The interaction of cholera toxin subunit A with cultured adrenal cells.

The interaction of cholera toxin subunit A with cultured adrenal cells

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by San Diego (UCSD) on 01/14/15 For personal use only.

Dc,ptrrtt)retrt ~$MecIiccilMicrohiolog.~,Ct.eig1zror1 Urri~.er..sit.v Scl~oolofMetlicir7e, O~)lcrhcr,NE, U.S.A. 681 78 Accepted April 26. 1978 KNOOP,F. C. 1978. The interaction ofcholera toxin subunit A withcultured adrenalcells.Can. J. Microbiol. 24: 915-921. Subunits A and B of cholera enterotoxin were isolated by chromatogrxphy on a Bio-Gel P-60 column in the presence of 4% formic acid. The purity and biological activity of the isolated subunits was assessed by polyacrylamide disc gel electrophoresis and mouse adrenal cell assay, respectively. The specific uptake of isolated "'I-labeled subunits A and B. peptides A , and A? and bovine serum albumin (BSA) by cultured adrenal cells was investigated. The results indicate that iodosubunit A, or peptide A, or A2, traverses the plasma membrane and is released to the cell cytosol. A significant portion of bound iodosubunits A or B was associated with the plasma membrane. suggesting the presence of specific membrane receptors. The biological activity of subunit A was determined by the mouse adrenal cell assay. The purified subunit c ~ u ~ s ea dcha~xcteristiccellular change from epithelioid to rounded morphology. A 30-fold higher concentration of subunit A (on a mole/mole basis) ascompared with native toxin was required for ;I maximum morphologic response. These res~rltsextendprevious observations related to the bioactivity of subunit A of the cholera enterotoxin molecule. KNOOP,F. C. 1978. The interaction ofcholera toxinsubunit A withcultured adrenalcells. Can. J Microbiol. 24: 91 5-92 1. Les sous-unites A et B de I'enterotoxine du cholera ont ete isolees par chromatographie sur colonne de Bio-Gel P-60en presence d'acide formique (4%). La purete et I'activite biologique des sous-unites isolees sont evaluees, respectivement parelectrophorese surge1 de polyacrylamide et par ces essais sur les cellules des capsules surrenales de solrrris. Nous avons examine I'assimilz~tionspecifique des sous-unites A et B marquees i 1'1"'. des peptides A, et A? et de I'albumine du serum de bovin (ASB) B I'aide des cultures decellules des capsules sul'renales. Les resultats indiquent que la sous-unite A ou les peptides A, ou A? traversent In membrane plasmique et sont liberes dans le cytosol. Une partie importante des sous-unites iodees liees A ou B, est associee i la membrane plasmique; ce qui suggere la presence de recepteurs membrans~iresspecifiques. L'activite biologique de la sous-unite A a etC determinee par des essais sur les cellules des c;lpsules surrenales de souris. La sous-unite purifieeliz~leh une forme arrondie. Comparee h la toxine native, une concentration 30 fois plus elevee de 121 sous-unite A (sur une mole/mole base) est necessaire pour obtenir une reponse morphologique maximum. Ces resultats sont une suite :lux observations precedentes apparentees B la bioactivite de la sous-unite A de la molecule d'enterotoxine du cholera. [Tladuit par le journal]

Introductory Statement The mammalian plasma membrane acts as a barI-ierto prevent biologically active macromolecules from reaching the cell cytosol. Recent investigations have shown that several macromolecules aDpear capable of traversing the host plasma membrane and gaining access to the cell cytosol without apparent loss of biological activity. In these instances, a portion of the native molecule (portion B) is responsible for the recognition of plasma membrane I-eceptors,and thus functions to bind the macromolecule firmly. In a second set of events, a pol-tion of the native molecule (portion A) traverses the plasma membrane and pelforms a specific biological function. Such events have been shown to occur with diphtheria toxin (Boquet and Pappenheimer 1976; Collier and Kandel 1971; Gill and Dinius 1971; Moehring and Moehring 1976), Pseudomonas exotoxin (Iglewski and Kabat 1975;

