Altered Guanine Nucleotide Hydrolysis as Basis for Increased Adenylate Cyclase Activity after Cholera Toxin Treatment

(Received for publication, September 30, 1976)

SUSAN L. LEVINSON* AND ARTHUR J. BLUME$

From the Department ofPh,ysiological Chemist?-y, Roche Institute ofMolecular Biolog,y, Nutley, New dersen, 07110

Cholera toxin activation of the adenylate cyclase of mouse neuroblastoma cells occurs in situ with intact cells or in uitro with cell membranes. The in vitro activation process requires X41) and nucleotide triphosphates, in addition to toxin. The cholera toxin-activated adenylate cyclase, when assayed at physiological concentrations of M&I, (10 mnl) in assay systems containing ATP and a nucleotide-regenerat- ing system, has a very high activity and is not significantly activated by 2-chloroadenosine, prostaglandin E,, guanyl-5’- yl imidodiphosphate, or NaF. Under similar assay condi- tions, the activity of the normal enzyme is low, and maximal activity requires the above activators. The normal enzyme after activation by guanyl-5’-yl imidodiphosphate closely resembles the toxin-activated enzyme when assayed at 10 ml1 MgCl,. However, these two enzymes can be distin- guished from each other. When the M&I, concentration exceeds 20 mM, the toxin-activated enzyme, but not the guanyl-5’-yl imidodiphosphate-activated enzyme, again re- quires exogenously added guanine nucleotide triphosphates, NaF, or prostaglandin E, for maximal activity.

Ry using adenyl-j’-yl imidodiphosphate as substrate (in

assays with and without a nucleotide-regenerating system) it was found that the high activity of the toxin-activated enzyme observed at 10 ml1 M&l, requires the presence of guanine nucleotide triphosphates. GTP or guanyl-5’-yl imi- dodiphosphate can fulfill this requirement. The source of the endogenous guanine nucleotides in the assays which contain 4TP and a regenerating system appears to be the membrane preparation itself. The normal enzyme can only use these endogenous guanine nucleotides as activators in the presence of 2-chloroadenosine or prostaglandin E,. 2- Chloroadenosine and prostaglandin E, do not activate the normal enzyme in the absence of guanine nucleotides. Anal- ysis of the interaction of guanyl-5’-yl imidodiphosphate and GDP with the normal and toxin-activated enzyme reveals no significant differences between the two enzymes.

We propose that at physiological concentrations of MgCl, the toxin-activated enzyme is “fixed” in a highly active state (E-GTP). The normal enzyme decays from such a GTP state

rapidly at physiological concentrations of MgCI,. Activation of the enzyme by cholera toxin results in a reduction or prevention of this decay mechanism. A general model of

* Present address, USV Pharmaceutical Corporation, Department of Biochemistry, Tuckahoe, New York 10707.

$ To whom all correspondence should be addressed.

control of the activity of adenylate cyclase by guanine nu- cleotides, 2-chloroadenosinc, prostaglandin E,, as well as other enzyme activators, is presented.

Cholera toxin, an enterotoxin produced by Vihrio cholerae, produces its diarrheaegenic effect by activating the adenylate cyclase (EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)) of the small intestine (l-5). The original suggestion that adenyl- ate cylase was activated by cholera toxin was made by Michael Field (5). Cholera toxin has been observed to stimulate adenyl- ate cyclase in a wide variety of intact cells and tissues to which

it has been added (see review, Ref. 6). Based on the work of King and Van Heyningen (7) and confirmed by others (8-131, such a wide range of effectiveness appears to be due to the fact that the primary recognition site on the cell for cholera toxin is the ubiquitous G,,, ganglioside in mammalian plasma mem- branes.

The CAMP concentration of tissues or isolated cells can be increased by specific hormones or cholera toxin (1, 14-16). When the adenylate cyclase from toxin-treated cells is assayed in uitro, it exhibits greatly elevated activities in the absence of any hormonal stimulators (17-24). Many of these toxin-acti-

vated enzymes have been found to show altered responses to hormones, guanine nucleotides, and MgCl, (17, 19, 24). How- ever, it remains unclear which, if any, of these alterations are responsible for the increased activity displayed by these en- zymes.

The mechanisms regulating the activity of the normal aden- ylate cyclase have also been difficult to elucidate. We have reported that increases in the CAMP concentration in intact mouse neuroblastoma cells can be elicited by adenosine and prostaglandin E, (25). The normal adenylate cyclase of these cells, when assayed in uitro, is activated by adenosine, certain adenosine analogues, i.e. 2-chloroadenosine (261, PGE,’ (271, guanyl-5’-yl imidodiphosphate (28), and NaF (27, 28). Based on the in vitro effects of various combinations of the above effecters, we have proposed that guanine nucleotides (GTP and GDP) are the primary regulators of the activity of adenyl- ate cyclase. PGE, and adenosine were considered secondary enzyme regulators since they were found to control the pri- mary regulatory process (28). Efforts to solubilize an unaltered

’ The abbreviations used are: PGE,, prostaglandin E,; ClAdo, Z- chloroadenosine; GMP-P(NH)P, guanyl-5’.yl imidodiphosphate; AMP-P(NH)P, adenyl-5’.yl imidodiphosphate.

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Cholera Toxin Modification of Adenylate Cyclase

enzyme from the plasma membranes of various cells, includ- ing the neuroblastoma cells, have not been successful. Because of the limitation of working with the normal adenylate cyclase in its membrane environment, a new approach was sought to confirm and further extend the previous novel proposals for regulation of adenylate cyclase. The studies reported here, on the characterization of the toxin-activated adenylate cyclase from mouse neuroblastoma cells, confirm the primary role of guanine nucleotides in regulation and suggest that cholera toxin alters this primary recognition process.

