:: ::

;;..,wF:;

':.'.

.... "'

.0,.

FIG. 7. SDS-gel electrophoresis of purified LTpreparations: (A) 286C2 LT, nonreduced; (B) 286C2LT, heated and reduced; (C) 263 LT, nonreduced;and (D) 263 LT, heated and reduced.

/lli-e Ph..plint...

--oi.. S.,-wAlb-t.

*; * * . . . . * *

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592 KUNKEL AND ROBERTSON

TABLE 2. Stability properties ofpurified 286C2 LTBiological activity

Treatmenty_lb PELc

Storage at -20 or -70'C for 2 5.0 320months

Heat (00)50 2.9 30070 1.4 22590 0.8 150

EnzymeTrypsin 6.0 380Pronase, proteinase K 0, 0.2 35, 15Ribonuclase A, deoxyribo- 4.9 300

nuclease I, phospholipaseD

pH1 0.2 803 1.7 1005 3.5 2407 6.5 3809 6.7 35011 5.9 300a 220 pg of LT was used for each assay.b Nanomoles of steroid per milligram of Y-1 protein.e Picomoles of cyclic adenosine 3',5'-monophos-

phate.

dues. To assess the state of purity and providefurther information on the number of subunits,it was of interest to identify the amino-terminalgroups of purified 286C2 LT. After dansylationand acid hydrolysis, two dansyl derivatives wereobserved which corresponded to dansyl threo-nine and dansyl aspartic acid.Amino acid analysis. The amino acid com-

position of purified 286C2 LT is shown in Table3. Six tryptophan residues per 74,000 daltonswere found by the spectrophotometric assay ofEdelhoch (11). When normalized to one prolineresidue, the values were close to whole integers.The levels of about one-half of the amino acidswere similar to those reported for cholera toxin(17). However, the composition was uniquewhen compared with previously described LTpreparations (49). It is interesting that hydro-phobic residues (valine, isoleucine, and leucine)constituted about 20% of the total amino acidsexpressed on a weight basis, which may explainthe hydrophobic properties of the protein andinteraction with norleucine-Sepharose.Biological activity of purified LT and

cholera toxin. The biological activity of puri-fied LT was similar or equivalent to that ofcholera toxin in four biological assays specificfor the two enterotoxins. The effect of eachenterotoxin on CHO cells is representative of

INFECT. IMMUN.

results obtained with all four assays (Fig. 8).Similar results were obtained with the Y-1 ad-renal tumor cell assay with respect to rounding;however, the dose-response curves were slightlydifferent when steroid production was measured.The steroid response to LT was less than withcholera toxin at low concentrations but similar

TABLE 3. Amino acid analysis ofpurified 286C2 LT

Amino acid g/100 g of Residues peramino acids proline residues

Lysine 7.09 3.00 (3)Histidine 3.54 1.40 (1)Arginine 5.33 1.80 (2)Aspartic acid 9.20 3.73 (4)Threonine 6.19 3.65 (4)Serine 4.4 3.10 (3)Glutamic acid 12.33 4.73 (5)Proline 1.82 1.0 (1)Half-cystine 5.4lb 2.73c(3)Glycine 2.44 2.3 (2)Alanine 3.50 2.7 (3)Valine 5.56 3.0 (3)Methionine 5.48 2.3 (2)Isoleucine 8.80 4.1 (4)Leucine 6.41 3.1 (3)Tyrosine 8.37 2.7 (3)Phenylalanine 4.44 1.73 (2)Tryptophan

aCalculated by dividing the amount of each aminoacid by the amount of proline (0.15,umol). Numbers inparentheses are values rounded to the nearest wholeinteger.

' Determined as cystine.e The value determined as S-carboxymethylcysteine

was 2.5.

50 /

z- /

0-J CTw ,~~~~~LT

30/

0 20

z

a10

° 0.0001 0.01 1.0 100

TOXIN (ng)FIG. 8. Biological activity of purified 286C2 LT

and cholera toxin against CHO cells. CT, Choleratoxin.

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PURIFICATION AND PROPERTIES OF LT 593

at toxin concentrations which gave greater than90% rounding. The specific activity of LT wasthe same as that of cholera toxin in the PELassay, and the response of LT was enhanced, aswith cholera toxin, by dilute detergent (21). Also,the activities of the two toxins were comparableby the skin permeability assay (5).