Vasil et 01. 1976), and the toxic proteins abrin and ricin (Lin et 01. 1970; OlsneS and Pihl 1973; OlSneS ef "1. 1974). Although cholera enterotoxin has not been shown to ~enetrate mammalian ~ l a s m amembranes, there are reasons for suspecting that it might. It is composed of two subunits, A and B. The B subunit has a molecular weight of 11 600 daltons, binds to a specific GM,gangliosidereceptor in a variety of mammalian plasma membranes, and lacks biological activity (Cuatl-ecasas 19736; Holmgren 1973; Holmgren et 01. 1973, 1974, 1975; Holmgren and Lonnroth 1975). However, a variety of biological responses have been reported when subunit A is associated with subunit B (Bhatiaet al. 1969; Burrows and Musteikis 1966; Carpenteret 01. 1969; Sharp and Hynie 1971). Subunit A has a molecular weight of 28 000 daltons and is the biologically active moiety of native


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C A N . J . MICROBIOL. VOL. 14. 1978

cholera enterotoxin (Berkenbile and Delaney 1976; Gill 1976; Holmgren and Lonnroth 1975; Klapper et (11. 1976; Lonnroth and Holmgren 1973). It is composed of two covalently linked peptides, A , and A,, with molecular weights of about 21 000 and 7000 daltons (Klapper et al. 1976). A weak response to subunit A alone has been reported for skin capillary permeability reaction (Lonnroth and Holmgren 1973; van Heyningen 1974) and diarrhea (Finkelstein et al. 1974). However, the recent studies of Donta (1976) and Donta et al. (1976) indicate that cultured mouse adrenal cells undergo morphologic and steroidogenic changes in response to low concentrations of purified subunit A. This finding is unique in that it allows a closer in vivo look at the ultimate fate of this protein moiety. In the present study, the binding and uptake of isolated I2'Ilabeled subunits A and B bv cultured mouse adrenal cells was investigated. Experimental Procedures Plrrified Cl~olercrErrterotoxirl All native cholera enterotoxin (choleragen) used in this study was purified by the method of Finkelstein and LoSpalluto (1970) and supplied by Schwarz-Mann (lot BZ-2487; Orangeburg, NY). Each sample of toxin was rehydrated in sterile deionized and distilled water to a final concentration of I .O mg per millilitre. Srrbrrrzit Sepnrc~tior~ crrld Purity Subunits A and B of native cholera enterotoxin were separated and purified by molecular sieve chromatography according to a revised procedure of Lai et (11. (1976). A sample of toxin ( I .O mg), to which 4% formic acid had been added. was fractionated on a column of Bio-Gel P-60 (82 X 1.5cm) equilibrated in 4% formic acid (Bio-Rad Laboratories, Richmond, CA). Elution was carried out at a flow rate of 4.0 ml per hour and eluates of I .O ml were collected with an automatic fraction collector(Buchler Instruments, Fort Lee, NJ). Protein concentration was measured by absorbance at 280nm. The purity of isolated subunits in each fraction was determined by polyacrylamide disc gel electrophoresis (PAGE) according to the procedure of Davis (1964). The molecular weights of purified subunits A and B were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Neville (1971). Reference proteins were DNase I, 31 000 (Worthington Biochemical Corp., Freehold, NJ); papain, 21000 (Worthington); trypsin, 23300 (Worthington); lipase, 38000 (Worthington); RNase, 13700 (Sigma Chemical Co., St. Louis, MO); insulin, 5700 (Sigma); cytochrome C , 1 1 700 (Sigma); BSA, 67000 (Sigma); and human transferrin, 88000 (Sigma). The complete dissociation of all protein samples closely followed the procedure of Neville (1971). Protein samples, except as noted, were dissolved in 0.05 M Na2C0, containing 10 M urea, 2 mM ethylenediaminetetraacetic acid (EDTA), and 8 mg SDS per milligram protein. Each sample was then denatured by heat (60°C for I h) and dialyzed against upper gel buffer containing 0.1% SDS, 2% sucrose, and a trace of bromphenol blue. Gel preparation, electrophoresis, and staining were carried out as described elsewhere (Neville 1971). Kndioiodinntiorz of Proteirls Subunit A or B from molecular sieve chromatography or bovine serum albumin (BSA) was dialyzed against two 1 5 4 changes of 0.01 M borate buffer (pH 8.5) for 24 h at 4°C. Each sample was then concentrated 10-fold by aspiration at 23°C and

