Quartz crystal microbalance investigation of the interaction of bacterial toxins with ganglioside containing solid supported membranes

The binding of cholera toxin, tetanus toxin and pertussis toxin to ganglioside containing solid supported membranes has been investigated by quartz crystal microbalance measurements. The bilayers were prepared by fusion of phospholipid-vesicles on a hydrophobic monolayer of octanethiol chemisorbed on one gold electrode placed on the 5 MHz AT-cut quartz crystal. The ability of the gangliosides G_M1, G_M3, G_D1a, G_D1b, G_T1b and asialo-G_M1 to act as suitable receptors for the different toxins was tested by measuring the changes of quartz resonance frequencies. To obtain the binding constants of each ligand-receptor-couple Langmuir-isotherms were successfully fitted to the experimental adsorption isotherms. Cholera toxin shows a high affinity for G_M1 (K_a = 1.8 ⋅ 10⁸M^–1), a lower one for asialo-G_M1 (K_a = 1.0 ⋅ 10⁷ M^–1) and no affinity for G_M3. The C-fragment of tetanus toxin binds to ganglioside G_D1a, G_D1b and G_T1b containing membranes with similar affinity (K_a∼10⁶ M^–1), while no binding was observed with G_M3. Pertussis toxin binds to membranes containing the ganglioside G_D1a with a binding constant of K_a = 1.6 ⋅ 10⁶ M^–1, but only if large amounts (40 mol%) of G_D1a are present. The maximum frequency shift caused by the protein adsorption depends strongly on the molecular structure of the receptor. This is clearly demonstrated by an observed maximum frequency decrease of 99 Hz for the adsorption of the C-fragment of tetanus toxin to G_D1b. In contrast to this large frequency decrease, which was unexpectedly high with respect to Sauerbrey's equation, implying pure mass loading, a maximum shift of only 28 Hz was detected after adsorption of the C-fragment of tetanus toxin to G_D1a. Received: 14 January 1997 / Accepted: 15 April 1997     

Study of the Functional Organization of a Novel Adenylate Cyclase Signaling Mechanism of Insulin Action

In this study we continued decoding the adenylate cyclase signaling mechanism that underlies the effect of insulin and related peptides. We show for the first time that insulin signal transduction via an adenylate cyclase signaling mechanism, which is attended by adenylate cyclase activation, is blocked in the muscle tissues of the rat and the mollusk Anodonta cygnea in the presence of: 1) pertussis toxin, which impairs the action of the inhibitory GTP-binding protein (G_i); 2) wortmannin, a specific blocker of phosphatidylinositol 3-kinase; and 3) calphostin C, an inhibitor of different isoforms of protein kinase C. The treatment of sarcolemmal membrane fraction with cholera toxin increases basal adenylate cyclase activity and decreases the sensitivity of the enzyme to insulin. We suggest that the stimulating effect of insulin on adenylate cyclase involves the following stages of hormonal signal transduction cascade: receptor tyrosine kinase → G_iprotein (βγ) → phosphatidylinositol 3-kinase → protein kinase C (ζ?) → G_sprotein → adenylate cyclase → cAMP.     

Part I: an x-ray scattering study of cholera toxin penetration and induced phase transformations in lipid membranes

Cholera toxin is a highly efficient biotoxin, which is frequently used as a tool to investigate protein-membrane interactions and as a reporter for membrane rafts. Cholera toxin binds selectively to gangliosides with highest affinity to GM₁. However, the mechanism by which cholera toxin crosses the membrane remains unresolved. Using x-ray reflectivity and grazing incidence diffraction, we have been able to monitor the binding and penetration of cholera toxin into a model lipid monolayer containing the receptor GM₁ at the air-water interface. Very high toxin coverage was obtained allowing precise measurements of how toxin binding alters lipid packing. Grazing incidence x-ray diffraction revealed the coexistence of two monolayer phases after toxin binding. The first was identical to the monolayer before toxin binding. In regions where toxin was bound, a second membrane phase exhibited a decrease in order as evidenced by a larger area per molecule and tilt angle with concomitant thinning of the monolayer. These results demonstrate that cholera toxin binding induces the formation of structurally distinct, less ordered domains in gel phases. Furthermore, the largest decrease in lateral order to the monolayer occurred at low pH, supporting a low endosomal pH in the infection pathway. Surprisingly, at pH = 8 toxin penetration by the binding portion of the toxin, the B₅ pentamer, was also observed.     