MATERIALS AND METHODS

Growth of Cells -All studies were performed on clonal line NS20 of mouse neuroblastoma Cl300 (29). Cells were maintained as stock cultures in Falcon flasks and grown in glass roller bottles for large preparations (26). The growth media was Dulbecco’s Modified Ea- gle’s Medium supplemented with 10% horse serum, penicillin G (10 unWml1, and streptomycin sulfate (10 Fg/mll. Cells were treated with cholera toxin by replacing the growth medium of confluent cells with an equal volume of serum-free medium containing 20 pg/ml of cholera toxin and incubating at 37” for various times up to 18 h (see text).

Preparations ofAdenylate Cyclase -Cells were harvested by shak- ing without the use of trypsin and were washed and homogenized as previously described to produce a crude homogenate (26). Unless otherwise indicated, a particulate fraction was isolated from the crude homogenate by centrifugation at 600 x g for 10 min at 4”, followed by centrifugation of this supernatant at 37,000 x g for 30 min at 4”. The resultant pellet was resuspended in 10 mM Tris/ maleate buffer, pH 7.4, and could be frozen and stored in liquid nitrogen without loss of activity for at least 6 months. The enzyme prepared from cells which were not treated with toxin will be re- ferred to as normal enzyme. Toxin-activated enzyme will refer to the enzyme prepared from cells which were incubated with 20 *g/ml of toxin for 18 h, unless noted otherwise.

Adenylate Cyclase Assays - Production of CAMP was assayed at 30” using either [a~‘“P]ATP or [&‘*PlAMP-P(NH)P according to the method of Salomon et al. (30) with modifications as previously de- scribed (26). Unless otherwise specified in the text, assays with ATP were performed with 50 mM Trislmaleate buffer, pH 7.4, 1 rnM ATP, 6 mM MgCl*, 0.5 mM CAMP, 0.3 mM R020-1724 (CAMP phosphodies- terase inhibitor), and an ATP-regenerating system consisting of 20 mM creatine phosphate and 0.1 mglml of creatine phosphokinase. Assays with AMP-P(NH)P were as above but do not contain a regenerating system (unless specified in text) and contain 0.2 mM AMP-P(NH)P. Proteins were determined by the method of Lowry et al. (31). All specific activities are reported as picomoles of CAMP produced/min/mg of protein. All activity values, unless otherwise specified, were averages of duplicate determinations, varying by less than 15%.

Materials -Cholera toxin was purchased from Schwarz/Mann. ATP, GDP, GTP, creatine phosphokinase, creatine phosphate, and ClAdo were purchased from Sigma Chemical Co. GMP-P(NH)P and AMP-P(NH)P were purchased from P-L Biochemicals, Inc. [a- “PIATP was obtained from ICN Corp. (50 to 100 Ci/mmol) and New England Nuclear (20 to 25 Ci/mmol). la-:“P]AMP-P(NH)P (7 to 12 Ci/ mmol) was purchased from ICN Corp. R020-1724 and PGE, were gifts from Hoffmann-La Roche, Inc., Nutley, N. J.

RESULTS

Activation of Adenylate Cyclase in Situ by Cholera Toxin -

Incubation of intact NS20 with cholera toxin leads to increased “basal” adenylate cyclase (e.g. activity measured with homog- enates at 10 mM Mg*+ in the absence of stimulators (Fig. 1)). The effects of the various known enzyme activators on the activity of the toxin-activated enzyme and normal enzyme have been compared (Table I). The activity of the normal adenylate cyclase is elevated by ClAdo, PGE,, GMP-P(NHlP, and NaF. None of these compounds significantly elevates the activity of the enzyme isolated from cells treated with toxin for 18 h. The activity of the toxin-activated enzyme is equal to the activity of the fully stimulated normal enzyme. The increases

3767

TIME (hours)

FIG. 1. Activation of adenylate cyclase during incubation of NS20 cells with cholera toxin. NS20 cells were incubated with 20 pglml of cholera toxin in serum-free growth medium at 37” for various times. Cells were then washed free of toxin, homogenized, and a nucleus- free particulate membrane preparation prepared (see “Materials and Methods”). The activity of the adenylate cyclase in these prepara- tions was assayed in the standard ATP assay system with 1 mM ATP with no other additions (0); 1.4 c(M PGE, (0); 100 jbM ClAdo (A); 5 rnM NaF (ml; or 100 PM GMP-P(NH)P CT). The time of the toxin incubation period is given along the ordinate. Panel B lists the ratio of the activity of the enzyme observed in the presence of one of the above compounds (V ,,l,,,uIB,op) to that observed in the absence of any of these compounds ( V “0 ,tLm”lalor . )

TABLE I

Comparison of normal and toxin-activated adenylate cyclase in ATP assay system

All assays were performed in the ATP assay system (see “Materi- als and Methods”) with 1 mM ATP. Additions were made as follows: ClAdo (100 PM); PGE, (1.4 PM); GMP-P(NH)P (100 PM); GTP (100 MM); GDP (100 a~); and NaF (5 mM1.

Adenylate cyclase

Assay additions Normal Toxin activated

10” 100” ion 100”

None 8.2 ClAdo 23.4 PGE, 89.3 NaF 43.6

GDP GTP GTP/ClAdo GTP/PGE, GTP/NaF

GMP-P(NH)P 45.4 GMP-P(NH)P/ClAdo 58.8 GMP-P(NH)P/PGE, 92.8 GMP-P(NH)P/NaF 54.1 GMP-P(NH)P/GTP 19.2 GMP-P(NH)P/GDP 18.8 GMP-P(NH)PIClAdo/GTP 33.4 GMP-P(NH)P/PGE,/GTP 74.2

6.9 6.5

20.9 70.8

pmollmmlmg protein

4.4 47.9 4.8 44.7

21.3 54.8 54.9 43.5

7.6 41.2 7.6 47.6 9.5 52.3

28.6 90.1 40.6

8.5 9.8

12.7 23.9

38.6 44.0 33.5 49.5 48.8

83.9 44.4 74.3 42.3 87.9 61.4 75.4 44.8 47.7 37.6 47.3 38.2 41.8 42.8 52.5 66.6

45.6

58.3 41.7 55.8 56.1 50.8 48.9 43.1 52.5 47.9 GMP-P(NH)P/NaF/GTP

” mM M&l,.

in “basal” activity and the decrease in the activation produced by the above activators are not seen immediately after addi- tion of toxin, but are subject to a characteristic lag of 1 h (2,8, 32-34). Both aspects of the in situ activation processes are virtually complete within 2 h after addition of toxin.