tested for purity by PAGE (Davis 1964) and SDS-PAGE (Neville 1971) a s previously described. The procedure for radiolabeling and calculation of specific activity followed that of Bolton and Hunter (1973). An aliquot of 5OkI ['251]-Radioligand (Schwarz-Mann) was evaporated to dryness in a 13 X 100 mm test tube with dry nitrogen. The tube was then chilled in an ice bath for 5 min. The reaction was initiated by the addition of 30kg of purified subunit A or B or reference protein (BSA) in 15-20kl 0.1 M borate buffer ( p H 8.5). The tube was stirred every 5 min and the reaction terminated after 30min by the addition of 0.5 ml of borate buffer (0. l M, pH 8.5) containing 0.2 M glycine. The reaction components were separated on a 25 X 0.9 cm column of Sephadex G-25 (Pharmacia Fine Chemicals Inc., Piscataway, NJ) equilibrated in 0.05 M phosphate buffer (pH 7.5) containing 0.25% gelatin. Fractions of 0.5 ml were collected every 15 min and aliquots of 10 kI from each fraction assayed for radioactivity in a gamma counter (model T1200, Pharmacia Fine Chemicals Inc.). Each radioactive peak was then pooled and concent~.atedby vacuum aspilxtion at 23°C for 18 h. All radiolabeled proteinswere stored at 4°C. Thespecific activity of iodinated proteinsvaried between 1 and 5 ~ C i l k (g1 Ci = 37GBq), depending on the concentration of [12'I]-Radioligand and toxin subunit used. Specific activities of 2-4 kCi/kg (subunit A), 3-5 pCi/kg (subunit B), and 2-5 kCi/ kg (BSA) were obtained. Protein concentration wasdetermined by the method of Lowry et crl. (1951), using bovine serum albumin (Fr. V , Sigma) as a standard. The separation of peptides A, and A? followed ladioiodination and reduction of purified subunit A. Labeled subunit A (30 kg) was completely reduced with 5 0 mM 2-mercaptoethanol (2-ME) in the presence of 10M urea, 2 m M EDTA, and dry nitrogen for 18 h at 23°C. Peptides A, and A? were then separated on a column of Sephadex G-50 equilibrated in 0.05 M Tris buffer (pH 7.5) containing lOmM 2-ME and 0.25% gelatin. Specific activities of 1-3 pCi/I*.g (peptide A , ) and 1-2kCi/kg (peptide A?) were obtained. Mornrr~ulic~r~ Cell C~rltures Mouse adrenal cell monolayers were grown at 37'C in a humidified atmosphere of 95% air and 5% CO,, and maintained on minimal essential medium F-15 (GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum. Cell monolayers for determination of radioactive uptake were routinely maintained in 30-mI disposable tissue culture flasks (Costal-, Cambridge, MA). Otherwise, dose-response studies for toxic activity were carried out in eight-chambered tissue culture slides (Lab-Tek Products, Naperville, IL). All tissue culture slides were challenged with serial twofold dilutions of isolated subunits A or B o r BSA and bioactivity determined after 6 h of incubation. The highest dilution showing in excess of 90% cell rounding was considered an end point. Uptuke of Rudiolabeled Proteirls Mouse adrenal cell monolayers were challenged with either iodoBSA, iodosubunits A or B, or iodopeptides A, or A,. Incubation was carried out for 6 and 18h at 37°C. After incubation, adrenal cell morphology was observed by light microscopy; biological activity of appropriate samples was indicated by a change in cell morphology according t o the procedure of Donta et ul. (1976). The cultures were then thrice washed with 15 ml Dulbecco's phosphate-buffered saline (pH 7.5). Each monolayer was removed from the flask by a rubber policeman and the cells gently dissociated at 4°C by Dounce homogenization (50 strokes) or acutely ruptured by ultrasonics using a Biosonik IV needle probeat 50%output; total sonication time was60s (VWR Scientific, Denver, CO). No difference in binding o r uptake between these two procedures was observed; samples of the suspending buffer before cell rupture were repeatedly negative for radioactivity. Other methods of cell disruption, such a s