CHOLERA TOXIN

1. Death in several infectious diseases is caused by protein toxins secreted by invading bacteria. Cholera toxin is a simple protein secreted by Vibrio cholerae colonizing the gut; it is responsible for the massive diarrhoea that is cholera. 2. The primary action of cholera toxin is an activation of adenylate cyclase, an enzyme found on the inner membrane of eukaryotic cells that catalyses the conversion of ATP to cyclic AMP. Consequent increases in the intracellular concentration of cyclic AMP are responsible for other manifestations of cholera toxin including the diarrhoea. The toxin is active on almost all eukaryotic cells. 3. The toxin can be purified from culture filtrates of V. cholera. It has a molecular weight of 82000; and is composed of one subunit A (itself two polypeptide chains joined by a disulphide bond: AI (22000) and A2 (5000)) and five subunits B (11500). These can be separated in dissociating solvents such as detergents or 6 M guanidine hydrochloride. An amino-acid sequence of subunit B has been published. The five B subunits (sometimes found by themselves in the filtrate and known as ‘choleragenoid’) are probably arranged in a ring with the subunit A in the middle joined to them non-covalently by peptide A2. 4. The first action of cholera toxin on a cell is to bind to the membrane strongly and irreversibly. Several thousand molecules of toxin bind to each cell and the binding constants are of the order of 10^-10 M. The binding is rapid, but is followed by a lag phase of about an hour before the intracellular cyclic AMP concentration begins to increase. 5. Ganglioside G_M1, a complex amphiphilic lipid found in cell membranes, binds tightly to the toxin which shows an enzyme-like specificity for this particular ganglioside. Toxin that has already bound ganglioside can no longer bind to cells and is therefore inactive. This and other experiments using cells depleted of endogenous ganglioside suggest that ganglioside G_M1 is the natural receptor of the toxin on the cell surface. The binding is followed by a lateral movement of the toxin-ganglioside complex in the cell surface forming a ‘cap’ at one pole of the cell. 6. The binding of ganglioside by toxin is a function exclusively of subunit B; Subunit A does not bind and can be eluted with 8 M urea from an insolubilized toxin-ganglioside complex. Subunit B is not by itself active, and so preincubation with B can protect cells or even whole gut from the action of toxin by occupying all the ganglioside binding sites. 7. Subunit A is responsible for activation of adenylate cyclase. Purified subunit A or just peptide AI is active by itself and this activity is not inhibited by ganglioside or by antisera to subunit B. In intact cells the activity is low and shows the characteristic lag phase but in lysed cells the subunit (or the whole toxin) is much more active and there is no lag phase. This suggests that the lag phase represents the time that subunit A takes to cross the cell membrane and get to its target. 8. Several cofactors are needed for toxin activity in lysed cells: NAD+, ATP, sulphydryl compounds and another unidentified cytoplasmic component. The activity of the cyclase is altered in a complex way generally rather similarly to the action of hormones such as adrenalin, but it is difficult to draw any general conclusions. 9. There are two chief theories of how cholera toxin acts. The first is that subunit A (or just peptide AI) enters the cell and there catalyses some reaction leading to activation of the cyclase. The cleavage of NAD+ into nicotinamide and adenosine diphosphoribose could be such a reaction; it is catalysed by high concentrations of cholera toxin. 10. The other theory is that part of the toxin binds directly to the adenylate cyclase or to some other molecule that can then interact with the cyclase, perhaps after the lateral movement of the toxin-ganglioside complex in the cell surface. This binding may be related to the known action of guanyl nucleotides on the cell surface. 11. The entry of peptide AI into the cell and its transport through the membrane is mediated by the binding of subunits B to the cell surface, perhaps just because the binding increases the local concentration of subunit A, or perhaps following specific conformational changes in the subunits and the formation of a tunnel of B subunits through the membrane. An experiment showing that the toxin remains active when the subunits are covalently bonded together suggests that peptide AI does not separate completely from the rest of the molecule. 12. There are several other proteins that resemble cholera toxin in structure and function. For example, glycoprotein hormones such as thyrotrophin also activate adenylate cyclase and have an apparently similar subunit structure with one type of subunit that binds to a ganglioside. There may also be analogies between the amino-acid sequences of toxin and hormones. 13. The enterotoxin made by some strains of Escherichia coli produces a similar diarrhoea to that of cholera. Several different toxic proteins have been prepared but they all seem to activate adenylate cyclase in the same sort of way as cholera toxin does and also to cross-react immunologically with it. The E. coli toxin also reacts with ganglioside G, but the reaction is weak and probably physiologically insignificant. Salmonella typhimurium secretes a similar toxin. 14. Tetanus toxin also reacts with a ganglioside receptor. This protein has two polypeptide chains of which only one reacts with the ganglioside; but the molecular activity is not yet known. 15. Diphtheria toxin has an A fragment that is directly responsible for the toxicity (by catalysing an NAD+ cleavage reaction leading to the total inhibition of protein synthesis) and a B fragment that gets the A fragment into the cells. This structure of active and binding components therefore seems to be common to many toxins. 16. The ability to produce toxin may confer some selective advantage on V. cholerae. The toxin may originate from accidental incorporation of DNA from an eukaryotic host, or alternatively from some material involved with the cyclic AMP metabolism of the bacterium.     