The neuroblastoma enzyme, like other adenylate cyclases (35-38), possesses a regulatory site which is specific for gua-

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Cholera Toxin Modification of Adenylate Cyclase

nine nucleotides (28). GMP-P(NH)P, an analogue of GTP which is not readily converted to GDP (39), activates the normal enzyme at 10 mM MgCl, (Table I). In contrast, GMP- P(NH)P does not stimulate the toxin-activated enzyme at 10 mM MgCl,. GDP and GTP alone do not influence the activity of either enzyme. An increase in the activity of the toxin- activated enzyme, but not the normal enzyme, is observed when GTP is added in combination with PGE,. The same qualitative difference between the activity of the normal and toxin-activated enzyme is observed when the concentration of ATP in the assay is either 1.0 or 0.1 mM (data not shown).

Effects of Mg”+, Mn”, and CCL’+-Mg”+ appears to be re- quired along with ATP to form the active metal. ATP sub- strate complex for the neuroblastoma (27) and other adenylate cyclases (40). Moreover, Mg’+ influences the rate of activation of the normal enzyme by GMP-P(NH)P (28). Mg*+ also affects the guanine nucleotide regulatory site of the toxin-activated enzyme (Fig. 2). The activity of the toxin-activated enzyme is progressively inhibited when MgCl, exceeds 20 mM. Addition of GMP-P(NH)P prevents any loss of activity at high MgCl, concentrations and actually results in higher activity than is seen in the absence of GMP-P(NH)P at lower Mg2+ concentra- tions. At 100 mM MgCl,, the activit,y of the toxin-activated enzyme, when assayed with GMP-P(NH)P, is between 6- and lo-fold higher than when assayed without GMP-P(NH)P. At 100 mM MgCl,, the K,,, for this effect of GMP-P(NH)P on the toxin-activated enzyme is 2.9 * 1 PM (data not shown). Con- centrations of Mg2+ in excess of 20 mM produce other striking changes in the toxin-activated enzyme (Table I). At 100 mM MgCll, the toxin-activated enzyme and the normal enzyme have comparable low “basal” activities. Both are stimulated by PGE, and NaF, as well as GMP-P(NH)P. Addition of GTP or GDP (resulting in an equal mixture of GTP and GDP in the assay under these conditions) elevates the activity of the nor- mal enzymes less than 2-fold and blocks activation of this enzyme by GMP-P(NH)P. In contrast, the GTP/GDP mixture stimulates the toxin-activated enzyme almost as much as does GMP-P(NH)P, and the GTP/GDP mixture only slightly in- hibits the activation of GMP-P(NH)P.

For the normal enzyme, Mn2+ can replace MgZ+ in the active metal. ATP substrate complex (27). The MgCl, and MnCl,

I I I I -1

,Or 7---K i

0 MgCl2 (mM)

FIG. 2. Effect of Mg*+ on the activity of the toxin-activated en- zyme. Activity was assayed with 1 rn~ ATP without GMP-P(NH)P (0) and with 100 rn~ GMP-P(NH)P (0).

dose-response curves for both the normal and toxin-activated adenylate cyclases are quite similar (Fig. 3). With both en- zymes, a bell-shaped activity curve is seen in the absence of GMP-P(NH)P. GMP-P(NH)P prevents the decline in activity with increasing divalent cation concentration for both en- zymes, although with Mn2+ the protection is not complete. The inhibition of the toxin-activated enzyme caused by high con- centrations of MgCl, and MnCl, does not appear to be due to a nonspecific increase in the ionic strength of the assay mixture. Addition of 200 mM sodium or ammonium chloride does not cause any inhibition (Table II). CaCl, will cause an inhibition of activity which is not prevented by GMP-P(NH)P (see be- low).

The activation of the normal neuroblastoma enzyme by GMP-P(NH)P appears to be irreversible. Similar findings

60

40

20 0 i-it

TOXIN ACTIVATED

60

40

20

MgCl2 (mM) MnC12 (mM)

FIG. 3. Comparison of normal and toxin-activated enzymes at various concentrations of Mg’+ or MI?+. Assays were performed in the ATP assay system with 1 rnM ATP with either MgCl, (Pnnels A and C) or MnClp (Pan& B and D). Normal enzyme assayed without GMP-P(NH)P (0) and with 100 FM GMP-P(NH)P (0). Toxin-acti- vated enzyme. assayed without GMP-P(NH)P (ml and with 100 ELM GMP-P(NH)P (0). G, GMP-P(NH)P. In each panel, maximal activ- ity was determined from the l/V versus l/ion plots shown in the inset.

TABLE II

Effects of various ions on actioity of toxin-activated adenylate cyclase

All assays were performed in the ATP assay system with 1 rnM ATP. Where indicated, GMP-P(NH)P was also present in the assay mixture at 100 uM.

Ions present m assay mixture

Adenylate cyclase activity

No GMP- Plus GMP- P(NH)P PCNHIP

pmollrnmlmg protein

M&I, (10 mM) 32.8 -t 3.6 30.4 i 0.3

M&l, (100 mM) 16.8 -t 1.1 44.3 -t 0.6

MgCl, (10 rnr4 + NaCl (200 rnM) 31.9 t 1.5 35.9 t- 0.6

MgCl, (10 mM) + NH,CI (200 rnM) 25.5 i- 2.5 30.8 ? 0.5

MgCl, (10 mM) + CaCl, (100 mM) 1.5 k 0.5 1.9 zk 0.2

MnCl, (1 mM) 26.6 -t 5.0 26.4 -t 1.3

MnCl, (50 mM) 8.3 t 0.9 40.6 i 1.3

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Cholera Toxin Modification of Adenylate Cyclase 3769

have been reported with other adenylate cyclases (41, 42). After incubation with GMP-P(NH)P and subsequent washing, the normal enzyme resembles the toxin-activated enzyme when assayed at 10 mM MgCl, (Table III); the activity of the GMP-P(NH)P-activated enzyme is maximal in the absence of any stimulators. PGE,, ClAdo, NaF, or GMP-P(NH)P do not stimulate activity any further and GDP, by itself or in the presence of GMP-P(NH)P, does not affect activity. CaCl, in- hibits the activity of this enzyme (see below). The GMP- P(NH)P and toxin-activated enzyme, however, can be distin- guished from each other. At 100 mM MgCL, the GMP-P(NH)P- activated enzyme, unlike the toxin-activated enzyme, is only slightly less active than it is at 10 mM MgCl,. Furthermore, the GMP-P(NH)P-activated enzyme is not stimulated by either NaF, PGE,, or GMP-P(NH)P at 100 mM M&l,.

