Proc. Nat. Acad. Sci. USA Vol. 72, No. 9, pp. 3438-3442, September 1975
Biochemistry
Mechanism of activation of adenylate cyclase by cholera toxin (plasma membranes/solubilized adenylate cyclase/immunoprecipitation/multivalent binding/membrane fluidity)
N. SAHYOUN AND P. CUATRECASAS Department of Pharmacology and Experimental Therapeutics, and Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore,
Maryland 21205 Communicated by Victor A. McKusick, July 3, 1975
ABSTRACT Cholera toxin (choleragen) can stimulate adenylate cyclase [EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)] activity in whole particulate fractions or purified plasma membranes of homogenates of isolated fat cells provided special precautions are taken to stabilize the enzyme during the required preincubation period. As observed with intact cells, the activation exhibits a protracted (about 25 min) lag phase, and it is blocked by ganglioside GM; and choleragenoid ("binding" subunit of toxin). The 36,000 molecular weight subunit ("active" subunit),.a hydrophobic polypeptide which does not block choleragen binding or action, can di. rectly activate the enzyme in intact cells without a lag phase. Its effects are not blocked by ganglioside GM; or choleragenoid, yet the stimulated activity exhibits reduced fluoride and enhanced isoproterenol sensitivity, properties characteristic of the choleragen-4ctivated enzyme. Binding of the 1251_ labeled 36,000 molecular weight subunit to cells is not saturable and is unaffected by gangliosides, choleragen, or choleragenoid, and the bound material behaves as an integral membrane protein; this protein may simply artition into the membrane matrix. With increasing time of incubation cellbound choleragen may dissociate into its component subunits, but these remain in the membrane. Using a double antibody imniunoprecipitin system, substantial precipitation of cyclase activity occurs with antisera against the 36,000 molecular weight subunit provided toxin activation has occurred. The normal process of activation may involve an initially inactive toxin-ganglioside complex which, as a result of lateral mobility and multivalent binding (lag phase), results in destabilization of the molecule with release of the "active" subunit into the membrane core where it can spontaneously associate with and perturb the cyclase complex.
Here, we report direct toxin activation of cyclase in particulate cell fractions and isolated plasma membranes by processes and with properties nearly identical to those of intact cells. Further, the "active" subunit* can also activate the cyclase, but by presumably short-circuiting the normal ganglioside binding and membrane rearrangement steps. Also, evidence is presented for the possible subunit dissociation of choleragen on the membrane after binding, and for the di-, rect interaction of the "active" subunit with the cyclase complex.
MATERIALS AND METHODS Cholera toxin was obtained from Dr. C. E. Miller, SEATO research program, choleragenoid and antiserum against 36,000 molecular weight subunit from Dr. R. Finkelstein, antiserum against 27,000 molecular weight polypeptide* from Dr. I. Parikh, and antiserum against cholera toxin from Dr. S. Craig. Iodination with carrier-free 125I has been described (1). Purified fat cell (25) plasma membranes were obtained (26) from the interphase of an 8-40% discontinuous sucrose gradient (1 ml of the particulate fraction, 45 min at 25,000 X g at 00 in an SW 25.1 rotor). The "whole homogenate" was obtained by homogenizing (Polytron) fat cells suspended in 3 volumes of 25 mM Tris-HCl, pH 7.7, 2.5% bovine serum albumin, followed by low-speed centrifugation (1000 X g, 5 min) to remove the "fat cake". Membranes were solubilized by sonication in 0.5% Lubrol PX in ice (8, 10). Adenylate cyclase was assayed (8, 10) for 12 min at 300 in a final volume of 0.1 ml containing 25 mM Tris-HCl, pH 7.7, 0.15 mM ATP, 2 to 5 X 106 cpm of [a-32P]ATP (300700 cpm/pmol) (8, 10), 50 ,gg GTP, 7 mM phosphoenolpyruvate, 6.5 mM MgCl2, 8 mM aminophylline, 50 gg/ml of pyruvate kinase, and 100-300 ,ug of membrane protein. The same ATP-regenerating system was used for assay and preincubation experiments.
Cholera toxin (choleragen) appears to interact initially with cell surface ganglioside GM1 (1-6), and.it stimulates adenylate cyclase [EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)] activity ubiquitously (7-10). The binding of toxin to cells and membranes (1, 6, 8-16), the kinetics of the process of activation (8), and the properties of the stimulated enzyme (8, 17, 18) have been described. Enzyme activation has until now required the exposure and subsequent incubation of intact cells with the toxin. After a characteristic lag phase (20-30 min), the ensuing activation of cyclase persists upon breaking the cells. It has been suggested (1, 8, 10, 12) that after membrane binding the toxin may undergo a rearrangement which leads to approximately stoichiometric interaction of a portion (36,000 molecular weight, "active" subunit)* of the toxin molecule with the cyclase complex by processes essentially independent of cytoplasmic events.
RESULTS
Activation by choleragen in membranes Incubation of the whole particulate fraction or of the sucrose-purified plasma membranes (5-10 mg of protein per ml) of fat cells for 30 min at 300 in 25 mM Tris-HCI, pH 7.7, results in virtually complete loss of the basal enzyme activity, and choleragen has no effect whether it is preincubated (3-120 min) with the membranes or added directly to the
* Studies (ref. 19 and unpublished) on the structure of choleragen, which in the essential features are in harmony with other studies (20-22), indicate the existence of two major components, the "binding" (molecular weight 66,000) and "active" (molecular weight 36,000) subunits. The former is identical to the derivative, choleragenoid (1, 19, 23, 24), which possesses unmodified membrane and ganglioside binding activity, yet is biologically inert (1, 10, 19); thus, it is an antagonist of toxin action. This subunit consists of 8000 molecular weight components (in acid or heating in sodium dodecyl sulfate). The "active" subunit, which is highly hydrophobic, does not compete for toxin binding or activity; it consists of very tightly but not covalently bound 27,000 and 8,000 molecular weight components. Antisera against choleragen crossreact with the "active" and "binding" subunits but not the 27,000 molecular weight component.
3438
Biochemistry: Sahyoun and Cuatrecasas
Proc. Nat. Acad. Sci. USA 72 (1975)
3439
Table 1. Binding of the 125I-labeled 36,000 molecular weight subunit to fat cells cpm 25I bound x 10-3 ± SD
36,000 molecular '25I-labeled ligand
No additions
Ganglioside GM,
Choleragenoid
Choleragen
weight subunit
Choleragen 36,000 Subunit
9.1 ± 0.3 8.0 ± 0.5
2.2 ± 0.2 8.2 ± 0.4
1.5 ± 0.3 9.0 ± 0.4
0.8 ± 0.1 9.6 ± 0.7
5.2 ± 0.5 9.2 ± 0.6
3.5 x 106 cpm of 125I-labeled 36,000 molecular weight (MW) subunit (50 qCi/lg) or labeled choleragen were incubated with fat cells for 10 min at 24°. The cells were washed three times by centrifugation, and 10 Ml of the floating fat cells were counted in triplicate for 1251. GM, was preincubated (30°, 10 min) with the labeled choleragen or 36,000 molecular weight subunit while choleragenoid was preincubated with the cells. Choleragenoid, GM1, the unlabeled chileragen, and 36,000 subunit were added at 100-fold molar excess over the labeled ligand. The inhibition of 125I-labeled choleragen binding by the 36,000 molecular weight subunit probably represents trace residual contamination with choleragenoid.