DISCUSSIONThis report describes the purification and

chemical characterization of LT produced by anENT' E. coli strain (286C2) isolated from ahuman patient in Mexico (41). Although strain286C2 produces only LT, the purification wasapplied to human and porcine ENT' strains thatproduce heat-stable enterotoxin and LT. Themain limitation in the applicability of this puri-fication scheme to other ENT' E. coli strains isthat, as with many ENT' E. coli strains exam-ined in other laboratories, 24 strains tested inour laboratory produced 10- to 15-fold less LTthan did strain 286C2. Some recent human iso-lates have been found to produce higher levelsof LT than do porcine strains, but, in general,ENT' strains release only 20 to 30% of the totalLT activity detected in sonic extracts (unpub-lished observations). Use of a defined mediumfacilitated attempts to purify LT. For example,a two- to fivefold increase in the yield of cell-associated LT was obtained when comparedwith growth in Casamino Acids-yeast extractmedium (14). Also, the amount of LT not com-plexed with outer membrane components wasincreased by hypotonic extraction with Tris-chloride, pH 8.5 (32). The combined effects of adefined growth medium and hypotonic extrac-tion (pH extracts) yielded enough purified LT,even from porcine strains, to raise antisera andexamine the subunit structure.Hydrophobic chromatography was considered

as a potential LT purification step based on thefollowing preliminary experiments: (i) chroma-tography on Bio-Gel A (diethylaminoethyl-aga-rose) and other anion-exchange resins yieldedthree peaks of LT, each with different biologicalactivities; (ii) the amount of LT activity in pHextracts from most porcine strains which ad-sorbed to anion-exchange resins was much lessthan the activity in material which remainedunbound; and (iii) LT appeared to be complexedwith other hydrophobic components of the outermembrane. Hydrophobic chromatography hasalso been a useful step in fractionation of proteinmixtures (19, 28, 43, 50, 60). In the present study,LT was one of the most hydrophobic proteins inpH extracts, but could be readily eluted with alinear decreasing (NH4)2SO4 gradient combinedwith an increasing ethylene glycol gradient or a

stepwise elution using decreasing increments of0.25M (NH4)2SO4 and increasing ethylene glycolby 10% with each decrease in salt concentration.The LT from strain 286C2 eluted at about 0.5 M(NH4)2SO4, whereas the toxin from strain 263eluted at 0.75 M (NH4)2SO4. In addition to dif-ferences in hydrophobicity, the two toxins havebeen shown to exhibit antigenic differences,measured by neutralization assays using specificantiserum raised against each toxin (Gilliganand Robertson, unpublished observations). Eth-ylene glycol was added to the decreasing saltgradient to decrease the effect of polar forcesand to minimize the spread of LT activity. Thealkyl residue norleucine immobilized on a non-charged but polar matrix (Sepharose 4B) likelybehaved as a solid-state detergent and preventedintermolecular protein aggregation betweeneither toxin or other hydrophobic macromole-cules (34, 38).

Further purification by hydroxylapatite col-umn chromatography resulted in elution of LTactivity associated with a single protein peak.The final purification step used Bio-Gel P-150since gel filtration of partially purified LT onagarose resins (e.g., Bio-Rad A-5m and UltrogelAcA 44) led to aggregation and low recoveries ofLT activity. These latter results were surprisingsince A-5m has been used in the purification ofcholera toxin and several LT purificationschemes (9, 18, 35).The specific activity (biological activity per

nanogram) of LT after Bio-Gel P-150 gel filtra-tion was similar to that of cholera toxin wheneach toxin was incubated with CHO cells and Y-1 adrenal cells. Similar results were observedwith the PEL assay and the skin permeabilitytest. Furthermore, the nicotinamide adenine di-nucleotide glycohydrolase and adenosine 5'-di-phosphate ribosyltransferase activities of chol-era toxin and purified LT were comparable (J.Moss, personal communication). It should benoted that purified 286C2 LT has not been testedfor enterotoxin activity with acute rabbit intes-tinal loops; however, to our knowledge, this re-port is the first example ofLT preparations withbiological activity comparable to that of choleratoxin.