The effect of cholera toxin on myogenesis in rat skeletal muscle cultures

Cholera toxin, when added to rat primary embryonic muscle cultures, stimulates intracellular cyclic AMP and cell fusion. The effect on cell fusion can be mimicked by daily addition of dibutyryl cyclic AMP, but not by choleragenoid, which like cholera toxin binds to the ganglioside G_M1, but does not stimulate adenyl cyclase. The effects on fusion of three other agents known to affect intracellular cyclic AMP levels, indomethacin, isobutylmethyl xanthine, and isoproterenol were also studied. It is concluded that intracellular cyclic AMP levels are important in the control of rat skeletal muscle cell fusion.     

The mechanism of action of cholera toxin in pigeon erythrocyte lysates.

The adenylate cyclase activity of intact pigeon erythrocytes begins to rise after about 20 min of exposure to cholera toxin. The maximum rate at which the cyclase activity increases appears to be limited by the number of toxin molecules which can reach an intracellular target. If the erythrocytes are made permeable to the toxin by a bacterial hemolysin, no such limit exists, and adenylate cyclase activity starts to rise immediately upon the addition of toxin, and continues to rise to a maximum at an initially constant rate which is dependent upon the concentration of toxin. On lysed erythrocytes, the addition of cholera antitoxin immediately prevents any further rise in adenylate cyclase activity, but does not reverse any activation already achieved. Erythrocyte lysates may also be activated by isolated peptide A1 of cholera toxin, although activation of adenylate cyclase of intact erythrocytes requires the complete toxin molecule. In the intact cells, toxin first attaches by its Component B to surface receptors of which there are about 30 per erythrocyte. Subsequently, peptide A1 but not Component B is inserted into the erythrocyte. It takes only about 1 min at 37 degrees for peptide A1 to be sufficiently deep within the cell membrane to be inaccessible to extracellular antitoxin, but its complete transit through the membrane appears to take longer. The surface receptors are used only once, for they remain blocked by Component B. The number of receptors available on the surface may be increased by soaking cells in ganglioside GM1. Cholera toxin also decreases the rate of apparently spontaneous loss of adenylate cyclase activity and increases the response to epinephrine. Theophylline inhibits the action of cholera toxin.     