assay mixture. Also, preincubation at 25°, 300, or 350 in the presence or absence of Mg9l2 (1-10 mM) or CaCl2 (0.5-2 mM), or substituting Krebs-Ringer bicarbonate as the buffer, does not lead to choleragen effects. No choleragen stimulation is elicited by adding 1 mM GTP, NAD, NADH, NADP, NADPH, or dithiothreitol alone or in combination during the preincubation, or by addition of phosphatidylcholine or phosphatidylserine or a 1:1 mixture of both at 1 mg/ml. Performing these experiments on whole cell homogenates does not lead to choleragen stimulation of cyclase of the particulate fraction separated at the end of the preincubation period. However, when 1-5 mM Mg-ATP together with an ATPregenerating system is added during the preincubation of the particulate cell fraction, consistent activation occurs (Fig. 1). The activation occurs after an apparent lag phase of about 25 min. Under these conditions, the phospholipid additions described above increase the activation slightly. After 2 hr at 330 the enzyme retains about 20-25% of its original basal activity and 30-35% of its original response to isoproterenol (10-5 M), which ordinarily disappears within 15-20 min at 330 in the absence of ATP and the regenerating system. Similar "stabilization" of adenylate cyclase has been described elsewhere recently (27, t). In various experiments (as in Fig. 1) the specific activity of cyclase in the particulate or purified membrane preparations declines similarly in the presence or absence of choleragen up to 20-25 min, after which the decline in the presence of choleragen diminishes relative to the control. The specific activity actually increases at about 80 min, but the values attained are not higher than that at zero-time. These effects are blocked by preincubating (10 min, 300) the toxin with a 100-fold molar excess of ganglioside GM1 or by pretreating (10 min, 330) the membranes with excess choleragenoid. * Binding of the 36,000 molecular weight subunit to fat cells Since the l25-Ilabeled 36,000 molecular weight subunit* binds extensively to various filters, all binding experiments were performed by centrifugal flotation of the cells (Table 1). In contrast to choleragen binding, the "binding" of the 36,000 molecular weight subunit is nonsaturable and is not blocked by choleragen, choleragenoid, or ganglioside GM1. Further, its binding is unaffected by unlabeled 36,000 molecular weight subunit. When the particulate fraction prepared from fat cells labeled with 125I-labeled 36,000 molecular weight subunit is subjected to conditions that release "pet S. Jacobs, V. Bennett, and P. Cuatrecasas (1975) submitted to J. Biol. Chem.
ripheral" proteins (28), such as 0.2 M KC1, 1-10 mM EDTA in 25 mM Tris-HCI, pH 7.7, and mild tryptic digestion, not more than 60% of the counts can be released from the membranes. Direct activation by the 36,000 molecular weight subunit The 36,000 molecular weight subunit can activate adenylate cyclase in intact cells, but the characteristic lag period is conspicuously absent (Fig. 2). As in the binding studies, neither ganglioside GM1 nor choleragenoid blocks cyclase activation (Table 2). Moreover, the activated enzyme shows diminished sensitivity to fluoride and potentiation of isoproterenol stimulation (Table 3), properties unique to the choleragen-stimulated enzyme (8, 10, 17). Activation is observed with concentrations of the subunit as low as 70 ng/ml. Dissociation of choleragen subunits after binding to cells In the absence of a direct assay for the dissociation of choleragen after binding to cells or membranes, an indirect approach was utilized. If washed toad erythrocytes are incubated with fat cells that have been exposed to 125I-labeled choleragen (followed by washing), transfer of counts from the fat cells to the erythrozytes occurs to an extent signifi.4'S
80
>-
70
-J U
60-J Z
50
u-
4
0
0
z
o 30
D- 20-
t0 0
20
40
60 80 TIME, min
100
120
140
FIG. 1. Time course of direct activation of adenylate cyclase by choleragen in the particulate membrane fraction of fat cells. The membranes (5-10 mg of protein per ml) were preincubated for various times at 330 in 25 mM Tris-HCI, pH 7.7, containing 2 mM ATP and the ATP-regenerating system with or without choleragen (0.5 ,g/ml). Before assay, the suspension was diluted with 30 volumes of ice-cold buffer and centrifuged at 40,000 X g for 30 min at 0°. Basal activity decreased from about 20 pmol of cAMP produced per min/mg of protein to about 20% of this value after 130 min. Assays were in triplicate, and the % stimulation (relative to the basal activity at each time) was calculated + SEM.
Biochemistry: Sahyoun and CuatrecasasPProc. Nat. Acad. Sci. USA 72 (1975)
3440
' 70 In
60-
0
0)
o 50 z
~,40 0)
-J
30
w
E
-J
-h
o-
r
20-
~~~~~~J 0
10
0.
Z 0
0
20
40
60
100
80
120
140
160
TIME, min
FIG. 2. Direct activation of adenylate cyclase by the 36,000 molecular weight subunit* of choleragen. Cells from 10 fat pads, suspended in 20 ml of Krebs-Ringer bicarbonate (pH 7.4), 2% bovine serum albumin, were incubated with the subunit (5 ,ug/ml) at 370. At various times the cells were washed twice with the same buffer and once with 25 mM Tris.HCl, pH 7.7, 2% albumin, followed by homogenization and preparation of the particulate fraction for assay. The 36,000 molecular weight subunit contained 0.05% Lubrol PX (in 50 mM phosphate buffer, pH 7.5) to maintain its solubility; the final concentration of detergent in the preincubation mixture was less than 0.0001%. Equal concentrations of Lubrol and phosphate were added to the control cells; a slight timedependent increase in activity resulted which was subtracted from the activity in the presence of the subunit.
cantly greater than expected from spontaneous dissociation of the label from the fat cells. This kind ot exchange is presumably mediated by the multivalent nature of toxin binding, as shown by its ability to redistribute (patching and capping) on cell surfaces and to cross-link lymphocytes to ganglioside-agarose beads (12). If cells containing bound 125Ilabeled choleragen are preincubated for 5 min at 370, 30% of the label is exchanged, whereas only about 7% is exchanged if the cells are incubated for 2 hr before measuring the exchange (conditions as in Fig. 3). The major portion (about 80%) of the 125I of iodocholeragen is localized in the 36,000 molecular weight subunit. Table 2. Effect of ganglioside and choleragenoid stimulation of adenylate cyclase by the 36,000 molecular weight subunit
on the
Adenylate cyclase activity*
Ganglioadditions side GM1 No
Membrane preparation Control
Choleragen-treated 36,000 molecular weight subunit-treated
17±2 87 7 ±
30
±
2
19±3 21 ± 3
28
±
1
Choleragenoid
2 gg/ml of the 36,000 molecular weight (MW) subunit
or
3 0.5
Ag/ml of choleragen were incubated with fat cells from six fat pads
for 85 min at 37' as described in the legends of Figs. 1 and 2. Ganglioside GM1 was incubated (30', 10 min) with the 36,000 molecular weight subunit or choleragen before preincubating with cells. The cells were washed and the particulate fraction of the homogenate was assayed. Stimulation by the subunit was calculated as in the legend of Fig. 2. * pmol of cAMP/min per mg of protein, mean of triplicate determinations 4f one standard deviation.