In addition to biological activity, the chemicalproperties of purified LT were remarkably sim-ilar to those of cholera toxin (17, 21, 33). Thenative molecular weight of LT was determinedto be 73,000. Purified LT could be dissociated inneutral SDS at room temperature into two com-ponents corresponding to molecular weights of44,000 and 30,000. The 30,000-molecular-weightsubunit has been observed in other LT prepa-rations obtained by polymyxin B treatment of aporcine ENT' strain (strain 1362) grown below

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594 KUNKEL AND ROBERTSON

280C or in early log phase at 370C (32) and likelycorresponds to the A subunit of cholera toxinsince it catalyzes the nicotinamide adenine di-nucleotide-dependent activation of adenylatecyclase in PELs and is active against Y-1 adrenalcells at 10- to 100-fold-higher concentrationsthan the holotoxin of LT. A species with amolecular weight of 22,000 has been observedupon SDS-gel electrophoresis of heated and re-duced LT preparations (32; Kunkel and Robert-son, manuscript in preparation). Although notdescribed in detail in this report, the LT pro-duced by ENT' E. coli strain 263 has beenpurified extensively and compared with 286C2LT. Two major bands with the same mobilitiesas the components of 286C2 LT, as well as six toeight faint bands, were detected by SDS-gelelectrophoresis. The 44,000-molecular-weightcomponent was reduced to a molecular weightof 11,000 by heating; thus, it appears that thesubunit structures of the two enterotoxin aresimilar.There are significant differences in the molec-

ular weight assigned to LT by various laborato-ries (8, 10, 15, 18, 31, 35, 40, 49, 53, 56). The datapresented in this report and other observationsmay offer an explanation. As noted earlier, theA subunit of LT is synthesized in early stages ofgrowth, followed by the appearance of the extra-cellular toxin released by pH adjustment (32).The A subunit may therefore be inserted intothe outer membrane to facilitate associationwith the polypeptides of the binding component.Thus, it might be expected that polymyxin Bextraction and disruption ofthe outer membranein early log phase would release a species witha molecular weight of less than 30,000, as ob-served by Evans et al. (15). In addition, a varia-ble number of B polypeptides might be presentto increase or alter immunogenicity. Hoekstra etal. (27) have clearly shown that E. coli strainsrelease outec membrane blebs during growth.These high-molecular-weight complexes re-leased by ENT' E. coli strains possess LT activ-ity (unpublished observations) and probably ex-plain the isolation of LT preparations with mo-lecular weights of greater than 106. It is thereforeadvisable to carry out (NH4)2SO4 fractionationon crude extracts to determine the amount ofLT activity which precipitates between 0 and40% and between 40 and 90% saturation. Theamount ofLT activity in the supernatant at 40%saturation provides an indication of the finalyield of LT activity. The fraction which precip-itates between 0 and 40% (NH4)2SO4 saturationhas LT activity with a molecular weight ofgreater than 106 and is heavily contaminatedwith lipopolysaccharide, as measured by the col-orimetric reaction for KDO. The amount of LT

INFECT. IMMUN.

activity which precipitates between 0 and 40%saturation was increased if the pH of the cellcultures was adjusted from 6.8 to 8.0 at 8 h andthe culture supernatant was concentrated byAmicon ultrafiltration instead of working withpH extracts. Such preparations have biologicalactivity probably because the A subunit candissociate from aggregates and interact with asusceptible cell. Molecular weights in the rangeof 35,000 to 100,000 with biological activity from103- to 106-fold less than the activity observedfor cholera toxin (8, 10, 18, 31, 49, 56) may reflectassociation of the A subunit with other mem-brane or periplasmic proteins. It is also possiblethat what appeared to be homogeneous prepa-rations contained traces of holotoxin. Gill et al.detected enzymatic activity similar to that as-sociated with the A subunit of cholera in LTsubunit preparations at different stages of puritywhich migrated during SDS-gel electrophoresiscorresponding to a molecular weight of 23,000 to24,000 (20; M. Gill, personal communication).The data are consistent if nonspecific associationof the A subunit occurs with other proteins.