Dissociation by cooling of hormone and cholera toxin activation of adenylate cyclase in intact cells

Cholera toxin, through adenylate cyclase activation reproduced cyclic AMP-mediated effects of thyroid-stimulating hormone (TSH) in dog thyroid slices, i.e protein iodination, [1-¹⁴C]glucose-oxidation and hormone secretion. Iodide and carbamylcholine decreased the cyclic AMP accumulation induced by cholera toxin as well as by TSH, which supports the hypothesis of an action of these agents beyond the steps of hormone-receptor and receptor-adenylate cyclase interaction. Cooling to 20°C did not impair the TSH induced cyclic AMP accumulation in thyroid slices, but completely suppressed the cholera toxin effect.This observation has been extended to other hormones and target tissues, such as the parathyroid hormone (PTH) (kidney cortex), adrenocorticotropic hormone (ACTH) (adrenal cortex)_and luteinizing hormone (LH) (ovary systems). As in thyroid, cooling dissociated the cholera toxin and hormonal effects on cyclic AMP accumulation. In homogenate, cooling decreased cyclic AMP generation in the presence of cholera toxin but at 20°C and 16°C a cholera toxin stimulation was still observed. These results bear strongly against the hypothesis that the glycoprotein hormones TSH and LH activate adenylate cyclase by a mechanism identical to cholera toxin.     

Enhanced GTP-dependent activities of the adenylate cyclase system: basis for increased hormonal responsiveness.

Normal rat kidney (NRK) cells growth arrested by picolinic acid and isoleucine deprivation exhibit an increased response to certain agents (i.e., prostaglandin E₁, (?)-isoproterenol, and cholera toxin) which elevate intracellular cyclic AMP levels. The enhanced hormonal response is apparently due, at least in part, to increased adenylate cyclase activity. Adenylate cyclase activities measured in the presence of GTP, GTP plus prostaglandin E₁, and GTP plus (?)-isoproterenol are increased two- to threefold in membranes prepared from treated cells. In contrast, basal activity is potentiated only 20 to 50% and activity determined in the presence of fluoride is only marginally altered. Also of interest is the increase in cholera toxin activation of cyclase activity in the treated cells. Lower concentrations of cholera toxin (5 ng/ml) are required to achieve maximal stimulation of cyclase activity from picolinic acid-treated and isoleucine-deprived cells; maximal stimulation of control cell adenylate cyclase is attained with 25 to 50 ng/ml cholera toxin. Picolinic acid treatment and isoleucine deficiency both have been shown to arrest NRK cell growth in the G₁ phase of the cell cycle. However, results with cells arrested in G₁ by serum starvation and by growth to high cell population density indicate that G₁ specific growth arrest does not appear to account for the increase in hormonal responsiveness. Chelation of inhibitory metals and proteolytic activation also do not appear to be involved in the mechanism by which picolinic acid enhances cyclic AMP formation. Rather, the results suggest that the treated cells have an increased amount of an active GTP-dependent function required for hormone and cholera toxin stimulation of adenylate cyclase. Thus, picolinic acid treatment and isoleucine deprivation may provide a useful means of modulating the GTP-dependent step required to potentiate hormonal responsiveness.     

Differential effects of cholera toxin on guanine nucleotide regulation of beta-adrenergic agonist high affinity binding and adenylate cyclase activation in frog erythrocyte membranes

The guanine nucleotide regulatory protein(s) regulates both adenylate cyclase activity and the affinity of adenylate cyclase-coupled receptors for hormones or agonist drugs. Cholera toxin catalyzes the covalent modification of the nucleotide regulatory protein of adenylate cyclase systems. Incubation of frog erythrocyte membranes with cholera toxin and NAD+ did not substantially alter the dose dependency for guanine nucleotide activation of adenylate cyclase activity. In contrast, toxin treated membranes demonstrated a 10 fold increase in the concentrations of guanine nucleotide required for a half maximal effect in regulating beta-adrenergic receptor affinity for the agonist (+/-) [3H]hydroxybenzylisoproterenol. The data emphasize the bifunctional nature of the guanine nucleotide regulatory protein and suggest that distinct structural domains of the guanine nucleotide regulatory protein may mediate the distinct regulatory effects on adenylate cyclase and receptor affinity for agonists.     