KNOOP

detergents or freeze-thawing, were not used for obvious deleterious reasons. These methods :we known to cause the dissociation of membrane-bound proteins. The lysates (3.0 ml) were centrifuged at 40000 x g for 30 min at 10°C to separate plasma membrane and cytosol fractions. After centrifugation, an aliquot of 1.0ml supernatant was removed from each tube: this sample represented the cell cytosol. An equal volume (2.0ml) of 4 N NaOH was then added to the remaining sample and the tube heated at 100°C for 30min to solubilize plasma membranes; an aliquot of 1.Oml was then removed from each tube and tested for radioactivity. This sample represented the amount of radioactivity associated with solubilized plasma membrane and cell cytosol. The data for plasma membrane activity were expressed a s a difference betweencell cytosol and solubilized sample. Thecontamination of cytosol with the plasma membrane fraction was monitored by phase-contrast microscopy.

Results Sclbc~nitIsolation and Bioactivity The fractionation of purified cholera enterotoxin by molecular sieve chromatography in the presence of 4% formic acid resulted in the appearance of two protein peaks, I and I1 (Fig. 1). The purity of protein within each peak is shown in Fig. 2. As indicated, both protein components migrate as distinct protein bands. The complete dissociation and SDS-gel migration (SDS-PAGE) of peaks I and I1 also resulted in the appearance of single discrete protein bands. The purity of these subunits from molecular sieve chromatography has been previously demonstrated (Lai et a / . 1976). Assessment of molecular weight by SDS-PAGE revealed that peak I contained an isolated subunit with a molecular weight of 23000 daltons; peak I1 protein was found to have a molecular weight of about 11 000 daltons. These data on molecular weight and purity are in close agreement with the results of other recent studies (Kurosky et nl. 1976, 1977; Lai et (11. 1976; Lonnroth and Holmgren 1973; Ohtomo et al. 1976; van Heyningen 1976). The biological activity of purified subunits A and B, peptides A, and A,, and native toxin was determined by the mouse adrenal cell assay (Donta et 01. 1976). Subunit A (0.4 pg) and native toxin (0.05 pg) caused a characteristic change of host cells from normal epithelioid to rounded morphology following challenge (Fig. 3). Analysis of dose-response curves for purified subunit A and native cholera toxin revealed that a 30-fold higher concentration of subunit A (on a mole/mole basis) was required for a maximum morphologic response. This data suggests that entry of the active subunit (A) is better facilitated when associated with the binding subunit (B). In contrast, subunit B and peptides A, and A, did not show morphologic responses at equimolar concentrations. These observations indicate that an intact A,-S-S-A, subunit (subunit A) is required to elicit a morphological response.

FRACTION NUMBER

FIG. 1. Molecular sieve fractionation of purified Vibrio cl~olercreenterotoxin on a column (84 x 1.5 cm) of Bio-Gel P-60 equilibrated in 4% (vlv) formic acid. Peaks 1 and 11 represent toxin subunits A and B , respectively. Protein concentration was measured by absorption of ultraviolet light at 280nm. See Experimental Procedures for details of the experiment.