It is likely that some protein purification stepspromote dissociation of the A and B subunits.Early in our experiments, anion-exchange chro-matography was omitted as a useful purificationstep as it resolved the limited amount of LTwhich bound to the resin into three peaks: twowith PEL activity and one with both PEL andY-1 adrenal tumor cell activities. These peakslikely correspond to subunits with molecularweights of 30,000 and 22,000 and the holotoxinwith a molecular weight of 73,000 (Kunkel andRobertson, in preparation). These data show theneed for using more than one bioassay to followthe course of a purification. Although LT andcholera toxin appear to have similar subunitstructures and mechanisms of action, it does notseem that purification methods applicable tocholera toxin can be applied to LT.

Purified LT preparations derived from 286C2proved to be unusually stable to extremes oftemperature and pH (Table 2). These resultswere surprising in view of the acid and temper-ature labilities of crude LT preparations. How-ever, the A fragment of cholera toxin is stable toheating (21) and, possibly, the holotoxin of LTmay be stabilized by the A fragment. The un-expected temperature and pH stabilities of LTmay require reconsideration of the conditionsused to inactivate LT and to show that only STis involved in disease. Heating at 650C did notdestroy biological activity associated with the Asubunit. Since the A subunit of E. coli is activeagainst Y-1 adrenal tumor cells at high concen-trations (32) and appears to be fairly heat stable,similar to the A subunit of cholera toxin (9),

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PURIFICATION AND PROPERTIES OF LT 595

more rigorous criteria than heating at 65CC seemto be necessary to eliminate LT activity in en-terotoxin preparations. The biological activity ofLT, destroyed by proteinase K and pronase, wasnot affected by deoxyribonuclease I, ribonucle-ase A, or phospholipase D. On the other hand,incubation of purified LT with trypsin enhancedthe biological activity of the toxin. Similar ob-servations have been made by Rappaport et al.(44).Preliminary experiments on the nature of the

receptor for 286C2 LT were performed using amixed ganglioside preparation purified from bo-vine brain (Sigma type III). The toxin was in-cubated for 30 min with increasing amounts ofganglioside, essentially as described by Van Hey-ningen et al. (55), and assayed for residual bio-logical activity, using Y-1 adrenal tumor cells.The activity of 286C2 LT decreased as the gan-glioside concentration increased, which was sim-ilar to the titration curves obtained with choleratoxin. One nanogram of ganglioside neutralized27.5 pg of purified LT (50% neutralization point).These data suggest that LT binds to a ganglio-side receptor; however, if LT and cholera toxincan compete for a receptor (54), it is not clearwhy Pierce could not inhibit the biological activ-ity ofLT after preincubation with choleragenoid(42).Toxin purified from strains 286C2 and 263 has

been used to raise high-titer antisera. Ouchter-lony analysis indicated that LT and choleratoxin share at least one antigenic determinantwhile exhibiting a minimum of one unique anti-genic determinant (P. H. Gilligan, J. C. Brown,and D. C. Robertson, manuscript in prepara-tion). Anti-286C2 efficiently neutralized 286C2LT, but only partially neutralized pH extracts ofthree other human ENT' strains and exhibitedlittle or no neutralization when incubated withpH extracts from porcine ENT' strains. Clearly,more extensive investigation will be required toestablish a common subunit structure of E. coliLT and whether there are antigenic differencesbetween LTs. However, methods described inthis report, coupled with isolation of hypertoxi-genic mutants (3) and availability of specificantisera, should significantly affect progress inunderstanding the structure and function of LTin diarrheal disease.

ACKNOWLEDGMENTSWe are grateful to Lee Bulla and John Hubbard of the

Grain Market Research Center, Kansas State University,Manhattan, for the amino acid analysis of LT. We are alsograteful to J. Peterson from the University of Texas, Galves-ton, for his gift of purified cholera toxin.

This work was supported by Public Health Service researchgrant AI-12357 under the U.S.-Japan Cooperative MedicalScience Program, administered by the National Institute ofAllergy and Infectious Diseases.

LITERATURE CITED1. Alderete, J. F., and D. C. Robertson. 1978. Purification

and chemical characterization of the heat-stable enter-otoxin produced by porcine strains of enterotoxigenicEscherichia coli. Infect. Immun. 19:1021-1030.

