Concentrations Of Cholera Toxin Research Articles

Isolation of nontoxinogenic mutants of Vibrio cholerae in a colorimetric assay for cholera toxin using the S49 mouse lymphosarcoma cell line.

A mouse lymphosarcoma (S49) cell line that is growth-inhibited by agents that elevate intracellular concentrations of adenosine 3':5'-cyclic phosphate was used in a sensitive and convenient colorimetric assay for cholera toxin. S49 cells suspended in Dulbecco's modified Eagle's minimal essential medium containing 10(-5)--10(-6) M RO 20-1724, an analogue of 4-(3,4-demethoxybenzyl)-2-imidazolidinone and a phosphodiesterase inhibitor, were growth-inhibited by subnanogram concentrations of cholera toxin. Effects of toxin were detected by the absence of a yellow pH change (phenol red indicator) which normally accompanies the production of acid metabolites by lymphoma cells. An assay using S49 cells grown in microtiter plates, which is capable of detecting 10 pg of cholera toxin or 0.01 units of cholera antitoxin, was used in screening for nontoxinogenic mutants of Vibrio cholerae strain 569B. The properties of two mutants of the Tox--phenotype, which lacked biologically and immunologically detectable toxin products, are described.

CHOLERA TOXIN

CHOLERA TOXIN

Ultrastructural localization of cell membrane G M1 ganglioside by cholera toxin

An immunoelectron microscopic method is described for sensitive high-resolution visualization of tissuebound cholera toxin. The principle is to incubate cells or tissue sections with toxin and then to localize the bound toxin with toxin-specific peroxidase (donor:hydrogen-peroxide oxidoreductase; EC 1.11.1.7)-conjugated antibody and enzyme substrate. Thin sections are examined for electron-opaque precipitates in a transmission electron microscope. Because of the specific binding of the toxin to membrane ganglioside G(M1), the method can be used for ultrastructural localization of this ganglioside. Semiquantitative data are obtained by titration of the limiting concentration of cholera toxin producing specific precipitates. The specificity of the method was controlled in various ways, including analyses of the correlation between the immunoelectron microscopy results and determinations of ganglioside G(M1) in tissues with different ganglioside concentrations, tissues hydrolyzed with Vibrio cholerae sialidase, tissues in which exogenous G(M1) has been incorporated, and lipid-extracted tissues. The immunoelectron microscopic method demonstrates that membrane G(M1) ganglioside is positioned on the external side exclusively. Cell-bound toxin remains in its original location on the plasma membrane surface of cells below 18 degrees , but appears to be redistributed both laterally and vertically in the membrane of cells incubated at 37 degrees for 30 min or longer. The results of this method indicate that in the central nervous system G(M1) is concentrated in the pre- and postsynaptic membranes of the synaptic terminals; a further increase in reactivity of these structures after hydrolysis of the nervous tissue with V. cholerae sialidase suggests that higher gangliosides of the same series are particularly increased in the pre- and postsynaptic junctions.

Mechanism of adenylate cyclase activation by cholera toxin: Inhibition of GTP hydrolysis at the regulatory site

Treatment of turkey erthrocyte membranes with cholera toxin caused an enhancement of the basal and catecholamine-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activities. Both of these activities required the presence of GTP. The toxin effect on the adenylate cyclase activity concided with an inhibition of the catecholamine-stimulated guanosinetriphosphatase activity. Inhibition of the guanosinetriphosphatase, as well as enhancement of the adenylate cyclase activity, showed the same dependence on cholera toxin concentrations, and the effect of the toxin on both activities was dependent on the presence of NAD. It is proposed that continuous GTP hydrolysis at the regulatory guanyl nucleotide site is an essential turn-off mechanism, terminating activation of the adenylate cyclase. Cholera toxin inhibits the turn-off guanosinetriphosphatase reaction and thereby causes activation of the adenylate cyclase. According to this mechanism GTP should activate the toxin-treated preparation of adenylate cyclase, as does the hydrolysis-resistant analog guanosine 5'-(beta,gamma-immino)triphosphate [Gpp(NH)p]. Indeed, the toxin-treated adenylate cyclase was maximally activated, in the presence of isoproternol, by either GTP or Gpp(NH)p, while adenylate cyclase not treated with toxin was stimulated by hormone plus GTP to only one-fifth of the activity achieved with hormone plus Gpp(NH)p. Furthermore, the toxin-treated adenylate cyclase activated by isoproterenol plus GTP remained active for and extended period (half-time of 3 min) upon subsequent addition of the beta-adrenergic blocker, propranolol. The native enzyme, however, was refractory to propranolol only if activated by Gpp(NH)p but not by GTP.