Binding of'Radiolobeled Proteins Experiments were conducted to determine the amount of iodosubunits A and B, iodopeptides A, and A,, and iodoBSA bound after incubation conditions (37°C for 6 h) that result in a change of cellular morphology. This time interval is the same as revealed by Donta et al. (1976) for maximum morphological effects. Subunit B has been previously shown to bind specifically to GM, ganglioside in plasma membranes, and was thus used as an indication for membrane-bound radioactivity (Table 1). These results were expected, as the specific interaction of this subunit with the G M ,ganglioside fraction of mammalian plasma membranes is well documented. Unexpectedly, however, a fraction of the radioactivity associated with subunit B was internalized. Aliquots of suspending buffer prior to cell disruption were repeatedly negative for radiolabel. The uptake of subunit A is supported by the high percentage of radioactivity remaining in the cell cytosol. This observation suggests that subunit A, or peptide A, or A,, enters the cell cytosol. In addition, a significant portion of iodosubunit A was associated with the plasma membrane. This finding suggests that a plasma membrane receptor binds the active moiety of cholera enterotoxin. The uptake of isolated iodopeptides A, and A, was negligible (Table l), indicating that the attachment and (or) entry of these proteins requires an intact (A,S-S-A,) molecule. Preliminary studies in this laboratory indicate that the uptake of isolated peptides A, or A, is not facilitated by either subunit B or natural cholera toxoid (choleragenoid). The specific uptake and binding of subunits A


C A N . J . MICROBIOL. VOL. 71. 1978

bound ~xdiolabelwas associated with the plasma membrane and 80% with the cell cytosol, regardless of incubation time. The data suggest that the active subunit of cholera enterotoxin is specifically bound to plasma membrane and s~~bsequently internalized by cultured mouse adrenal cells. AIthough sequential time-uptake studies were not performed, an early intl-acellular increase followed by maximal uptake would be expected prior to 6 h of incubation. This would be consistent with other studies on the early (3 h) activation of this cell line by cholera enterotoxin (Donta 1976; Donta et crl. 1976). Preliminary studies on the kinetics of uptake are in progress. Bound iodopeptide A , , although negligible when compared with iodosubunit A, was associated with the plasma membrane; a smaller fraction was internalized (Table 1). The bioactivity of peptides A, and A, was consistently negative.

FIG.2. Electrophoretic analysisof peaks I and I1 from molecular sieve chromatography on 7.5% polyacrylamide disc gels (pH 8.9). Ammonium persulfate (0.0007%) was the catalyst for gel polymerization. The gels represent 6 0 k g each of subunits A (left) and B (right) obtained from peaks I and 11, respectively. Bothgels were stained for protein withcoomassie brilli21nt blue. The procedure followed that of Davis (1964).

and B at two time intervals was also examined. These results did not show any significant difference in the amount of bound subunit A in plasma membrane or cytosol at the end of 6- and 18-h incubation periods. As indicated, about 20% of