2. Ames, G. F. 1974. Resolution of bacterial proteins bypolyacrylamide gel electrophoresis on slabs. J. Biol.Chem. 249:634-644.

3. Bramucci, M. G., and R. K. Holmes. 1978. Radialpassive immune homolysis assay for detection of heat-labile enterotoxin produced by individual colonies ofEscherichia coli or Vibrio cholerae. J. Clin. Microbiol.8:252-255.

4. Chervenka, C. H. 1973. A manual of methods for theanalytical ultracentrifuge, p. 39-55. Spinco Division ofBeckman Instruments, Inc., Palto Alto, Calif.

5. Craig, J. P. 1971. Cholera toxin, p. 189-254. In S. Kadis,T. C. Minfic, and S. J. Ajl (ed.), Academic Press Inc.,New York.

6. Crestfield, A. M., S. Moore, and W. H. Stein. 1963. Thepreparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J. Biol. Chem. 238:622-627.

7. Cuatrecasas, P. 1970. Protein purification by affinitychromatography. Derivatizations of agarose and poly-acrylamide beads. J. Biol. Chem. 245:3059-3065.

8. Dafni, Z., R. B. Sack, and J. C. Craig. 1978. Purificationof heat-labile enterotoxin from four Escherichia colistrains by affinity immunoadsorbent: evidence for sim-ilar subunit structure. Infect. Immun. 22:852-860.

9. Donta, S. T., H. W. Moon, and S. C. Whipp. 1974.Detection of heat-labile Escherichia coli enterotoxinwith the use of adrenal cells in tissue culture. Science183:334-336.

10. Dorner, F. 1975. Escherichia coli enterotoxin purificationand partial characterization. J. Biol. Chem. 250:8712-8719.

11. Edelhoch, H. 1967. Spectroscopic determination of tryp-tophan and tyrosine in proteins. Biochemistry 6:1948-1954.

12. Efling, W., N. Henn-ich, M. Klockow, H. Metz, H. D.Orth, and H. Lang. 1977. Proteinase K from Tritir-achium album limber. Eur. J. Biochem. 47:91-97.

13. Ellman, G. L 1959. Tissue sulfhydryl groups. Arch. Bio-chem. Biophys. 82:70-77.

14. Evans, D. J., Jr., D. G. Evans, and S. L Gorbach.1973. Production of vascular permeability factor byenterotoxigenic Escherichia coli isolated from man.Infect. Immun. 8:725-730.

15. Evans, D. J., Jr., D. G. Evans, S. H. Richardson, andS. L Gorbach. 1976. Purification of the polymyxin-Breleased heat-labile enterotoxin of Escherichia coli. J.Infect. Dis. 133(Suppl.):S97-S108.

16. Field, M., L H. Graf, Jr., W. J. Laird, and P. L Smith.1978. Heat-stable enterotoxin of Escherichia coli: invitro effects on guanylate cyclase activity, cyclic GMPconcentration, and ion transport in small intestine.Proc. Natl. Acad. Sci. U.S.A. 75:2800-2804.

17. Finkelstein, R. A. 1973. Cholera. Crit. Rev. Microbiol. 2:553-563.

18. Finkelstein, R. A., M. K. LaRue, D. W. Johnston, M.L. Vasil, G. J. Cho, and J. R. Jones. 1976. Isolationand properties of heat-labile enterotoxins from entero-toxigenic Escherichia coli. J. Infect. Dis. 133(Suppl.):S120-S137.

19. Geren, C. R., S. C. Magee, and K. D. Ebner. 1976.Hydrophobic chromatography of galactosyltransferase.Arch. Biochem. Biophys. 172:149-155.

20. Gill, D. M., D. J. Evans, Jr., and D. G. Evans. 1976.Mechanism of activation of adenylate cyclase in vitroby polymyxin released, heat-labile enterotoxin of Esch-erichia coli. J. Infect. Dis. 133(Suppl.):S103-S107.

21. Gill, D. M., and C. A. King. 1975. The mechanism of

VOL. 25, 1979

on April 19, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

596 KUNKEL AND ROBERTSON

action of cholera toxin in pigeon erythrocyte lysates. J.Biol. Chem. 250:6424-6432.