The activation of adenylate cyclase from small intestinal epithelium by cholera toxin

ADP-ribosylation of membrane proteins from rabbit small intestinal epithelium was investigated following incubation of membranes with [32P]NAD and cholera toxin. Cholera toxin catalyzes incorporation of 32P into three proteins of 40 kDA, 45 kDa and 47 kDa located in the brush-border membrane. In contrast, basal lateral membranes do not contain any protein which becomes labeled in a toxin-dependent manner when incubated with cholera toxin and [32P]NAD. The modification of membrane proteins from brush border occurred in spite of the virtual absence in these membranes of adenylate cyclase activatable either by cholera toxin, vasoactive intestinal peptide (VIP) or fluoride. The three agents activated adenylate cyclase when crude plasma membrane were used. Cholera toxin activated fivefold at 10 micrograms/ml. Vasoactive intestinal peptide activated at concentrations from 10-300 nM, the maximal stimulation being sixfold. Fluoride activated 10-fold at 10 mM. When basal lateral membranes were assayed for adenylate cyclase it was found that, with respect to the crude membranes, the specific activity of fluoride-activated enzyme was 3.3-fold higher, VIP stimulated enzyme was maintained while cholera-toxin-stimulated enzyme showed half specific activity. Moreover, while fluoride stimulated ninefold and VIP stimulated fivefold, cholera toxin only stimulated twofold at the highest concentration. The results suggest that the activation by cholera toxin of adenylate cyclase located at the basal lateral membrane requires ADPribosylation of proteins in the brush border membrane.     

Isolation ofEscherichia coli heat-labile enterotoxin by affinity chromatography: Characterization of subunits

This paper describes the isolation ofEscherichia coli heat-labile enterotoxin (LT) by affinity chromatography on an anti-cholera toxin immunoglobulin-Sepharose column, and the subunit composition of crude and affinity-isolated LT. LT and its subunits were assayed with ganglioside (G_M1)-ELISA, immunodiffusion, skin toxicity, and broken cell adenylate cyclase activation methods. The results show that the immunoaffinity method, applied to LT of different strains and batches, yielded about 100-fold purification with approximately 50% recovery of LT antigen. LT was shown to contain a G_M1-ganglioside binding subunit as well as another subunit which does not bind to G_M1 but activates adenylate cyclase. Immunodiffusion tests showed that the two LT subunits were immunologically related to but not identical with, respectively, the B and A subunits of cholera toxin. The LT “A” and “B” subunits were present in similar proportions in the affinity-isolated and crude LT preparations, but in the purified fraction they had only partially reassociated into holotoxin.     

Cholera toxin and membrane gangliosides: Binding and adenylate cyclase activation in normal and transformed cells

Summary A virally transformed, ganglioside GM₁-deficient cell line binds 2% of the cholera toxin (choleragen) bound by the parent, line and is less responsive to choleragen with respect to adenylate cyclase stimulation. This biological response is maximal when 10% of choleragen-binding sites in the transformed line, or 0.5% in the parent line, are occupied. In contrast, in isolated fat cells saturation of binding and adenylate cyclase stimulation are seen at very similar concentrations.Incubation of ganglioside GM₁ with intact cells increases choleragen binding (defined here as ganglioside incorporation) in the transformed cell line but does not enhance the biological response to choleragen. Stimulation of adenylate cyclase is enhanced in isolated fat cells, however, by exogenous ganglioside GM₁. The binding and cyclase response in fat cells can be reduced by the addition of the inactive analog and competitive antagonist, choleragenoid, and there is recovery of the enzyme response and binding upon subsequent addition of exogenous GM₁. Failure of enhancement in the transformed cell line is explained by the presence of a five- to tenfold excess of binding sites over the number required for the full biological effect of choleragen. Cells with a large excess of toxin receptors are relatively refractory to the blocking effects of choleragenoid on biological responses. Notably, untransformed cells, which contain large quantities of toxin receptor, cannot incorporate exogenously added ganglioside GM₁. These findings suggest the possible existence in the cytoplasmic membrane of specific molecular structures, present in finite and limited number, for recognizing and accepting ganglioside molecules exposed to the external medium.     