Activation of adenylate cyclase by cholera toxin in rat liver homogenates.

The effect of cholera toxin on adenylate cyclase from rat liver has been studied in a broken cell preparation. The activation of the enzyme in this in vitro preparation requires the addition of nicotinamide adenine dinucleotide (NAD) to the incubation medium and the presence of cell components other than the membrane-bound adenylate cyclase. Once the activation of the cyclase is produced, the effect persists despite repeated washing or solubilization of the enzyme. The effect can be obtained with concentrations of cholera toxin as low as 0.4 nM after 15 min of incubation at 22 degrees C, and stimulation can be detected after only 5 min of incubation at 37 degrees C. The activation of the enzyme is still apparent after at least 2 h at 0 degrees C. Preincubation with choleragenoid in vitro does not interfere with this effect of the toxin. Animals pretreated by an intravenous injection of cholera toxin do not respond to the in vitro addition of cholera toxin and NAD to the same extent as untreated animals; i.e., the effects overlap to suggest that the in vitro effect is the same as that in vivo. Responses to isoproterenol, glucagon, and NaF were also similar in the in vitro broken cell-activated system, as previously reported for the enzyme activated in vivo.

Mechanism of action of Vibrio cholerae enterotoxin. Effects on adenylate cyclase of toad and rat erythrocyte plasma membranes.

The characteristics of the cholera toxin-stimulated adenylate cyclase of toad (Bufus marinus) and rat erythrocyte plasma membranes have been examined, with special emphasis on the response to purine nucleotides, fluoride, magnesium and catecholamine hormones. Toad erythrocytes briefly exposed to low concentrations of cholera toxin (40,000 to 60,000 molecules per cell) and incubated 2 to 4 hr at 30 degrees C exhibit dramatic alterations in the kinetic and regulatory properties of adenylate cyclase. The approximate Km for ATP, Mg++ increases from about 1.8 to 3.4 mMin the toxin-stimulated enzyme. The stimulation by cholera toxin increases with increasing ATP, Mg++ concentrations, from 20 percent at low levels (0.2 mM) to 500 percent at high concentrations (greater than 3 mM). Addition of GTP, Mg++ (0.2 mM) restores normal kinetic properties to the toxin-modified enzyme, such that stimulation is most simply explained by an elevation of Vmax. GTP enhances the toxin-treated enzyme activity two- to fourfold at low ATP concentrations, but this effect disappears at high levels of the substrate. At 0.6 mM ATP and 5 mM MgC12 the apparent K alpha for GTP, Mg++ is 5 to 10 muM. The control(unstimulated) enzyme demonstrates a very small response to the guanyl nucleotide, 5'-ITP also stimulates the toxin-treated enzyme but cGMP, guanine, and the pyrimidine nucleotides have no effect. Cholera toxin also alters the activation of adenylate cyclase by free Mg++, decreasing the apparent K alpha from about 25 to 5 mM. (minus)-Epinephrine sensitizes the toad erythrocyte adenylate cyclase to GTP and also decreases the apparent K alpha for free metal. Sodium fluoride, which causes a 70- to 100-fold activation of enzyme activity, has little effect on sensitivity to GTP, and does not change the apparent K alpha for Mg++; moreover,it prevents modulation of these parameters by cholera toxin. Conversely, cholera toxin severely inhibits NaF activation, and in the presence of fluoride ion the usual three to fivefold stimulation by toxin becomes a 30 to 60 percent inhibition of activity. The toxin-stimulated enzyme can be further activated by catecholamines; in the presence of GTP the (minus)-epinephrine stimulation is enhanced by two- to threefold. The increased catecholamine stimulation of toad erythrocyte adenylate cyclase induced by cholera toxin is explained primarily by an increase in the maximal extent of activation by the hormones. Rat erythrocyte adenylate cyclase is also modified by cholera toxin. In the mammalian system the apparent affinity for the hormone appears to be increased. Cholera toxin thus induces profound and nearly permanent changes in adenylate cyclase by a unique process which mimics the stimulation by hormones in important ways, and which also accentuates the normal hormonal response. The relevance of these findings to the mechanism of action of cholera toxin is considered.