0
After initial separation of the fat cells from the erythrocytes, the small fraction of erythrocytes that remains bound to the fat cells usually sediments after one or two more washes of the fat cells; these contain a disproportionately large fraction of the total counts exchanged. By phase contrast microscopy, the erythrocytes in the pellet are free of fat cells. By sodium dodecyl sulfate disc gel electrophoresis, the proportion of the label in the erythrocytes that corresponds to the "binding" subunit (choleragenoid) increases with increasing length of incubation of the 125I-labeled toxin-fat complex (Fig. 3). The material exchanged during the early phases appears to correspond to the intact toxin while at later times the exchanged "binding" subunit appears to have become dissociated from the 36,000 molecular weight component. Table 3. Effect of fluoride and isoproterenol on adenylate cyclase activity stimulated by 36,000 molecular weight subunit
Adenylate cyclase activity*
±
±
0
FIG. 3. Preferential exchange of the "binding" subunit* of cholera toxin from fat cells to toad erythrocytes with increasing length of incubation of the 1251-labeled toxin-fat cell complex. 1251-Labeled choleragen was added to fat cells and incubated for 10 min at 240. The cells were washed thoroughly in Krebs-Ringer bicarbonate buffer, pH 7.4, 2% albumin, and incubated for various intervals at 370 (cells from 0.5 to 1 fat pad per ml). Packed, washed toad erythrocytes (0.3 ml) were added, and the mixture was shaken slowly at 24° for 10-15 min. The erythrocytes were separated by centrifugation (1500 X g, 5 min, 240), the fat cells were washed twice, and all the sedimented erythrocytes were pooled. The erythrocyte ghosts (8) were solubilized in 0.7% Lubrol PX (8, 10) and aliquots were subjected to 0.1% sodium dodecyl sulfate/polyacrylamide (10%) disc gel electrophoresis (19) under conditions (samples heated 30 min at 600 in 1% sodium dodecyl sulfate) that separate the "active" and "binding" subunits. The gels were sliced and counted. The control reflects the ratio of 125I-label in the active and binding subunits of the native molecule after binding to erythrocytes or fat cells without any cell mixing.
20± 2 22 4
27
o
Membrane
preparation
Control 36,000 molecular weight subunit-treated
No addition
Fluoride
Isoproterenol
16 ± 3
77 ± 6
40 ± 5
32 ± 4
80 ± 6
68 ± 4
Fat cells were incubated with 2 ug/ml of the 36,000 molecular weight (MW) subunit for 100 min at 370, and the enzyme activity of the particulate fraction of the cell homogenates was assayed with 10 mM NaF or 50 AM isoproterenol. * pmol of cAMP per min/mg of protein, mean of triplicate determinations ± one standard deviation.
Biochemistry: Sahyoun and Cuatrecasas
Proc. Nat. Acad. Sci. USA 72 (1975)
3441
Precipitation of activated cyclase by rabbit antisera against toxin followed by goat antiserum against
rabbit y-globulin
It has been shown that small quantities of toxin-stimulated adenylate cyclase can be precipitated with rabbit antiserum against "active" subunit (36,000 molecular weight) (10). When a second antibody (goat antiserum against rabbit yglobulin) is used to facilitate complete precipitation, substantial precipitation of cyclase activity occurs under optimal conditions of temperature and time of incubation (Fig. 4). Furthermore, immunoprecipitation appears to parallel activation by toxin, as indicated by the dependence on the time of preincubation and by the failure of control sera to precipitate significant activity. Quantitative precipitation is complicated by the lability of the enzyme, which limits the length of incubation with antiserum. Of the three types of antisera tested, the order of potency is anti-36,000 molecular weight subunit > anti-27,000 molecular weight subunit > anti-choleragen, while the order of potency for precipitating total radioactivity is anti-choleragen > anti-36,000 molecular weight subunit > anti-27,000 molecular weight subunit. The antisera against choleragen and 36,000 molecular weight subunit crossreact with each other's antigens but not with the 27,000 molecular weight subunit; antisera to the latter do not crossreact with the former antigens. DISCUSSION Previous studies (1, 8, 10) have suggested that toxin activation of adenylate cyclase can occur independently of cytoplasmic processes, despite the fact that heretofore direct stimulation in isolated membrane systems has not been possible. For example, stimulation occurs in the presence of inhibitors of metabolism and of protein, RNA, and prostaglandin synthesis (1, 8). The kinetics of the dependence of enzyme activation on toxin concentration are not consistent with catalytic mechanisms (i.e., one toxin molecule activating increasing numbers of cyclase molecules with time) but suggest instead a bimolecular-type of reaction between membrane-bound toxin molecules and other components, presumably also membrane-bound (8). Further, under conditions where about 2000 molecules of toxin bound per cell (in toad erythrocytes) are needed to achieve half-maximal activation, not more than 50 molecules are present intracellularly (8). The present studies demonstrate directly that intracellular proteins or cofactors are not obligatory requirements for
Cl)
w4
Control Serum (al I times)
5 min
80min
180min
FIG. 4. Immunoprecipitation of adenylate cyclase with antiserum to the 36,000 molecular weight subunit of choleragen. Fat cells were incubated with choleragen at 370 for various periods, the particulate cell fractions were solubilized, and 200-300 jAl were incubated with 50 Ml of immune or normal rabbit serum for 12 min at 30° followed by 2 hr in ice. Goat antiserum against rabbit gamma globulin (200 Il) was added and the mixture incubated at 300 for 5 min and in ice for 1 hr. The precipitate was washed twice with 1 ml of 25 mM Tris.HCl, pH 7.7, suspended in 200 Al, and assayed (50 Ml). 15-20% of the 1251 label was precipitated when 125I-labeled choleragen was used. Activity is in pmol of cAMP produced per 50 Ml/12 min at 370 I SEM.
toxin activation since this can be shown to occur in simple, isolated membrane preparations (Fig. 1). This activation demonstrates the lag phase and sensitivity (blockade) to gangliosides and choleragenoid which characterize toxin action in intact cells. The principal difficulty in demonstrating toxin activation in isolated membranes appears to reside in the inherent instability of the enzyme, since ordinarily the activity in membranes cannot tolerate incubation for even the minimal length (about 30 min) at the temperature known to be required for the. lag phase to pass and for activation to begin in intact cells. Only by special maneuvers designed to stabilize the enzyme, such as by adding ATP and a regenerating system (27, t), can activation of the type seen in intact cells be shown to occur in isolated membranes.
-
t ¶s
FASTj +
SLOW (LAG) t
FAST
.....
"BINDING"
~rOUT~l~lIL-'GANGLIOSIDE 11
_j
0.
rOU
T
IEXPONENTIAL PHASE
"4AITIVE" SUBUNIT SUBUNIT
tiiNO LAG
0 L IN v .. + A
IL EXPONENTIA
_
1
PHASE ACTIVATED
FIG. 5. Diagrammatic summary of postulated sequence of events in the activation of adenylate cyclase (AC) by native choleragen and by the "active" subunit* (see text). The specific mechanism by which the "active" subunit modifies the cyclase complex is unknown and is therefore unspecified. Although the "binding" subunit is fundamentally not an essential component, it serves to give specificity and orientation, to enhance the affinity for the toxin, as well as to provide a specialized water-soluble vehicle for direct delivery of the active molecular species.