CaY’ is an equally effective inhibitor of the normal and toxin-activated neuroblastoma adenylate cyclase. With the normal enzyme, Ca” is noncompetitive with the enzyme sub- strate, ClAdo (271, PGE,, and GMP-P(NH)P (data not shown). In all experiments with Cay+, the K, for calcium is the same, i.e. 340 PM. Cari inhibits the toxin-activated enzyme when assayed at low or high MgCl, with or without GMP-P(NH)P (Fig. 41, PGE,, or ClAdo with the same K, (data not shown).

Requirements for ATP for Normal and Toxin-activated En- a,yymes-For the normal enzyme, the apparent K,,, for ATP increases as the MgCl, concentration increases (Fig. 5). In- creases in the K,,, for ATP are also seen with the toxin- activated enzyme as the MgCl, concentration is increased. For the toxin-activated enzyme, these changes are more dramatic than those seen for the normal enzyme. With both enzymes, GMP-P(NH)P prevents a large part of these elevations in the K,,, (Fig. 5). For the toxin-activated enzyme, the Mg”’ concen- tration at which the change in K,,, for ATP becomes dramatic

corresponds to the concentration of MgCl, which begins to inhibit activity as well as allow stimulation of activity by GMP-P(NH)P.

Enz,yme Stahilitn, - Several investigators have reported that

TABLE III

Con~parison of normal and GMP-PiNHiP-activated ntler!?/lnte c,yclase

GMP-P(NH)P-activated enzyme was obtained as follows. Normal

adenylate cyclase was incubated at 30” for 10 min in 10 IIIM Trisi maleate buffer, pH 7.4, with 5 rnM MgY’ and 50 GM GMP-P(NH)P.

The enzyme preparation was then washed twice in the buffer/Mg”

solution and resuspended in same. Normal enzyme was carried through the above procedure with the exception that GMP-P(NH)P

was not present. All assays were performed in the ATP assay system with 0.1 rnM ATP at 5 or 100 rnM Mg” as indicated. Assay additions

were as follows: ClAdo (100 PM); PGE, (1.4 FM); NaF (5 mM); GDP (100 qvr); and GMP-P(NH)P (100 PM).

Adenylate cyclase activity

Assay additions

None

ClAdo PGE ,

NaF

GDP GMP-P(NH)P

GMP-P(NH)P/ClAdo GMP-P(NH)P/PGE,

GMP-P(NH)P/GDP

GMP-P(NH)P-actrvated enzyme

5 IIIM ME’+ 100 NIM ME”+

prnolllnln/rng protein

0.9 22.6 17.3

19.6 15.4 22.3 13.0

24.0 17.9 0.9 23.1 16.6

6.6 22.5 17.1

7.3 18.5 9.9 24.7 16.9

2.0 24.5 16.6

activation by guanine nucleotides, NaF, and hormones stabi- lizes the activity of various adenylate cyclases (35, 43-46). We observe that increasing M&l, concentration increases the decay rate of the toxin-activated enzyme (Fig. 6). Inclusion of GMP-P(NH)P not only prevents any loss in activity, but ac- tually results in the recovery of an enzyme which is more active after incubation than before incubation. Although add- ing GMP-P(NH)P to a complete assay mixture which contains less than 10 mM MgCl, does not result in activation, adding GMP-P(NH)P to an incubation mixture containing only buffer and 10 mM MgCl, does result in a 50% increase in activity of the toxin-activated enzyme. The activity of the toxin-activated enzyme does not become more dependent upon added guanine nucleotides as the incubation period increases from 5 to 60 min. The following observations were made in similar experi- ments performed on the normal enzyme. (a) Decay is faster at higher than lower MgY’ concentrations; (h) GMP-P(NH)P pre- vents decay; and (c) the decay rates of “basal” activity of the normal enzyme are not significantly different from those seen for the toxin-activated enzyme, at either low or high Mg” concentrations.

Other nucleotides have been tested for their ability to effect the stability of the toxin-activated enzyme (Fig. 7). At 10 mM MgCl,, decay is prevented by GTP or ATP when they are added in combination with the nucleotide-regenerating sys- tem. AMP-P(NH)P alone will also prevent decay, whereas, the regenerating system does not effect decay. Although ATP, GTP, and AMP-P(NH)P prevent decay at 10 mM MgCl,, they do not result in the appearance of a more active enzyme. At 30 mM MgCL, only GMP-P(NH)P is completely effective in pre- venting enzyme decay. GTP, ATP, and AMP-P(NH)P, in this order, are only partially effective.

Studies with AMP-P(NH)P As Enzyme Suhstrntr - We have reported previously (281 that AMP-P(NH1P can be used as a substrate by the normal adenylate cyclase from neuro- blastoma cells. In an adenylate cyclase assay which contains 10 mM MgCl, and AMP-P(NH)P as substrate but which does not contain a nucleotide-regenerating system, the activity of the toxin-activated enzyme is similar to that of the normal enzyme (Table IV). The activity of both enzymes is very low, both are stimulated by NaF and GMP-P(NH)P, but neither are stimulated by PGE, or ClAdo. When GTP is added in combination with PGE,, stimulation of the toxin-activated enzyme occurs. The combination of GMP-P(NH)P and PGE, results in greater activity for the toxin-activated enzyme than is seen with GMP-P(NHlP alone. Addition of ATP, with or without PGE,, does not effect the activity of either enzyme. The K,,, for GMP-P(NH)P for the toxin-activated and normal enzyme in this assay system is 1.3 & 0.3 pM (data not shown) and 0.5 ? 0.2 PM (281, respectively.