Discussion The action of native cholera enterotoxin on a variety of mammalian cells involves several timedependent events. In the first set of events, s ~ ~ b u n i t B specifically interacts with a G,,, ganglioside receptor within the plasma membrane. This interaction results in the specific attachment of the toxin molecule to plasma membrane (Cuatrecasas 1973u, 19736; Halt 1975; Holmgren 1973; Holmgren et ul. 1973, 1974, 1975; King and van Heyningen 1973; van Heyningen 1974; van Hey ningen et crl. 1971). Berkenbile and Delaney (1976) have shown that the native toxin moiety undergoes an "apparent biological modification" following attachment. In a second set of events Gill suggests that the active moiety of cholera enterotoxin (peptide A,), facilitated by the molecular events of the binding subunit and juxtaposition of peptide A,, is released to the host cell cytosol (Gill and King 1975). Such internalization leads to an increase in adenylate cyclase activity and consequent intracellular elevation of cyclic AMP (Beckman et 01. 1974; Bilezikian and Aurbach 1973; Cuatrecasas 1 9 7 3 ~ ;Field 197 1 ; Holmgren and Lonnroth 1976; Kimberget crl. 1971; Schafer er ul. 1970; Sharp and Hynie 1971; Wodnar-Filipowicz and Lai 1976). These observations are consistent with the view that the host cell plasma membrane functions a s a time-dependent barrier which the active subunit of cholera enterotoxin must traverse. Wheeler et al. (1976) have demonstrated that the activation of adenylate cyclase in vitro involves three discrete phases, each with specific time, tempel-ature, and ~0factorrequirements. Other studies have suggested that the active subunit of cholera enterotoxin traverses the pigeon erythrocyte membrane into the c ~ t o s o l ,


'8 I!unqns ~ I ! M[l:J!luap! a.lan\ iv pu~:Iv sapydad put: 1o.11uo~ ~ I ! Ms a s u w l s a ~'v I!unqns q l ! 11?:,!1uap! ~ a.lah\ u!xol aq!!u o l uasuodsa.~[[as :q () 11: p1:a.l a.raM sllnsa.1 aqj. .sa.lnpaso.r,[ [l:luaw!.~adxg.lapun pal!:s!pu! st: iun!paw a.rni[ns anss!] a[!.lals u! (1qfQ.1) 8 .lo ( p i ) v I!unqns .laq)!a ~ I ! M pall!a.ll a.ram s.~aA~:louow[[asal!!.r!?dag "[[a:, [I:U~.IPI: a ~ m i pa.~n~[ri:, u uo 8 ~ U I : v syunqns pall:los!jo s1sa.p 11::,!3oloqd.lop~ '[ '!II.J

CAN. J . MICROBIOL. VOL.

14. 1978

TABLE1 . Percentage of binding of 1251-labeledproteins by cultured mouse adrenal cells


Percentage of bound 1251-labeledproduct Culture constituent

Subunit A

Peptide A t

Peptide A,

Subunit B

BSA

Plasma membrane Cell cytosol

0.9" (22)b 3.3 (78)

0.04 ( 7 2 ) 0.02 (28)

NGc NG

6 . 6 (79) 2.8 (21)

NG NG

aFigures represent Y, o f total '251-labeled rotei ins added remaining within culture constituent afier 6 h o f incubation; similar results were obtained following an 18-h incubation period. Cultured mouse adrenal cell monolayers were challenged with purified subunils A (2.12 x l o 6 cpm) or B (20.58 x l o 6 cpm), bovine serum albumin (BSA, 23.39 x lob cpm) or peptides A1.(1.20 x 106cpm) or A , (1.00 x 106cpm). See Experimental Procedures for details o f the experiment. bFigures In parentheses represent distribution (9,)o f bound 1251-labeledproteins remaining in culture constituent arter 6 h o r incubation; similar results were obtained aftcr an 18-h incubation period. < N G = negligible binding.