3442
Biochemistry: Sahyoun and Cuatrecasas
There is ample evidence that the "binding" subunit (choleragenoid)* alone cannot activate adenylate cyclase (1, 10, 23, 24). On the basis of kinetic and chemical studies (8, 10), it has been suggested that after initial binding, changes occur within the membrane which lead to direct interaction between the "active" subunit* and a component of the cyclase complex, thus determining activation (after the lag phase). Here it is shown that the "active" subunit (solubilized in Lubrol PX) can, at relatively high concentrations, directly activate the cyclase in the absence of the "binding" subunit (Fig. 2). Since the enzyme activated by this subunit exhibits properties known to be unique for the choleragenactivated cyclase [i.e., diminished fluoride and enhanced isoproterenol responses (8, 10, 17)], the activation is probably a genuine reflection of a toxin-like response. The absence of a lag phase and the lack of inhibition by ganglioside GM1, choleragenoid, and choleragen suggest that in this case activation is bypassing the initial steps of the normal sequence of events, i.e., ganglioside binding and the ensuing process responsible for the lag phase. Together with the absence of detectable specific, saturable binding of the 125I-labeled "active" subunit, these observations suggest that this hydrophobic peptide may be passively inserting (i.e., partitioning) into the membrane core, perhaps by a process facilitated by the presence of small quantities of detergent. Although direct activation of cyclase by choleragen and "active" subunit in pigeon erythrocyte ghosts has recently been reported (29), these studies are difficult to assess because the preincubation conditions (temperature, buffer) and activities of the controls are not fully specified, no effects of ganglioside or choleragenoid on toxin activity occurred, no lag phase was observed, and Mg++ (known to be required for adenylate cyclase activity) was apparently not used in the enzyme assay. The fact that antisera against toxin can precipitate the activated but not the native cyclase (Fig. 4, ref. 10) is evidence which, together with other data (10), strongly suggests direct interaction between the toxin and the enzyme complex. Since the "active" subunit alone appears to contain the necessary structural information for enzyme activation, and since much better immunoprecipitation of enzyme activity but not of radioactivity occurs with antisera against the "active" or 27,000 molecular weight subunits than with antiserum against choleragen, it is suggested that after binding and toward the end of the lag phase the surface-bound toxin dissociates in a way that releases the "active" subunit into the hydrophobic core of the membrane. Once in the membrane, this subunit may, by lateral diffusion within the plane of the membrane, encounter, interact with, and thus perturb the cyclase complex. This process, which could in principle explain the exponential phase of activation occurring after the lag phase (8), would be analogous to that occurring when the "active" subunit alone is used for activation. The fact that the erythrocyte exchange experiments (Fig. 3) show that with increasing time of incubation there is increasing, preferential extraction of surface-bound "binding" relative to "active" subunit suggests that the toxin may indeed be dissociating in the manner suggested above. The molecular basis of the lag phase has remained enigmatic. This process is zero order with respect to toxin concentration, suggesting that each molecule is independently undergoing a change that is rate-limiting for the overall activation (8). A possible mechanism for this process is suggested by the recent demonstration (12) that choleragen can bind to cells or gangliosides multivalently, that this can lead to cell surface redistribution (patching and capping) by lateral mo-
Proc. Nat. Acad. Sci. USA 72 (1975)
bility in the plane of the membrane, that this is temperature dependent, and that this mobility may be necessary for cyclase activation. As a result of progressive, multivalent binding or crosslinking with membrane gangliosides, a destabilization or loosening of the toxin molecule may occur which leads to the release and slippage of the "active" subunit into the membrane. The exchange studies (Fig. 3) are also consistent with the "strengthening" of binding of the "binding" subunit with time. An overall mechanism of toxin action that is consistent with all the available data, and that reflects the above considerations, is depicted in Fig. 5. This work was supported by grants from NSF (GB34300), NIH (AM14956), and the Kroc Foundation. N.S. is supported by a Commonwealth Exchange Fellowship Program Grant, and P.C. is a recipient of USPHS RCDA (AM 31464). 1. Cuatrecasas, P. (1973) Biochemistry 12, 3547-3558; 35583566; 3567-3576; 3577-3581. 2. Holmgren, J., Lonnroth, I. & Svennerholm, L. (1973) Scand. J. Infect. Dis. 5,77-78. 3. Holmgren, J., Lonnroth, I. & Svennerholm, L. (1973) Infect. Immun. 8,208-214. 4. van Heyningen, S. (1974) Science 183,656-657. 5. King, C. A. & van Heyningen, W. E. (1973) J. Infect. Dis. 127,639-647. 6. Hollenberg, M. ., Fishman, P., Bennett, V. & Cuatrecasas, P. (1974) Proc. Nat. Acad. Sci. USA 71, 4224-4228. 7. Finkelstein, R. A. (1973) Crit. Rev. Microbiol. 2,553-623. 8. Bennett, V. & Cuatrecasas, P. (1975) J. Memb. Biol. 22, 1-28; 29-52. 9. O'Keefe, E. & Cuatrecasas, P. (1974) Proc. Nat. Acad. Sci. USA 71, 2500-2504. 10. Bennett, V., O'Keefe, E. & Cuatrecasas, P. (1975) Proc. Nat. Acad. Sci. USA 72,33-37. 11. Hollenberg, M. D. & Cuatrecasas, P. (1973) Proc. Nat. Acad. Sct. USA 70,2964-2968. 12. Craig, S. W. & Cuatrecasas, P. (1975) Proc. Nat. Acad. Sci. USA 72, in press. 13. Boyle, J. M. & Gardner, J. D. (1974) J. CGn. Invest. 53, 1149-1158. 14. Holmgren, J., Lindholm, L. & Lonnroth, I. (1974) J. Clhn. Invest. 139, 801-819. 15. Peterson, J. W. & Verivey, W. F. (1974) Proc. Soc. Exp. Biol. Med. 145, 1187-1191. 16. Walker, W. A., Field, M. & Isselbacher, K. J. (1974) Proc. Nat. Acad. Sci. USA 71, 320-324. 17. Field, M. (1974) Proc. Nat. Acad. Sci. USA 71,3299-302. 18. Beckman, B., Flores, J., Witkum, P. A. & Sharp, G. W. G. (1974) J. Clhn. Invest. 53, 1202-1205. 19. Cuatrecasas, P., Parikh, I. & Hollenberg, M. D. (1973) Biochemistry 12, 4253-4264. 20. Lonnroth, I. & Holmgren, J. (1973) J. Gen. Microbiol. 76, 417-427. 21. Holmgren, J. & Lonnroth, I. (1975) J. Gen. Microbiol. 86, 49-65. 22. Finkelstein, R. A., Boesman, M., Neoh, S. H., La Rue, M. K. & Delaney, R. (1974) J. Immunol. 113, 145-150. 23. Finkelstein, R. A., LaRue, M. K. & LoSpalluto, J. J. (1972) J. Infect. Immun. 6,934. 24. Finkelstein, R. A., Boesman, M., Neoh, S. H., La Rue, M. & Delaney, M. (1974) J. Immunol. 113, 145-150. 25. Rodbell, M. (1964) J. Biol. Chem. 239,375-380. 26. Chang, K.-J., Bennett, V. & Cuatrecasas, P. (1975) J. Biol. Chem. 250,488-500. 27. Cuatrecasas, P., Jacobs, S. & Bennett, V. (1975) J. Memb. Biol., in press. 28. Singer, S. J. (1974) Annu. Rev. Biochem. 43,805-833. 29. van Heyningen, S. & King, C. A. (1975) Biochem. J. 146, 269-271.