The addition of the regenerating system to the AMP- P(NH)P assay system causes a 7-fold increase in the basal activity of the toxin-activated enzyme; no change in the activ- ity of the normal enzyme is observed. Further, small increases

in activity of the toxin-activated enzyme occur when PGE,, PGE, plus ATP, PGE, plus GTP, or ClAdo plus ATP are added in the presence of the regenerating system. In the presence of the regenerating system, ClAdo and PGE, do stimulate the activity of the normal enzyme (28).

In an AMP-P(NH)P assay system devoid of a regenerating system (Table V), both enzymes exhibit the same activity at

100 mM MgCl, as they do at 10 mM MgCl,, and both enzymes are stimulated by NaF and GMP-P(NH)P at 100 mM MgCl?. The regenerating system does not stimulate the activity of

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3770 Cholera Toxin Modification of Adenylate Cyclase

GMP-P(NH)P (PM) MsC12 (mM)

FIG. 4 (left). Noncompetitive interaction of Ca2+ and GMP- P(NH)P with the toxin-activated adenylate cyclase. Assays were performed in the presence of 1 mM ATP and 100 mM Mg*+. Activity was assayed with increasing concentrations of GMP-P(NHIP and the following amounts of CaCl,, none (O), 0.2 mM (A), 0.05 mM (01, or 1 rnM (Oh

FIG. 5 (center). Changes in apparent ATP K,,, with increasing Mg2+ concentrations. The concentration of ATP required for half- maximal enzyme activity at various Mg*+ concentrations was deter- mined for both the normal (0, 0) and toxin-activated enzymes (A, Al. Apparent ATP K, values were determined in the absence and presence of 100 PM GMP-P(NH)P. Normal without GMP-P(NH)P (0); normal with GMP-P(NH)P (0); toxin-activated without GMP(NH)P (A); and toxin-activated with GMP-P(NH)P (A).

FIG. 6 (right). Effect of Mg2+ on the decay of the toxin-activated enzyme. The enzyme was incubated at 30” for varying times in 10 mM

-I- B. A mM Mg

IO ’ ’ 3 ’ ’ I ,l, II 0 20 40 600 20 40 60

TIME (minutes)

FIG. 7. Protection against decay by various nucleotides. Toxin- activated enzyme was incubated at 30” in 10 mM Tris/maleate buffer, pH 7.4, and either 10 mM MgCl, (Panel A) or 50 mM MgCl, (PanelB) and the following: 100 PM GMP-P(NH)P (0); 100 PM GTP plus regenerating system (A); 100 PM ATP plus regenerating system (V); regenerating system (0); 100 pM AMP-(NH)P (0); or nothing (01. After incubation, enzyme was washed once in the buffer/Mg2+ solu- tion, resuspended in same, and assayed in the ATP assay system with 10 rnM Mg2+ and 0.1 rnM ATP. Initial activities are the same as stated in legend to Fig. 6.

either enzyme at 100 mM MgClp, yet GTP and ATP added

together with the regenerating system selectively stimulates the activity of the toxin-activated enzyme. In the absence of a regenerating system, when AMP-P(NH)P is used as substrate,

\ \ \ IO 0 20 40 60

TIME (minutes)

Tris/maleate buffer, pH 7.4; and either 10 mM MgCl, (0, 0); 100 mM MgCl, (0, W); or 100 mM MgCl, plus 100 ,u~ GMP-P(NHlP (A). The activity remaining was then determined in the ATP assay system at 10 mM MgCl,; 0.1 mM ATP; and with either no GMP-P(NHIP (0, 0) Or loo &&M GMP-P(NH)P (a, n ). When GMP-P(NH)P Was present in the incubation mixture, it was also present at 10 pM in the assays. Enzyme preparations were assayed directly after incubation. If after incubation, the enzyme is first washed twice before being assayed, the same results are obtained. Where the activities are assayed in the absence of GMP-P(NH)P, the initial activity is that seen without incubation. Where the activities are assayed with GMP-P(NH)P, the initial activity is that observed after a lo-min incubation with 10 mM Mg2+ and 100 PM GMP-P(NH)P. Initial activities for normal and toxin-activated enzymes are 4.5 and 33.4 pmol/min/mg of protein, respectively.

TABLE IV

Comparison of normal and toxin-activated adenylate cyclase in AMP- P(NH)P assay system

Assays were performed in the AMP-P(NH)P assay system (see Materials and Methods”) with 0.2 mM AMP-P(NH)P. Where indi- cated, a nucleotide triphosphate-regenerating system (Reg. System) consisting of 20 mM creatine phosphate and 0.1 mglml of creatine phosphokinase was also added to the assay mixture. Other assay additions were as follows: ClAdo (100 WM); PGE, (1.4 PM); NaF (5 mM); GTP (200 PM); ATP (200 &; and GMP-P(NH)P (IO0 CLM).

Adenylate w&se activity

Aasay additions No. reg. system Plus reg. syst&m

NOITlld TOXill- Normal Toxin- activated activated

None 0.88 ClAdo 0.38 l=El 0.46 NtlF 8.70

GTP GTPlClAdo GTP/PGE, ATP ATP/ClAdo ATPffGE,

GMP-P(NH)P GMP-P(NH)P/ClAdo GMP-P(NH)P/PGE,

0.78 0.74 1.76

0.80 0.81

3.50

- pmollminlmg protein

0.67 0.64 4.01 0.60 1.04 3.70 0.50 2.18 5.50 4.90

0.98 0.43 2.71 1.20 0.81 3.60 2.94 2.29 7.20 0.50 0.30 3.22

6.10 0.40 7.40

4.71 4.90 7.00

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Cholera Toxin Modification of Adenylate Cyclase

TABLE V

Effect of high Mg” on enzyme activity in AMP-P(NH)P assay system

Assays were performed in the AMP-P(NH)P assay system with 0.2 rn~ AMP-P(NH)P at either 10 or 100 rn~ MgCl, as noted. Other assay additions were as follows: ClAdo (100 PM); PGE, (1.4 PM); NaF (5 mM); GTP (200 FM); ATP (200 PM); GMP-P(NH)P (100 FM); and Reg. System, the nucleotide-regenerating system as described in the legend to Table IV.