The detection of picomolar quantities of GD1b,GT1b and GQ1b on thin-layer chromatograms by the direct binding of125I-labeled tetanus toxin,fragment C

The¹²⁵I-labeled fragment C of tetanus toxin was found to bind specifically to the gangliosides G_D1b, G_T1b, and G_Q1b when applied to thin-layer chromatograms on which a mixture of gangliosides had been resolved. As little as 2.5 pmoles of these gangliosides could be detected by this method. In addition to factors determined by the sample, namely the amount and species of gangliosides present, optimal binding of the¹²⁵I-labeled fragment C also depended upon the iodination procedure used to generate the probe, the toxin concentration, and the concentration, buffer type, pH, and ionic strength of the binding solution. This new technique was shown to be a sensitive method for the detection and identification of specific gangliosides originating from extraneural or neural cells.Nomenclature: The gangliosides follow the nomenclature system of Svennerholm [Eur J Biochem (1977) 79:11–21] G_M3 II³NeuAc-LacCer - G_D3 II³(NeuAc)₂-LacCer - G_M1 II³NeuAc-GgOse₄Cer - G_D1a IV³NeuAc, II³NeuAc-GgOse₄Cer - G_D1b II³(NeuAc)₂-GgOse₄Cer - G_T1b IV³NeuAc, II³(NeuAc)²-GgOse₄Cer - G_Q1b IV³(Neu-Ac)₂, II³(NeuAc)₂-GgOse₄Cer - G_P1b IV³(NeuAc)₃, II³(NeuAc)₂-GgOse₄Cer     

Analysis of transmembrane dynamics of cholera toxin using photoreactive probes

Using sodium dodecyl sulfate--polyacrylamide gel electrophoresis and autoradiography, we have shown that ¹²⁵I-labeled cholera toxin binds to Newcastle disease virus. Pretreatment of Newcastle disease virus with “cold” cholera toxin (at 37°C for 30 minutes) inhibits the binding of ¹²⁵I-labeled toxin in a subsequent incubation (at 37°C for 30 minutes). These results suggest that cholera toxin binds to Newcastle disease virus in a specific manner. The precise receptor for toxin is unknown in Newcastle disease virus but it is presumed to be the ganglioside GM₁. We have previously shown that the photoreactive probe 12-(4-azido-2-nitrophenoxy)stearoylgucosamine[1-¹⁴C] labels the membrane proteins of Newcastle disease virus. Since the reactive group of the probe, ie, N₃, resides within the membrane bilayer, studies were initiated to determine which, if any, of the subunits of cholera toxin cross the membrane of Newcastle disease virus and become radioactively labeled upon photoactivation of the probe at 360 nm. After a 15-minute incubation of cholera toxin with Newcastle disease virus containing the photoreactive probe, irradiation effected the ¹⁴C-labeling of the active A₁ subunit of cholera toxin. Irradiation of cholera toxin in solution with an equivalent amount of probe but without virus resulted in no labeling of toxin subunits.     

Effects of cholera toxin and 5'-guanylylimidodiphosphate on human spermatozoal adenylate cyclase activity

The effects of cholera toxin and 5′-guanylylimidodiphosphate (Gpp(NH)p) on human spermatozoal adenylate cyclase activity were tested. Cholera toxin had no demonstrable effect on adenylate cyclase activity in human spermatozoa at concentrations between 5 and 20 μg/ml, whether the toxin was preincubated with intact spermatozoa between 5 min and 5 h prior the adenylate cyclase assay, or was added to lysed spermatozoa, where the adenylate cyclase would be accessible to the toxin. In contrast, Gpp(NH)p at concentrations between 10 and 100 μM was effective in activating human spermatozoal adenylate cyclase activity.     