Biochemistry
Mechanism of activation of adenylate cyclase by cholera toxin (plasma membranes/solubilized adenylate cyclase/immunoprecipitation/multivalent binding/membrane fluidity)
N. SAHYOUN AND P. CUATRECASAS Department of Pharmacology and Experimental Therapeutics, and Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore,
Maryland 21205 Communicated by Victor A. McKusick, July 3, 1975
ABSTRACT Cholera toxin (choleragen) can stimulate adenylate cyclase [EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)] activity in whole particulate fractions or purified plasma membranes of homogenates of isolated fat cells provided special precautions are taken to stabilize the enzyme during the required preincubation period. As observed with intact cells, the activation exhibits a protracted (about 25 min) lag phase, and it is blocked by ganglioside GM; and choleragenoid ("binding" subunit of toxin). The 36,000 molecular weight subunit ("active" subunit),.a hydrophobic polypeptide which does not block choleragen binding or action, can di. rectly activate the enzyme in intact cells without a lag phase. Its effects are not blocked by ganglioside GM; or choleragenoid, yet the stimulated activity exhibits reduced fluoride and enhanced isoproterenol sensitivity, properties characteristic of the choleragen-4ctivated enzyme. Binding of the 1251_ labeled 36,000 molecular weight subunit to cells is not saturable and is unaffected by gangliosides, choleragen, or choleragenoid, and the bound material behaves as an integral membrane protein; this protein may simply artition into the membrane matrix. With increasing time of incubation cellbound choleragen may dissociate into its component subunits, but these remain in the membrane. Using a double antibody imniunoprecipitin system, substantial precipitation of cyclase activity occurs with antisera against the 36,000 molecular weight subunit provided toxin activation has occurred. The normal process of activation may involve an initially inactive toxin-ganglioside complex which, as a result of lateral mobility and multivalent binding (lag phase), results in destabilization of the molecule with release of the "active" subunit into the membrane core where it can spontaneously associate with and perturb the cyclase complex.
Here, we report direct toxin activation of cyclase in particulate cell fractions and isolated plasma membranes by processes and with properties nearly identical to those of intact cells. Further, the "active" subunit* can also activate the cyclase, but by presumably short-circuiting the normal ganglioside binding and membrane rearrangement steps. Also, evidence is presented for the possible subunit dissociation of choleragen on the membrane after binding, and for the di-, rect interaction of the "active" subunit with the cyclase complex.
MATERIALS AND METHODS Cholera toxin was obtained from Dr. C. E. Miller, SEATO research program, choleragenoid and antiserum against 36,000 molecular weight subunit from Dr. R. Finkelstein, antiserum against 27,000 molecular weight polypeptide* from Dr. I. Parikh, and antiserum against cholera toxin from Dr. S. Craig. Iodination with carrier-free 125I has been described (1). Purified fat cell (25) plasma membranes were obtained (26) from the interphase of an 8-40% discontinuous sucrose gradient (1 ml of the particulate fraction, 45 min at 25,000 X g at 00 in an SW 25.1 rotor). The "whole homogenate" was obtained by homogenizing (Polytron) fat cells suspended in 3 volumes of 25 mM Tris-HCl, pH 7.7, 2.5% bovine serum albumin, followed by low-speed centrifugation (1000 X g, 5 min) to remove the "fat cake". Membranes were solubilized by sonication in 0.5% Lubrol PX in ice (8, 10). Adenylate cyclase was assayed (8, 10) for 12 min at 300 in a final volume of 0.1 ml containing 25 mM Tris-HCl, pH 7.7, 0.15 mM ATP, 2 to 5 X 106 cpm of [a-32P]ATP (300700 cpm/pmol) (8, 10), 50 ,gg GTP, 7 mM phosphoenolpyruvate, 6.5 mM MgCl2, 8 mM aminophylline, 50 gg/ml of pyruvate kinase, and 100-300 ,ug of membrane protein. The same ATP-regenerating system was used for assay and preincubation experiments.
Cholera toxin (choleragen) appears to interact initially with cell surface ganglioside GM1 (1-6), and.it stimulates adenylate cyclase [EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)] activity ubiquitously (7-10). The binding of toxin to cells and membranes (1, 6, 8-16), the kinetics of the process of activation (8), and the properties of the stimulated enzyme (8, 17, 18) have been described. Enzyme activation has until now required the exposure and subsequent incubation of intact cells with the toxin. After a characteristic lag phase (20-30 min), the ensuing activation of cyclase persists upon breaking the cells. It has been suggested (1, 8, 10, 12) that after membrane binding the toxin may undergo a rearrangement which leads to approximately stoichiometric interaction of a portion (36,000 molecular weight, "active" subunit)* of the toxin molecule with the cyclase complex by processes essentially independent of cytoplasmic events.
RESULTS
Activation by choleragen in membranes Incubation of the whole particulate fraction or of the sucrose-purified plasma membranes (5-10 mg of protein per ml) of fat cells for 30 min at 300 in 25 mM Tris-HCI, pH 7.7, results in virtually complete loss of the basal enzyme activity, and choleragen has no effect whether it is preincubated (3-120 min) with the membranes or added directly to the
* Studies (ref. 19 and unpublished) on the structure of choleragen, which in the essential features are in harmony with other studies (20-22), indicate the existence of two major components, the "binding" (molecular weight 66,000) and "active" (molecular weight 36,000) subunits. The former is identical to the derivative, choleragenoid (1, 19, 23, 24), which possesses unmodified membrane and ganglioside binding activity, yet is biologically inert (1, 10, 19); thus, it is an antagonist of toxin action. This subunit consists of 8000 molecular weight components (in acid or heating in sodium dodecyl sulfate). The "active" subunit, which is highly hydrophobic, does not compete for toxin binding or activity; it consists of very tightly but not covalently bound 27,000 and 8,000 molecular weight components. Antisera against choleragen crossreact with the "active" and "binding" subunits but not the 27,000 molecular weight component.
3438
Biochemistry: Sahyoun and Cuatrecasas
Proc. Nat. Acad. Sci. USA 72 (1975)
3439
Table 1. Binding of the 125I-labeled 36,000 molecular weight subunit to fat cells cpm 25I bound x 10-3 ± SD
36,000 molecular '25I-labeled ligand
No additions
Ganglioside GM,
Choleragenoid
Choleragen
weight subunit
Choleragen 36,000 Subunit
9.1 ± 0.3 8.0 ± 0.5
2.2 ± 0.2 8.2 ± 0.4
1.5 ± 0.3 9.0 ± 0.4
0.8 ± 0.1 9.6 ± 0.7
5.2 ± 0.5 9.2 ± 0.6
3.5 x 106 cpm of 125I-labeled 36,000 molecular weight (MW) subunit (50 qCi/lg) or labeled choleragen were incubated with fat cells for 10 min at 24°. The cells were washed three times by centrifugation, and 10 Ml of the floating fat cells were counted in triplicate for 1251. GM, was preincubated (30°, 10 min) with the labeled choleragen or 36,000 molecular weight subunit while choleragenoid was preincubated with the cells. Choleragenoid, GM1, the unlabeled chileragen, and 36,000 subunit were added at 100-fold molar excess over the labeled ligand. The inhibition of 125I-labeled choleragen binding by the 36,000 molecular weight subunit probably represents trace residual contamination with choleragenoid.