22. Gilligan, P. H., and D. C. Robertson. 1979. Nutritionalrequirements for synthesis of heat-labile enterotoxin byenterotoxigenic Escherichia coli. Infect. Immun. 23:99-107.

23. Gray, W. R. 1967. Dansyl chloride procedure. MethodsEnzymol. 11:139-150.

24. Guerrant, R. L., L. L. Brunton, T. C. Schnaitman, L.I. Rebhun, and A. G. Gilman. 1974. Cyclic adenosinemonophosphate and alterations of Chinese hamsterovary cell morphology: a rapid, sensitive in vitro assayfor the enterotoxins of Vibrio cholerae and Escherichiacoli. Infect. Immun. 10:320-327.

25. Gyles, C. L. 1971. Heat-labile and heat-stable forms ofthe enterotoxin from Escherichia coli strains entero-pathogenic for pigs. Ann. N.Y. Acad. Sci. 176:314-322.

26. Hedrick, J. L., and A. J. Smith. 1968. Size and chargeisomer separation and estimation of molecular weightsof proteins by disc gel electrophoresis. Arch. Biochem.Biophys. 126:155-164.

27. Hoekstra, D., J. W. Van der Laan, L. de Leig, and B.Wilholt. 1976. Release of outer membrane fragmentsfrom normally growing Escherichia coli. Biochim. Bio-phys. Acta 455:889-899.

28. Hofstee, B. H. J. 1973. Hydrophobic affinity chromatog-raphy of proteins. Anal. Biochem. 52:430-448.

29. Keleti, G., and W. H. Lederer. 1974. Handbook ofmicromethods for the biological sciences, p. 74. VanNostrand Reinhold Co., New York.

30. Kohler, E. M. 1968. Enterotoxic activity of Escherichiacoli in young pigs. Am. J. Vet. Res. 29:2263-2274.

31. Konowalchuk, J., N. Dickie, S. Stavric, and J. I.Speirs. 1978. Comparative studies of five heat-labiletoxic products of Escherichia coli. Infect. Immun. 22:644-648.

32. Kunkel, S. L., and D. C. Robertson. 1979. Factorsaffecting release of heat-labile enterotoxin by entero-toxigenic Escherichia coli. Infect. Immun. 23:652-659.

33. Kurosky, A., D. E. Markel, B. Touchstone, and J. W.Peterson. 1976. Chemical characterization of the struc-ture of cholera toxin and its natural toxoid. J. Infect.Dis. 133:514-522.

34. Laas, T. 1975. Agar derivatives for chromatography, elec-trophoresis and gel-bound enzymes. J. Chromatogr.111:373-387.

35. Lariviere, S., C. L. Gyles, and D. A. Barnum. 1973.Preliminary characterization of the heat-labile entero-toxin of Escherichia coli F11(P155). J. Infect. Dis. 128:312-320.

36. Lugtenberg, G., J. Mijers, R. Peters, P. Hock, and L.Alphen. 1975. Electrophoresis resolution of the majorouter membrane protein of Escherichia coli K-12 intofour bands. FEBS Lett. 58:254-258.

37. Mancini, G., A. 0. Cargonara, and J. F. Jeremans.1965. Immunochemical quantitation of antigens by sin-gle radial immunodiffusion. Immunochemistry 2:235-254.

38. Memoli, V. A., and G. J. Doellgast. 1975. Hemoglobinand serum albumin: salt mediated hydrophobic chro-matography. Biochem. Biophys. Res. Commun. 66:1011-1016.

39. Mohler, H., K. Decker, and W. A. Wood. 1972. Struc-ture of 2-keto-3-deoxy-6-phosphogluconate aldolase. IV.Structural feature revealed by treatment with urea andEllman's reagent. Arch. Biochem. Biophys. 151:251-260.

40. Mollby, R., S. G. Hjalmarsson, and T. Wadstrom.1975. Separation of Escherichia coli heat-labile enter-otoxin by preparative isotachophoresis. FEBS Lett. 56:30-33.

41. Morris, G. K., M. H. Merson, D. A. Sack, J. G. Wells,W. T. Martin, W. E. DeWitt, J. C. Feeley, R. B.Sack, and D. M. Bessudo. 1976. Laboratory investi-

INFECT. IMMUN.

gation of diarrhea in travelers to Mexico: evaluation ofmethods for detecting enterotoxigenic Escherichia coli.J. Clin. Microbiol. 3:486-495.