Inhibition of murine leukemia (WEHI-3B and L1210) proliferation by cholera toxin B subunit

Cholera holotoxin produces both stimulation and inhibition of the growth of different cell populations. These opposite effects were both attributed to the enzymatic activity of the subunit A that activates adenylate cyclase, increasing the intracellular level of cAMP. We observed that the B subunit of cholera toxin produced by itself an inhibition of the 'in vitro' growth of two murine leukemia cell lines (L1210 limphoid leukemia and WEHI-3B myelomonocytic leukemia). The sensitivity of WEHI-3B cells towards cholera toxin was about 5000-times higher than that of the L1210 cells, whereas the two leukemias showed an identical sensitivity to the B subunit (IC50 = 5.10(-10) M for L1210 and 10(-10) M for WEHI-3B). The inhibition produced by the B subunit was neutralized by GM1 and in a minor degree by type II gangliosides. The two leukemias showed a remarkable difference in their gangliosides contents (L1210 cells contained GM1 (80.6%) and GM2 (19.4%), while WEHI-3B cells contained GM1 (28.2%), Fuc-GM1 (44.9%) and a band (26.9%) with a chromatographic mobility between GD1a and GD1b). The inhibition could be explained by a competitive mechanism between the B subunit and some autocrine factor binding GM1-containing receptors. Our data strengthen the suggestion to consider gangliosides as very important pleiotropic biomodulators.     

Biphasic regulation of adenylate cyclase by cholera toxin in neuroblastoma x glioma hybrid cells is due to the activation and subsequent loss of the alpha subunit of the stimulatory GTP binding protein (GS)

Exposure of neuroblastoma x glioma hybrid (NG108-15) cells to low concentrations of cholera toxin produced a stimulation of both basal and forskolin-amplified adenylate cyclase activity in membranes prepared from these cells. Higher concentrations of cholera-toxin reversed this effect. Mn2+ activation of adenylate cyclase indicated that this effect was not due to a modification of the intrinsic activity of this enzyme. Cholera toxin was demonstrated to produce a concentration and time-dependent loss of GS alpha from membranes of these cells. Loss of GS alpha from membranes of these cells was preceded by its ADP-ribosylation. The effects of cholera toxin were specific for GS alpha, as no alterations in levels of the pertussis toxin-sensitive G-proteins Gi2, Gi3 and Go, were noted in parallel. Equally, no alteration in levels of G-protein beta-subunit were produced by the cholera toxin treatment. These experiments demonstrate that cholera toxin-catalysed ADP-ribosylation does not simply maintain an activated population of GS at the plasma membrane and that alterations in levels of GS at the plasma membrane can modify adenylate cyclase activity.     

Polarized distribution of GM1-ganglioside in human duodenal absorptive enterocytes as visualized with cholera toxin-gold complex

Cholera toxin bound to particles of colloidal gold was used to investigate by electron microscopy the binding of the toxin in human duodenum. Cholera toxin binding was detected only in the apical (brush border) plasma membrane domain suggesting that the ganglioside G_M1 is absent from the basolateral plasma membrane domain. Intracellularly, toxin binding became detectable in thetrans side of the Golgi apparatus. Labeling of endosomes may indicate that the non toxin-occupied G_M1-ganglioside becomes internalized.     