assay mixture. Also, preincubation at 25°, 300, or 350 in the presence or absence of Mg9l2 (1-10 mM) or CaCl2 (0.5-2 mM), or substituting Krebs-Ringer bicarbonate as the buffer, does not lead to choleragen effects. No choleragen stimulation is elicited by adding 1 mM GTP, NAD, NADH, NADP, NADPH, or dithiothreitol alone or in combination during the preincubation, or by addition of phosphatidylcholine or phosphatidylserine or a 1:1 mixture of both at 1 mg/ml. Performing these experiments on whole cell homogenates does not lead to choleragen stimulation of cyclase of the particulate fraction separated at the end of the preincubation period. However, when 1-5 mM Mg-ATP together with an ATPregenerating system is added during the preincubation of the particulate cell fraction, consistent activation occurs (Fig. 1). The activation occurs after an apparent lag phase of about 25 min. Under these conditions, the phospholipid additions described above increase the activation slightly. After 2 hr at 330 the enzyme retains about 20-25% of its original basal activity and 30-35% of its original response to isoproterenol (10-5 M), which ordinarily disappears within 15-20 min at 330 in the absence of ATP and the regenerating system. Similar "stabilization" of adenylate cyclase has been described elsewhere recently (27, t). In various experiments (as in Fig. 1) the specific activity of cyclase in the particulate or purified membrane preparations declines similarly in the presence or absence of choleragen up to 20-25 min, after which the decline in the presence of choleragen diminishes relative to the control. The specific activity actually increases at about 80 min, but the values attained are not higher than that at zero-time. These effects are blocked by preincubating (10 min, 300) the toxin with a 100-fold molar excess of ganglioside GM1 or by pretreating (10 min, 330) the membranes with excess choleragenoid. * Binding of the 36,000 molecular weight subunit to fat cells Since the l25-Ilabeled 36,000 molecular weight subunit* binds extensively to various filters, all binding experiments were performed by centrifugal flotation of the cells (Table 1). In contrast to choleragen binding, the "binding" of the 36,000 molecular weight subunit is nonsaturable and is not blocked by choleragen, choleragenoid, or ganglioside GM1. Further, its binding is unaffected by unlabeled 36,000 molecular weight subunit. When the particulate fraction prepared from fat cells labeled with 125I-labeled 36,000 molecular weight subunit is subjected to conditions that release "pet S. Jacobs, V. Bennett, and P. Cuatrecasas (1975) submitted to J. Biol. Chem.
ripheral" proteins (28), such as 0.2 M KC1, 1-10 mM EDTA in 25 mM Tris-HCI, pH 7.7, and mild tryptic digestion, not more than 60% of the counts can be released from the membranes. Direct activation by the 36,000 molecular weight subunit The 36,000 molecular weight subunit can activate adenylate cyclase in intact cells, but the characteristic lag period is conspicuously absent (Fig. 2). As in the binding studies, neither ganglioside GM1 nor choleragenoid blocks cyclase activation (Table 2). Moreover, the activated enzyme shows diminished sensitivity to fluoride and potentiation of isoproterenol stimulation (Table 3), properties unique to the choleragen-stimulated enzyme (8, 10, 17). Activation is observed with concentrations of the subunit as low as 70 ng/ml. Dissociation of choleragen subunits after binding to cells In the absence of a direct assay for the dissociation of choleragen after binding to cells or membranes, an indirect approach was utilized. If washed toad erythrocytes are incubated with fat cells that have been exposed to 125I-labeled choleragen (followed by washing), transfer of counts from the fat cells to the erythrozytes occurs to an extent signifi.4'S
80
>-
70
-J U
60-J Z
50
u-
4
0
0
z
o 30
D- 20-
t0 0
20
40
60 80 TIME, min
100
120
140
FIG. 1. Time course of direct activation of adenylate cyclase by choleragen in the particulate membrane fraction of fat cells. The membranes (5-10 mg of protein per ml) were preincubated for various times at 330 in 25 mM Tris-HCI, pH 7.7, containing 2 mM ATP and the ATP-regenerating system with or without choleragen (0.5 ,g/ml). Before assay, the suspension was diluted with 30 volumes of ice-cold buffer and centrifuged at 40,000 X g for 30 min at 0°. Basal activity decreased from about 20 pmol of cAMP produced per min/mg of protein to about 20% of this value after 130 min. Assays were in triplicate, and the % stimulation (relative to the basal activity at each time) was calculated + SEM.
Biochemistry: Sahyoun and CuatrecasasPProc. Nat. Acad. Sci. USA 72 (1975)
3440
' 70 In
60-
0
0)
o 50 z
~,40 0)
-J
30
w
E
-J
-h
o-
r
20-
~~~~~~J 0
10
0.
Z 0
0
20
40
60
100
80
120
140
160
TIME, min
FIG. 2. Direct activation of adenylate cyclase by the 36,000 molecular weight subunit* of choleragen. Cells from 10 fat pads, suspended in 20 ml of Krebs-Ringer bicarbonate (pH 7.4), 2% bovine serum albumin, were incubated with the subunit (5 ,ug/ml) at 370. At various times the cells were washed twice with the same buffer and once with 25 mM Tris.HCl, pH 7.7, 2% albumin, followed by homogenization and preparation of the particulate fraction for assay. The 36,000 molecular weight subunit contained 0.05% Lubrol PX (in 50 mM phosphate buffer, pH 7.5) to maintain its solubility; the final concentration of detergent in the preincubation mixture was less than 0.0001%. Equal concentrations of Lubrol and phosphate were added to the control cells; a slight timedependent increase in activity resulted which was subtracted from the activity in the presence of the subunit.
cantly greater than expected from spontaneous dissociation of the label from the fat cells. This kind ot exchange is presumably mediated by the multivalent nature of toxin binding, as shown by its ability to redistribute (patching and capping) on cell surfaces and to cross-link lymphocytes to ganglioside-agarose beads (12). If cells containing bound 125Ilabeled choleragen are preincubated for 5 min at 370, 30% of the label is exchanged, whereas only about 7% is exchanged if the cells are incubated for 2 hr before measuring the exchange (conditions as in Fig. 3). The major portion (about 80%) of the 125I of iodocholeragen is localized in the 36,000 molecular weight subunit. Table 2. Effect of ganglioside and choleragenoid stimulation of adenylate cyclase by the 36,000 molecular weight subunit
on the
Adenylate cyclase activity*
Ganglioadditions side GM1 No
Membrane preparation Control
Choleragen-treated 36,000 molecular weight subunit-treated
17±2 87 7 ±
30
±
2
19±3 21 ± 3
28
±
1
Choleragenoid
2 gg/ml of the 36,000 molecular weight (MW) subunit
or
3 0.5
Ag/ml of choleragen were incubated with fat cells from six fat pads
for 85 min at 37' as described in the legends of Figs. 1 and 2. Ganglioside GM1 was incubated (30', 10 min) with the 36,000 molecular weight subunit or choleragen before preincubating with cells. The cells were washed and the particulate fraction of the homogenate was assayed. Stimulation by the subunit was calculated as in the legend of Fig. 2. * pmol of cAMP/min per mg of protein, mean of triplicate determinations 4f one standard deviation.