where it has an enzymatic function (Gill 1976; Gill and King 1975; Wodnar-Filipowicz and Lai 1976). The present studies indicate that the active subunit of cholera enterotoxin is capable of traversing the plasma membrane and entering the cytosol of mouse adrenal cells. The cytosol interaction of subunit A with internalized membrane structures cannot be excluded. Entry was followed by a complete alteration of adrenal cell morphology. The internalization of iodosubunit A was apparent at 6 h post challenge, a time interval noted for maximum morphologic change (Donta et al. 1976). The attachment of native cholera enterotoxin to plasma membrane is mediated by a specific reaction between the receptor, G,,, ganglioside, and toxin subunit B. Since this reaction is an exlusive function of subunit B, the binding of subunit A could likewise be predicted to rely on a specific membrane receptor molecule prior to entry. The recent studies by D~~~~et (1976), where purified subunit A induced morphologic changes in mouse adrenal cells, indicates the possibility of separate receptors for the binding and active portions of cholera enterotoxin. Biological responses were not when cholera was used to challenge adrenal cells prior to the administration of native toxin. These results are supported by the current investigation in that a significant quantity of iodosubunitA remained in the plasma membrane fraction. Moreover, the bulk of this subunit was found in the cell cytosol. These observations, and others (Cuatrecasas 19736; Gill and King 1975; H ~ 1973; H] ~~ 1973; ]~ ~~l~~~~~ ~~ and 1976), suggest that the tive moiety of cholera enterotoxin (subunit A) is first positioned in the plasma membrane by subunit B. The recent studies of Gill (1976) suggest that peptide A, effects positioning of the active moiety for the disulfide between and A, peptides. Following reduction, the active peptide enters the cell cytosol and stimulates adenylate cyclase ( ~ ~ ~ and ~ ~~i ~ 1976). ~ ~h~~~ ~ events are time-dependent and thus account for the characteristic lag in adenylate cyclase activation in other cell systems (Berkenbile and Delaney 1976;

Cuatrecasas 19736; Gill and King 1975). Several important aspects on the bioactivity of subunit A remain unanswered. Peptides A , and A, will have to be further evaluated for mechanism of cell entry and mode of adenylate cyclase activation. Likewise, the intrasubunit relationship of peptides A , and A, and the role of cytosol components in the stimulation process will have to be further determined. These data will provide answers as to the specificity and function of peptides A, and A, relevant to the role of membrane association and biological expression. Acknowledgements I am indebted to Gary L. Madsen for excellent technical assistance. This work was supported in part by a Biomedical Research Grant (5S07RR05390) at Creighton University. BECKMAN, B., J. FLORES,P. A. WITKUM, andG. W. G . SHARP. 1974. Studies on the mode of action of cholera toxin. Effects on solubilized adenylate cyclase. J. Clin. Invest. 53: 1202-1205, BERKENBILE, F., and R. DELANEY. 1976. Stimulation of adenylate cyclase by Vibrio cholerae toxin and itsactive subunit. J. Infect. Dis. 133 (Suppl.): S582-S588. BHATIA,R. Y. P., A. L. BHATIA, and A. K. THOMAS. 1969. Isolation and concentration of V . cl~olernetoxinand study of its effect on skin permeability i n guinea-pigs, Ind, J. Med, Res. 57: 2018-2029. B I L E Z I K I AJ.N ,P., and G. D. AURBACH. 1973. A P-adrenergi~ receptor of the turkey erythrocyte. I. Bindings of catecholamines and relationship to adenylatecyclase activity. J , Biol, Chem. 248: 5577-5583, BOLTON,A . E., and W. M. HUNTER.1973. The labelling of proteins to high specific radioactivities by conjugation to ~~ 1251-containingacylatingagent. ~~ ~ Biochem. ~ J. 133: 529-539. BOQUET,P., and A. M. PAPPENHEIMER, JR. 1976. Interaction of diphtheria toxin with mammalian cell membranes. J , Biol. them. 251: 5770-5778, BURROWS, w.. and G. M. MUSTEIKIS.1966. Cholera infection and toxin in the rabbit ileal loop. J. Infect. Dis. 116: 183-190. CARPENTER, C. C. J., G. T. C U R L I Nand , W. B. GREENOUCH. 1969. Response of canine Thiry-VeIlajejunal loops tocholera exotoxin and its modification by ethacrynic acid. J . Infect. Dis. 120: 332-338. COLLIER R., J., and J . KANDEL.1971. Structure and activity of diphtheria i ~ toxin. i I . Thiol-dependent ~ ~ ~ dissociation i ~of a fraction ~ of toxin into enzymatically active and inactive fragments. J. BioI, Chem, 246: 1496-1503, cuATREcAsAs,p, 1 9 7 3 ~ Interaction . of Vibrio cholerae enterotoxin withcell membranes. Biochemistry, 12: 3547-3558.