42. Pierce, N. F. 1973. Differential inhibitory effects of chol-era toxoid and ganglioside on the enterotoxins of Vibriocholerae and Escherichia coli. J. Exp. Med. 137:1009-1023.

43. Raibaud, O., A. Hogberg-Raibaud, and M. E. Gold-berg. 1975. Purification of Escherichia coli enzymes bychromatography on amphiphilic gels. FEBS Lett. 50:130-134.

44. Rappaport, R. S., J. F. Sagin, W. A. Piezchala, G.Bonde, B. A. Rachin, and H. Zint. 1976. Activationof heat-labile Escherichia coli enterotoxin by trypsin.J. Infect. Dis. 133(Suppl.):S41-S45.

45. Ryder, R. W., I. K. Wachsmuth, A. E. Buxton, H. L.DuPont, E. Mason, and F. F. Barrett. 1976. Infantilediarrhea produced by heat-stable enterotoxigenic Esch-erichia coli. N. Engl. J. Med. 295:849-853.

46. Sack, D. A., M. H. Merson, J. G. Wells, R. B. Sack,and G. K. Morris. 1975. Diarrhea associated with heat-stable enterotoxin-producing strains of Escherichiacoli. Lancet ii:235-241.

47. Sack, R. B. 1975. Human diarrheal disease caused byenterotoxigenic Escherichia coli. Annu. Rev. Microbiol.29:333-353.

48. Sack, R. B., S. L. Gorbach, J. G. Banwell, B. Jacobs,B. D. Chatterjee, and R. C. Mitra. 1971. Enterotoxi-genic Escherichia coli isolated from patients with se-vere cholera-like disease. J. Infect. Dis. 123:378-385.

49. Scheinkein, I., R. F. Green, D. S. Santos, and W. K.Maas. 1976. Partial purification and characterization ofa heat-labile enterotoxin of Escherichia coli. Infect.Immun. 13:1710-1720.

50. Shaltiel, S., and A. Er-El. 1973. Hydrophobic chroma-tography: use for purification of glycogen synthetase.Proc. Natl. Acad. Sci. U.S.A. 70:778-781.

51. Smith, H. W., and S. Halls. 1967. Studies on Escherichiacoli enterotoxin. J. Pathol. Bacteriol. 93:531-543.

52. Smyth, D. G., and D. F. Elliot. 1964. Some analyticalproblems involved in determining the structure of pro-teins and peptides. Analyst (London) 89:81-94.

53. Soderlind, O., R. Mollby, and T. Wadstrom. 1974.Purification and some properties of a heat-labile enter-otoxin from Escherichia coli. Zentralbl. Bakteriol. Par-asitenkd. Infectionskr. Hyg. Abt. 1. Orig. Reihe A 229:190-204.

54. Svennerholm, A. M., and J. Holmgren. 1978. Identifi-cation of Escherichia coli heat-labile enterotoxin bymeans of a ganglioside immunosorbent assay (Gn,1-ELISA) procedure. Curr. Microbiol. 1:19-23.

55. Van Heyningen, W. C., C. C. J. Carpenter, N. E.Pierce, and W. B. Greenough III. 1971. Deactivationof cholera toxin by gangliosides. J. Infect. Dis. 124:415-418.

56. Wadstrom, T., R. Mollby, and 0. Soderlind. 1977.Heat-labile enterotoxins of Escherichia coli. Toxicon15:511-519.

57. Warburg, O., and W. Christian. 1942. Isolation andcrystallization of enolase. Biochem. Z. 310:384-421.

58. Weber, K., and M. Osborn. 1969. The reliability ofmolecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412.

59. Woods, K. R., and K. T. Wang. 1967. Separation ofdansyl-amino acids by polyamide layer chromatogra-phy. Biochim. Biophys. Acta 133:369-379.

60. Yon, R. J., and R. J. Simmonds. 1975. Protein chro-matography on adsorbents with hydrophobic and ionicgroups: some properties of N-(3-carboxypropionyl)-aminodecyl-Sepharose and its interaction with wheat-germ aspartate transcarbamylase. Biochem. J. 151:281-290.

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