0
After initial separation of the fat cells from the erythrocytes, the small fraction of erythrocytes that remains bound to the fat cells usually sediments after one or two more washes of the fat cells; these contain a disproportionately large fraction of the total counts exchanged. By phase contrast microscopy, the erythrocytes in the pellet are free of fat cells. By sodium dodecyl sulfate disc gel electrophoresis, the proportion of the label in the erythrocytes that corresponds to the "binding" subunit (choleragenoid) increases with increasing length of incubation of the 125I-labeled toxin-fat complex (Fig. 3). The material exchanged during the early phases appears to correspond to the intact toxin while at later times the exchanged "binding" subunit appears to have become dissociated from the 36,000 molecular weight component. Table 3. Effect of fluoride and isoproterenol on adenylate cyclase activity stimulated by 36,000 molecular weight subunit
Adenylate cyclase activity*
±
±
0
FIG. 3. Preferential exchange of the "binding" subunit* of cholera toxin from fat cells to toad erythrocytes with increasing length of incubation of the 1251-labeled toxin-fat cell complex. 1251-Labeled choleragen was added to fat cells and incubated for 10 min at 240. The cells were washed thoroughly in Krebs-Ringer bicarbonate buffer, pH 7.4, 2% albumin, and incubated for various intervals at 370 (cells from 0.5 to 1 fat pad per ml). Packed, washed toad erythrocytes (0.3 ml) were added, and the mixture was shaken slowly at 24° for 10-15 min. The erythrocytes were separated by centrifugation (1500 X g, 5 min, 240), the fat cells were washed twice, and all the sedimented erythrocytes were pooled. The erythrocyte ghosts (8) were solubilized in 0.7% Lubrol PX (8, 10) and aliquots were subjected to 0.1% sodium dodecyl sulfate/polyacrylamide (10%) disc gel electrophoresis (19) under conditions (samples heated 30 min at 600 in 1% sodium dodecyl sulfate) that separate the "active" and "binding" subunits. The gels were sliced and counted. The control reflects the ratio of 125I-label in the active and binding subunits of the native molecule after binding to erythrocytes or fat cells without any cell mixing.
20± 2 22 4
27
o
Membrane
preparation
Control 36,000 molecular weight subunit-treated
No addition
Fluoride
Isoproterenol
16 ± 3
77 ± 6
40 ± 5
32 ± 4
80 ± 6
68 ± 4
Fat cells were incubated with 2 ug/ml of the 36,000 molecular weight (MW) subunit for 100 min at 370, and the enzyme activity of the particulate fraction of the cell homogenates was assayed with 10 mM NaF or 50 AM isoproterenol. * pmol of cAMP per min/mg of protein, mean of triplicate determinations ± one standard deviation.
Biochemistry: Sahyoun and Cuatrecasas
Proc. Nat. Acad. Sci. USA 72 (1975)
3441
Precipitation of activated cyclase by rabbit antisera against toxin followed by goat antiserum against
rabbit y-globulin
It has been shown that small quantities of toxin-stimulated adenylate cyclase can be precipitated with rabbit antiserum against "active" subunit (36,000 molecular weight) (10). When a second antibody (goat antiserum against rabbit yglobulin) is used to facilitate complete precipitation, substantial precipitation of cyclase activity occurs under optimal conditions of temperature and time of incubation (Fig. 4). Furthermore, immunoprecipitation appears to parallel activation by toxin, as indicated by the dependence on the time of preincubation and by the failure of control sera to precipitate significant activity. Quantitative precipitation is complicated by the lability of the enzyme, which limits the length of incubation with antiserum. Of the three types of antisera tested, the order of potency is anti-36,000 molecular weight subunit > anti-27,000 molecular weight subunit > anti-choleragen, while the order of potency for precipitating total radioactivity is anti-choleragen > anti-36,000 molecular weight subunit > anti-27,000 molecular weight subunit. The antisera against choleragen and 36,000 molecular weight subunit crossreact with each other's antigens but not with the 27,000 molecular weight subunit; antisera to the latter do not crossreact with the former antigens. DISCUSSION Previous studies (1, 8, 10) have suggested that toxin activation of adenylate cyclase can occur independently of cytoplasmic processes, despite the fact that heretofore direct stimulation in isolated membrane systems has not been possible. For example, stimulation occurs in the presence of inhibitors of metabolism and of protein, RNA, and prostaglandin synthesis (1, 8). The kinetics of the dependence of enzyme activation on toxin concentration are not consistent with catalytic mechanisms (i.e., one toxin molecule activating increasing numbers of cyclase molecules with time) but suggest instead a bimolecular-type of reaction between membrane-bound toxin molecules and other components, presumably also membrane-bound (8). Further, under conditions where about 2000 molecules of toxin bound per cell (in toad erythrocytes) are needed to achieve half-maximal activation, not more than 50 molecules are present intracellularly (8). The present studies demonstrate directly that intracellular proteins or cofactors are not obligatory requirements for
Cl)
w4
Control Serum (al I times)
5 min
80min
180min
FIG. 4. Immunoprecipitation of adenylate cyclase with antiserum to the 36,000 molecular weight subunit of choleragen. Fat cells were incubated with choleragen at 370 for various periods, the particulate cell fractions were solubilized, and 200-300 jAl were incubated with 50 Ml of immune or normal rabbit serum for 12 min at 30° followed by 2 hr in ice. Goat antiserum against rabbit gamma globulin (200 Il) was added and the mixture incubated at 300 for 5 min and in ice for 1 hr. The precipitate was washed twice with 1 ml of 25 mM Tris.HCl, pH 7.7, suspended in 200 Al, and assayed (50 Ml). 15-20% of the 1251 label was precipitated when 125I-labeled choleragen was used. Activity is in pmol of cAMP produced per 50 Ml/12 min at 370 I SEM.
toxin activation since this can be shown to occur in simple, isolated membrane preparations (Fig. 1). This activation demonstrates the lag phase and sensitivity (blockade) to gangliosides and choleragenoid which characterize toxin action in intact cells. The principal difficulty in demonstrating toxin activation in isolated membranes appears to reside in the inherent instability of the enzyme, since ordinarily the activity in membranes cannot tolerate incubation for even the minimal length (about 30 min) at the temperature known to be required for the. lag phase to pass and for activation to begin in intact cells. Only by special maneuvers designed to stabilize the enzyme, such as by adding ATP and a regenerating system (27, t), can activation of the type seen in intact cells be shown to occur in isolated membranes.
-
t ¶s
FASTj +
SLOW (LAG) t
FAST
.....
"BINDING"
~rOUT~l~lIL-'GANGLIOSIDE 11
_j
0.
rOU
T
IEXPONENTIAL PHASE
"4AITIVE" SUBUNIT SUBUNIT
tiiNO LAG
0 L IN v .. + A
IL EXPONENTIA
_
1
PHASE ACTIVATED
FIG. 5. Diagrammatic summary of postulated sequence of events in the activation of adenylate cyclase (AC) by native choleragen and by the "active" subunit* (see text). The specific mechanism by which the "active" subunit modifies the cyclase complex is unknown and is therefore unspecified. Although the "binding" subunit is fundamentally not an essential component, it serves to give specificity and orientation, to enhance the affinity for the toxin, as well as to provide a specialized water-soluble vehicle for direct delivery of the active molecular species.