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-19736. Ganglioside and membrane receptors for cholera

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KLAPPER, D. G., R. A. FINKELSTEIN, and J . D. CAPRA1976. toxin. Biochemistry, 12: 3559-3566. Subunit stlucture and N-terminal amino acid sequence of the 1973~. Cholera toxin-fat cell interaction and the mechathree chains of cholera enterotoxin. Immunochemistry, 13: nism of activation of the lipolytic response. Biochemistry, 12: 605-61 1. . KUROSKY, A., D. E. MARKEL, B. J. W. PETERSON, and W. M. 3567-3577. FITCH.1977. Primary structure of cholera toxin B-chain: a DAVIS,B. J . 1964. Discelectrophoresis. 11. Method and applicaglycoprotein hormone analog. Science, 195: 299-30 1. tion to human serum proteins. Ann. N.Y. Acad. Sci. 121: KUROSKY, A,, D. E. MARKEL,B. TOUCHSTONE, and J. W. 404-427. DONTA,S . T. 1976. Interactions of choleragenoid and GhII PETERSON.1976. Chemical characterization of the structure ganglioside with enterotoxins of Vibrio clzolerae and Esof cholera toxin and its natural toxoid. J . Infect. Dis. 133 chericlzia coli in cultured adrenal cells. J . Infect. 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Interaction of cholera enterotoxin with cultured adrenal tumor cells.

Morphological and steroidogenic changes in cultured adrenal tumor cells induced by a subunit of cholera enterotoxin.

Interaction of ganglioside GM1 with the B subunit of cholera toxin modulates growth and differentiation of neuroblastoma N18 cells.

Interaction of cultured mammalian cells with [125I] diphtheria toxin.

A Conformational Shift in the Dissociated Cholera Toxin A1 Subunit Prevents Reassembly of the Cholera Holotoxin.

Interaction of diphtheria toxin and its active subunit, fragment A, with toxin-sensitive and toxin-resistant cells.

Mapping epitopic regions of cholera toxin B-subunit protein.

The interaction of tetanus toxin with intact bovine adrenal chromaffin cells: binding of toxin and subsequent inhibition of catecholamine release.

Similarity of responses of cultured Sertoli cells to cholera toxin and FSH.

Cautionary note on the use of the B subunit of cholera toxin as a ganglioside GM1 probe: detection of cholera toxin A subunit in B subunit preparations by a sensitive adenylate cyclase assay.

Cholera toxin modulation of angiotensin II-stimulated inositol phosphate production in cultured vascular smooth muscle cells.

Cholera toxin stimulates division of 3T3 cells.

Small fragments from the A subunit of cholera toxin capable of activating adenylate cyclase.

Interaction of tetanus toxin and toxoid with cultured neuroblastoma cells. Analysis by immunofluorescence.

A protein-based pentavalent inhibitor of the cholera toxin B-subunit.

Regulation of bradykinin receptor level by cholera toxin, pertussis toxin and forskolin in cultured human fibroblasts.

Involvement of antigen-presenting cells in the enhancement of the in vitro antibody responses by cholera toxin B subunit.

Production of cholera toxin subunit B by a mutant strain of Vibrio cholerae.

Recombinant toxin-coregulated pilus A (TcpA) as a candidate subunit cholera vaccine.

Functions of cholera toxin B-subunit as a raft cross-linker.

Oral immunization of mice with a live recombinant Yersinia enterocolitica O:9 strain that produces the cholera toxin B subunit.

Seed Metabolome Analysis of a Transgenic Rice Line Expressing Cholera Toxin B-subunit.

Short communications. Subunit A from cholera toxin is an activator of adenylate cyclase in pigeon erythrocytes.

Adrenal cortex adenylate cyclase. In vitro modification of the enzyme by cholera toxin.