Adenylate cyclase

Assay additions NOlTd Toxin-activated

10” 100” 10” 100”

pmollminlmg protein

None 0.4 0.2 1.5 1.4 ClAdo 1.3 PGE , 0.2 0.1 1.0 0.5 NaF 11.6 8.7 4.9 2.9 Reg. System 3.6 1.6

Reg. SystemiGTP 0.3 0.4 3.3 4.1 Reg. System/GTP/ClAdo 5.4 3.7 Reg. SystemiGTPiPGE, 1.4 0.9 8.0 2.8

Reg. SystemiATP Reg. SystemiATPiClAdo Reg. System/ATP/PGE,

3.5 2.5 6.1 2.4

0.4 c.4 7.4

GMP-P(NH)P GMP-P(NH)P/ClAdo GMP-P(NH)P/PGE,

(I rnM MgCl,.

2.7 3.5 4.1 9.7 4.9 9.4 7.0 4.8

PGE, and ClAdo increase the concentration of GDP required to block activation of the normal enzyme by GMP-P(NH)P (28). We find that 25 PM GDP, which inhibits 50% of the GMP- P(NH)P activation of the normal enzyme, also inhibits 50% of the GMP-P(NH)P activation of the toxin-activated enzyme. Furthermore, PGE, increases the concentration of GDP re- quired to block the activation of the toxin-activated enzyme by GMP-P(NH)P (Fig. 8).

In Vitro Activation of Adenylate C,yclase by Cholera Toxin -Recently, the successful in vitro activation of other adenylate cyclases by cholera toxin has been demonstrated. NAD, ATP, and cytoplasmic components of the cells, as well as dissociated cholera toxin, were reported to be necessary for the in vitro activation process (47-52). In general, the in vitro activation of neuroblastoma adenylate cyclase by cholera toxin appears to have the same requirements as were found for other adenylate cyclases. The in vitro activation results in an en- zyme with properties similar to those of the enzyme activated in situ by the toxin.

In vitro activation of the normal neuroblastoma enzyme occurs when a cell-free membrane preparation is incubated with cholera toxin (after treatment with dithiothreitol, NAD, and nucleotide triphosphates) (Table VI). Half-maximal acti- vation occurs at 6.5 pg/ml of toxin and 30 PM NAD (data not shown). We do not observe any effect of the cytoplasm on this process. The requirement for a nucleotide is partially fulfilled by the inclusion of a nucleotide-regenerating system in the incubation mixture. Activation is maximal when ATP or GTP is added along with the regenerating system. In the absence of a regenerating system, GDP, ADP, GTP, ATP, or AMP- P(NH)P do not support the activation process (Table VI). The time course of the activation process in the complete incuba- tion mixture has a t,,, of 3 min (Fig. 9A). Based on the disappearance of stimulation by NaF, the activation process

GDP (pW

FIG. 8. GDP inhibition to activation of the toxin-activated aden- ylate cyclase by GMP-P(NH)P. Assays were performed in the AMP- P(NH)P assay system with 5 rnM Mgy+, 0.1 rnM AMP-P(NH)P, 50 pM GMP-P(NH)P, and without any regenerating system present. As- says were run for 20 min at 30”. Maximal adenylate cyclase activity is the activity observed under this assay condition in the absence of any added GDP. No PGE, present, 0 and 1.4 PM PGE, present, 0. Maximal activity after activation by GMP-P(NH)P under these as- say conditions is 3.5 pmoliminimg of protein.

TABLE VI

In vitro activation of adenylate c,yclase Adenylate cycl& activity

Additions to incubation mixture” Without NaF Plus NaF

pmollminlrn~ protein

Experiment A None 1.9 17.7 Reg. System 4.1 32.5 Reg. SystemiGTP (0.1 mM) 11.7 36.3 AMP-P(NH)P (0.1 mM) 2.4 18.6

Experiment B None 1.6 15.0 GDP (1.0 mivr) 1.4 17.6 ADP (1.0 mM) 2.3 20.8 Reg. System 10.5 32.9 Reg. SystemiGTP (0.1 mM) 13.5 24.3 Reg. SystemiATP (0.1 mM) 13.1 32.2

Experiment C None 2.6 21.2 GTP*(O.l mix) 4.0 17.6 ATP (0.1 mM) 3.5 25.4 Reg. System Reg. SystemiGTP (0.1 mivr) 28.5 35.3

(’ The basic incubation mixture contains normal adenylate cy- clase, 10 rnM Trisimaleate buffer, pH 7.4, 10 rnM Mg’+, 0.1 mM NAD,

and 50 pg/ml of cholera toxin which had been treated with 4 mM dithiothreitol at 22” for 50 min. Incubations were conducted at 30” and terminated by dilution with a buffer/Mgs+ solution. The enzyme preparations were then washed twice and resuspended in the above buffer/MgZ+ solution and then assayed. Assays were performed in the ATP assay system with 0.1 rn~ ATP. Other additions to the

incubation mixture were as noted above. Reg. system, nucleotide- regenerating system as described in legend to Table IV.

appears to be complete after 20 min. Toxin activation will also take place during an adenylate cyclase assay if NAD is present (Fig. 9B). Under such conditions, the activation process has a similar tjjP and after about 12 min the activity of the enzyme reaches a constant rate. The rate of the in vitro activation has

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3772 Cholera Toxin Modification of Adenylate Cyclase

- IO r,, / , I/ ,J&/4,/ / a/ .,* __.- I.--.*‘.-.-@ 0

_rl 100 2 g IO 20 30 0 IO 20

0 5 TIME (minutes)

FIG. 9. Time course of activation of normal adenylate cyclase in vitro by cholera toxin. A, normal enzyme was incubated at 30” in 10 mu Trisimaleate buffer, pH 7.4, 10 m”M MgCl,, 0.1 mM NAD, 0.1 mM GTP, regenerating system (see “Materials and Methods”), and 50 /*g/ml of cholera toxin (pretreated with 4 mM dithiothreitol at 22” for 30 min). After the incubation time noted, the enzyme was washed twice and then assayed in the ATP assay system with 1 rn~ ATP, 10 mM MgCl,, and either no NaF (0) or 5 mM NaF (0). B, activity of the normal enzyme assayed at 30” in 50 mM Tris/maleate buffer, DH 7.4.