3442
Biochemistry: Sahyoun and Cuatrecasas
There is ample evidence that the "binding" subunit (choleragenoid)* alone cannot activate adenylate cyclase (1, 10, 23, 24). On the basis of kinetic and chemical studies (8, 10), it has been suggested that after initial binding, changes occur within the membrane which lead to direct interaction between the "active" subunit* and a component of the cyclase complex, thus determining activation (after the lag phase). Here it is shown that the "active" subunit (solubilized in Lubrol PX) can, at relatively high concentrations, directly activate the cyclase in the absence of the "binding" subunit (Fig. 2). Since the enzyme activated by this subunit exhibits properties known to be unique for the choleragenactivated cyclase [i.e., diminished fluoride and enhanced isoproterenol responses (8, 10, 17)], the activation is probably a genuine reflection of a toxin-like response. The absence of a lag phase and the lack of inhibition by ganglioside GM1, choleragenoid, and choleragen suggest that in this case activation is bypassing the initial steps of the normal sequence of events, i.e., ganglioside binding and the ensuing process responsible for the lag phase. Together with the absence of detectable specific, saturable binding of the 125I-labeled "active" subunit, these observations suggest that this hydrophobic peptide may be passively inserting (i.e., partitioning) into the membrane core, perhaps by a process facilitated by the presence of small quantities of detergent. Although direct activation of cyclase by choleragen and "active" subunit in pigeon erythrocyte ghosts has recently been reported (29), these studies are difficult to assess because the preincubation conditions (temperature, buffer) and activities of the controls are not fully specified, no effects of ganglioside or choleragenoid on toxin activity occurred, no lag phase was observed, and Mg++ (known to be required for adenylate cyclase activity) was apparently not used in the enzyme assay. The fact that antisera against toxin can precipitate the activated but not the native cyclase (Fig. 4, ref. 10) is evidence which, together with other data (10), strongly suggests direct interaction between the toxin and the enzyme complex. Since the "active" subunit alone appears to contain the necessary structural information for enzyme activation, and since much better immunoprecipitation of enzyme activity but not of radioactivity occurs with antisera against the "active" or 27,000 molecular weight subunits than with antiserum against choleragen, it is suggested that after binding and toward the end of the lag phase the surface-bound toxin dissociates in a way that releases the "active" subunit into the hydrophobic core of the membrane. Once in the membrane, this subunit may, by lateral diffusion within the plane of the membrane, encounter, interact with, and thus perturb the cyclase complex. This process, which could in principle explain the exponential phase of activation occurring after the lag phase (8), would be analogous to that occurring when the "active" subunit alone is used for activation. The fact that the erythrocyte exchange experiments (Fig. 3) show that with increasing time of incubation there is increasing, preferential extraction of surface-bound "binding" relative to "active" subunit suggests that the toxin may indeed be dissociating in the manner suggested above. The molecular basis of the lag phase has remained enigmatic. This process is zero order with respect to toxin concentration, suggesting that each molecule is independently undergoing a change that is rate-limiting for the overall activation (8). A possible mechanism for this process is suggested by the recent demonstration (12) that choleragen can bind to cells or gangliosides multivalently, that this can lead to cell surface redistribution (patching and capping) by lateral mo-
Proc. Nat. Acad. Sci. USA 72 (1975)
bility in the plane of the membrane, that this is temperature dependent, and that this mobility may be necessary for cyclase activation. As a result of progressive, multivalent binding or crosslinking with membrane gangliosides, a destabilization or loosening of the toxin molecule may occur which leads to the release and slippage of the "active" subunit into the membrane. The exchange studies (Fig. 3) are also consistent with the "strengthening" of binding of the "binding" subunit with time. An overall mechanism of toxin action that is consistent with all the available data, and that reflects the above considerations, is depicted in Fig. 5. This work was supported by grants from NSF (GB34300), NIH (AM14956), and the Kroc Foundation. N.S. is supported by a Commonwealth Exchange Fellowship Program Grant, and P.C. is a recipient of USPHS RCDA (AM 31464). 1. Cuatrecasas, P. (1973) Biochemistry 12, 3547-3558; 35583566; 3567-3576; 3577-3581. 2. Holmgren, J., Lonnroth, I. & Svennerholm, L. (1973) Scand. J. Infect. Dis. 5,77-78. 3. Holmgren, J., Lonnroth, I. & Svennerholm, L. (1973) Infect. Immun. 8,208-214. 4. van Heyningen, S. (1974) Science 183,656-657. 5. King, C. A. & van Heyningen, W. E. (1973) J. Infect. Dis. 127,639-647. 6. Hollenberg, M. ., Fishman, P., Bennett, V. & Cuatrecasas, P. (1974) Proc. Nat. Acad. Sci. USA 71, 4224-4228. 7. Finkelstein, R. A. (1973) Crit. Rev. Microbiol. 2,553-623. 8. Bennett, V. & Cuatrecasas, P. (1975) J. Memb. Biol. 22, 1-28; 29-52. 9. O'Keefe, E. & Cuatrecasas, P. (1974) Proc. Nat. Acad. Sci. USA 71, 2500-2504. 10. Bennett, V., O'Keefe, E. & Cuatrecasas, P. (1975) Proc. Nat. Acad. Sci. USA 72,33-37. 11. Hollenberg, M. D. & Cuatrecasas, P. (1973) Proc. Nat. Acad. Sct. USA 70,2964-2968. 12. Craig, S. W. & Cuatrecasas, P. (1975) Proc. Nat. Acad. Sci. USA 72, in press. 13. Boyle, J. M. & Gardner, J. D. (1974) J. CGn. Invest. 53, 1149-1158. 14. Holmgren, J., Lindholm, L. & Lonnroth, I. (1974) J. Clhn. Invest. 139, 801-819. 15. Peterson, J. W. & Verivey, W. F. (1974) Proc. Soc. Exp. Biol. Med. 145, 1187-1191. 16. Walker, W. A., Field, M. & Isselbacher, K. J. (1974) Proc. Nat. Acad. Sci. USA 71, 320-324. 17. Field, M. (1974) Proc. Nat. Acad. Sci. USA 71,3299-302. 18. Beckman, B., Flores, J., Witkum, P. A. & Sharp, G. W. G. (1974) J. Clhn. Invest. 53, 1202-1205. 19. Cuatrecasas, P., Parikh, I. & Hollenberg, M. D. (1973) Biochemistry 12, 4253-4264. 20. Lonnroth, I. & Holmgren, J. (1973) J. Gen. Microbiol. 76, 417-427. 21. Holmgren, J. & Lonnroth, I. (1975) J. Gen. Microbiol. 86, 49-65. 22. Finkelstein, R. A., Boesman, M., Neoh, S. H., La Rue, M. K. & Delaney, R. (1974) J. Immunol. 113, 145-150. 23. Finkelstein, R. A., LaRue, M. K. & LoSpalluto, J. J. (1972) J. Infect. Immun. 6,934. 24. Finkelstein, R. A., Boesman, M., Neoh, S. H., La Rue, M. & Delaney, M. (1974) J. Immunol. 113, 145-150. 25. Rodbell, M. (1964) J. Biol. Chem. 239,375-380. 26. Chang, K.-J., Bennett, V. & Cuatrecasas, P. (1975) J. Biol. Chem. 250,488-500. 27. Cuatrecasas, P., Jacobs, S. & Bennett, V. (1975) J. Memb. Biol., in press. 28. Singer, S. J. (1974) Annu. Rev. Biochem. 43,805-833. 29. van Heyningen, S. & King, C. A. (1975) Biochem. J. 146, 269-271.