Mechanism of action of choleragen andE. coli heat-labile enterotoxin: activation of adenylate cyclase by ADP-ribosylation

Summary Choleragen exerts its effects on cells through the activation of adenylate cyclase. The initial event appears to be the binding of the B subunit of the toxin to ganglioside G_M1 on the cell surface, following which there is a delay prior to activation of adenylate cyclase. Patching and capping of the toxin on the cell surface, perhaps involved in the internalization of the enzymatically active subunit, may be occuring during this time. The activation of adenylate cyclase, which is catalyzed by the A₁ peptide of choleragen, does not require the B subunit or ganglioside G_M1. The A₁ peptide catalyzes the transfer of ADP-ribose from NAD to an amino acid, probably arginine, in a 42 000 dalton membrane protein. This protein appears to be the GTP-binding component (or G/F factor) of the adenylate cyclase system and is cruical to the regulation of cyclase activity by hormones such as epinephrine. ADP-ribosylation of the G/F factor is enhanced by GTP and, in some systems, by a cytosolic factor. GTP is also required for stabilization and optimal catalytic function of the choleragen-activated cyclase. Calmodulin, a calcium-binding protein, is necessary for expression of catalytic activity of the toxin-activated adenylate cyclase in brain and other tissues. The ADP-ribosyltransferase activity required for activation of the cyclase is an intrinsic property of the A₁ peptide of choleragen which is expressed only after the peptide is released from the holotoxin by reduction of a single disulfide bond. In the absence of cellular components, choleragen catalyzes the ADP-ribosylation of small guanidino compounds such as arginine as well as peptides and proteins that contain arginine. It is assumed, therefore, that the site of ADP-ribosylation in the natural acceptor protein is an arginine or similar amino acid. When guanidino compounds are not present as ADP-ribose acceptors, choleragen hydrolyzes NAD to ADP-ribose and nicotinamide at a considerably slower rate. E. coli heat-labile enterotoxin (LT) is very similar to choleragen in structure and function. It consists of two types of subunits, A and B, with sizes comparable to those of the A and B subunits of choleragen. Binding of LT to the cell surface is enhanced by prior incorporation of G_M1 but not other gangliosides; the oligosaccharide of G_M1 specifically interacts with LT and its B subunit. The A subunit of LT exhibits ADP-ribosyltransferase activity following activation by thiol to release the A₁ peptide. The A subunit of LT can be isolated in an ‘unnicked’ form and thus requires, in addition to reduction by a thiol, proteolytic cleavage to generate the active A₁ peptide. Like choleragen, LT uses guanidino compounds as model ADP-ribose acceptors and catalyzes the ADP-ribosylation of a 42 000 dalton protein in cell membrane prepatations. ADP-ribosyltransferases that use arginine as ADP-ribose acceptors are not restricted to bacterial systems; such an enzyme has been purified to apparent homogeneity (>500 000-fold) from turkey erythrocytes. Based on a subunit molecular weight of 28 000, its turnover number with arginine as the ADP-ribose acceptor is considerably higher than that of either toxin. Although with low molecular weight guanidino derivatives the substrate specificity of the enzyme is similar to that of choleragen, with protein substrates it clearly differs. The physiological role of the turkey erythrocyte transferase remains to be established.     

Activation of pigeon erythrocyte adenylate cyclase by cholera toxin partial purification of an essential macromolecular factor from horse erythrocyte cytosol

A cytosolic, macromolecular factor required for the cholera toxin-dependent activation of pigeon erythrocyte adenylate cyclase and cholera toxin-dependent ADP-ribosylation of a membrane-bound 43 000 dalton polypeptide has been purified 1100-fold from horse erythrocyte cytosol using organic solvent precipitation and heat treatment. This factor, 13 000 daltons, does not absorb to anionic or cationic exchange resins, is sensitive to trypsin or 10% trichloroacetic acid and is not extractable by diethyl ether. Activation of adenylate cyclase by cholera toxin requires the simultaneous presence of ATP (including possible trace GTP), NAD⁺, dithiothreitol, cholera toxin, membranes and the cytosolic macromolecular factor. Reversal of cholera toxin activation of adenylate cyclase, and of the toxin-dependent ADP-ribosylation, requires the presence of the cytosolic factor. The ability of the purified cytosolic factor to influence the hormonal sensitivity of liver membrane adenylate cyclase may provide clues to its physiological functions.