10 rnM MgCl,, 1 mM ATP, regenerating system, 0.1 rnM NkD plus the following additions: nothing (WI; 50 pg/ml of cholera toxin (pre- treated with dithiothreitol (0) or 5 mM NaF (0).

been noted by others (51) to be a function of the amount of free A, subunit of cholera toxin present. Dithiothreitol treatment of the toxin does not apparently fully dissociate the toxin as evidenced by the fact that additional treatment of t,he toxin with sodium dodecyl sulfate causes both the rate of in. vitro activation and extent of activation of the neuroblastoma aden- ylate cyclase to increase (data not shown).

DISCUSSION

Previous studies on the normal neuroblastoma adenylate cyclase (28) led us to propose the model of enzyme regulation which is diagrammatically shown in Fig. 10. In our model, guanine nucleotides are the primary regulators of enzyme activity. When GTP or GMP-P(NH)P is bound to the guanine nucleotide site, the enzyme (E-GTP orE-GMP-P(NH)P) is in a state of high catalytic activity. When the guanine nucleotide site is unoccupied (E-O) or filled with GDP@-GDP), the en- zyme has little or no activity. GDP functions as an inhibitor by preventing the binding of the activator guanine nucleotides. When assayed at physiological concentrations of Mg2+ (10 mM) and with ATP as substrate, the basal activity of the normal neuroblastoma adenylate cyclase is low unless ClAdo, PGE,, GMP-P(NH)P, or NaF is present. ClAdo and PGE, are second- ary enzyme regulators and do not activate the enzyme in the absence of guanine nucleotides. They cause an increase in activity by reducing the ability of GDP to pr&ent the binding of GTP or GMP-P(NH)P (28).

The studies reported here show that the interaction of chol-

era toxin with intact neuroblastoma cells or membranes iso- lated from these cells leads to an activation of adenylate cyclase. The toxin-activated enzyme exists, in the standard ATP assay system at physiological concentration of Mg’+ (10 mM), in a fully active state in the absence of ClAdo, PGE,, NaF, or GMP-P(NH)P. According to our model, the toxin- activated enzyme should exist, under these assay conditions, in an E-GTP state in the absence of ClAdo, PGE,, GMP- P(NH)P, or NaF. The primary source of the endogenous gua- nine nucleotide triphosphate in these assays appears to be the membrane preparation itself. When AMP-P(NH)P is used as enzyme substrate in assays devoid of a nucleotide-regenerat- ing system but with 10 mM Mg2+, the toxin-activated enzyme

\E UEGDP

it PGE ,

ClAdo

FIG. 10. Model for guanine nucleotide regulation of adenylate cyclase.

and normal enzyme both have the same low “basal” activity, ClAdo and PGE, do not stimulate either enzyme, and both require the addition of GMP-P(NH)P or NaF in order to ex-

press maximal catalytic activity. Addition of a regenerating system to these assays results in the toxin-activated enzyme once again displaying, in the absence of ClAdo or PGE,, an elevated catalytic activity as compared with the normal en- zyme. In addition, there is now a return in the ability of PGE, and ClAdo to activate. We have observed that the amount of membrane protein routinely used in these assays contains sufficient phosphatase activity to completely convert 1 mM GTP to GMP within minutes in the presence of 10 mM MgCl, at 30”. It appears that the regenerating system contained in the standard ATP assays maintains the endogenous guanine nucleotides of the membrane preparations as triphosphates. However, only the toxin-activated enzyme has the ability to use them as activators in the absence of ClAdo or PGE,.

We have found that at 10 mM MgCl,, the toxin-activated enzyme state is similar to the state in which the normal enzyme exists at 10 mM MgCl, after incubation with GMP- P(NH)P. Washing the enzyme after GMP-P(NH)P activation does not reverse the process. The fact that GMP-P(NH)P, unlike GTP, cannot readily be converted to GDP implies that some type of metabolism of GTP, which GMP-P(NH)P cannot undergo, may be required for conversion of the E-GTP to the E-GDP or E-O state. In our model, we propose that a GTP conversion to GDP must occur in order for the enzyme to return to E-GDP. The GMP-P(NH)P-activated enzyme state, however, is not identical with the toxin-activated state. Eleva-

tion of the MgCl, concentration above 20 mM converts the toxin-activated enzyme, but not the GMP-P(NH)P-activated enzyme, to a state which again requires exogenously added guanine nucleotides or secondary enzyme regulators for maxi- mal catalytic activity. High MgCl, inhibits the “basal” activity of the normal enzyme, and the MgCl, inhibition of both en- zymes is prevented by GMP-P(NH)P. The most simple expla- nation for these findings is that the conversion of GTP to GDP at the enzyme’s regulatory site is controlled by a Mg2+-depend- ent GTPase. Filling the guanine nucleotide site with GMP- P(NH)P yields an enzyme which is not inhibited by MgCl, as no conversion of GMP-P(NH)P to GDP is possible. The inhibi- tion of both enzymes by MnClp which we have observed is more complex. Some of the MnClt inhibition occurs at the guanine nucleotide regulatory site as it is partially prevented by GMP-P(NH)P. However, MnCl, also inhibits by binding to the separate Ca’+ sites on the enzyme. Evidence for the fact that Ca”+ and Mn” can interact at the same site and thereby inhibit activity has been presented previously (27). Ca2+ and GMP-P(NH)P do not interact at the same sites on the enzyme,

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Cholera Toxin Modification of Adenylate Cyclase 3773

and no change in the sensitivity of the enzyme to CaZ+ has been observed after toxin activation.

At physiological concentrations of Mg”+, the normal and toxin-activated enzymes have similar K,,l values for ATP. As the MgC12 concentration increases, there is an increase in the K,,, for both enzymes. This increase is much more dramatic for the toxin-activated enzyme than for the normal enzyme. GMP- P(NH)P prevents most of the increase in apparent ATP K,,, for the toxin-activated enzyme and about half the increase ob- served with the normal enzyme. This indicates that the added

ATP, or a contaminant therein, affects both enzymes through their guanine nucleotide site. Recently, commercial sources of ATP have been shown to contain GTP (53). The apparent increased dependence upon ATP for the toxin-activated en- zyme at high MgCl, concentrations is most likely due to its increased requirement for exogenously added GTP.

While toxin activation specifically affects the process by which the E-GTP state is converted to the E-GDP state, the two other steps in our model in which guanine nucleotides function are unaltered after toxin activation. Although the direct interaction of GTP with E-O cannot be assessed, the interaction of E-O with GMP-P(NH)P can be assessed for both enzymes. The K,,, for GMP-P(NH)P for the normal enzyme at 100 mM MgCl, in the standard ATP assay system is 2.9 PM and is not significantly different for the toxin-activated enzyme. In the AMP-P(NH)P assay system at 10 mM MgCL, the K,,, for GMP-P(NH)P and normal and toxin-activated enzymes is 0.5 2 0.2 pM (28) and 1.3 ? 0.3 FM, respectively. The toxin- activated enzyme, as well as the normal enzyme, can bind GMP-P(NH)P at low Mg2+ as evidenced by the fact that GMP- P(NH)P will stabilize both enzymes at low Mg*+. An indirect assessment of the interaction of GDP with these two enzymes was made by comparing the relative affinities of GDP and GMP-P(NH)P with both enzymes. The amount of GDP re- quired to block 50% of the activation of either enzyme by GMP- P(NH)P was found to be similar. Moreover, the interaction of GDP with the toxin-activated enzyme is decreased by second- ary regulators (i.e. PGE, and ClAdo) as it is with the normal enzyme. The lack of an effect of PGE, and ClAdo on the toxin- activated enzyme assayed with ATP as substrate and with 10 mM Mg”+ and a regenerating system is due to the fact that this enzyme exists in the E-GTP state in the absence of ClAdo or PGE,.

A number of questions can be raised concerning the require- ment for nucleotide triphosphates for the in vitro activation process catalyzed by cholera toxin. We have shown that gua- nine nucleotide triphosphates are required for expression of maximal catalytic activity after the in situ toxin activation process is completed. It is possible, therefore; that nucleotides do not play a direct role in the in vitro toxin activation process but instead are required at the nucleotide regulatory site for the subsequent expression of maximal activity in the assay. Since ATP normally will not substitute for GTP as an enzyme activator, its role in the in vitro activation by toxin may be to prevent hydrolysis of the endogenous GTP and GDP. The remaining endogenous GTP or GDP would be subsequently maintained, or converted to GTP, in the assay by the nucleo-

tide-regenerating system. In summary, all of the properties of the toxin-activated

adenylate cyclase can be accounted for by a decrease in the conversion of enzyme-bound GTP to GDP at physiological concentrations of Mg2+. This conversion of GTP to GDP ap- pears to be controlled by a divalent ion-dependent GTPase. Secondary enzyme regulators like ClAdo, PGE,, and perhaps

molecules such as catecholamines, glucagon, etc., exert their influence on a different regulatory process, i.e. the affinity of GDP for the nucleotide regulatory site.

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24

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26.

27 28.

29

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36. 37

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41.

REFERENCES

Schafer. D. E.. Lust. W. D.. Sircar. B.. and Goldberg, N. D. (1970jProc. N&l. &ad. Sci. U. S. A. 67, 851-856 -’

Sharp, G. W. G., and Hynie, S. (1971) Nature 229, 266-269 Sharp, G. W. G. (1973) Annu. Reu. Med. 24, 19-51 Field, M.. Fromm. D., Al-Awqati. Q., and Greenoueh, W. B.. III

(1972) J. Clin. Inuest. 51, 796-804 Field, M. (1971) N. EngZ. J. Med. 284, 1137-1144 Finkelstein, R. A. (1973) Cril. Rev. Muobiol. 2, 553-623 King, C. A., and Van Heyningen, W. E. (197315. Infect. Dis. 127,

639-647 Cuatrecasas, P. (1973) Biochemistry l2, 3547-3558 Cuatrecasas, P. (1973) Biochemistry 12, 3558-3566 Cuatrecasas, P. (1973) Biochemistr,y 12, 3567-3576 Cuatrecasas, P. (1973) Biochemistr,y 12, 3577-3581 Holmgren, J., Lonnruth, I., and Svennerholm, L. (1973) Stand.

J. Infect. Dis. 5, 77-78 Moss, J., Fishman, P. H., Manganiello, V. C., Vaughan, M., and

Brady, R. 0. (1976) Proc. N&Z. Acnd. Sci. U. S. A. 73, 1034- 1037

Sate. K.. Mivachi. Y.. Ohsawa. N.. and Kosaka. K. (1975) Bio- chtbn. Bio;hys. kes: Cornmu;. 62, 696-703

Haksar, A.. Maudslev. D. V., and P&on. F. G. (1975) Biochim. Biophys. Acta 381,308-323

Palfreyman, J. W., and Schulster, D. (1975) Biochim. Biophys. Acta 404. 221-230.

Gill, D. M.; and King, C. A. (1975) J. Biol. Chem. 250,6424-6432 Bitenskv, M. W., Wheeler, M. A., Mehta. H.. and Miki, N.

(1975jProc. N&Z. Acad. Sci. U. S. A. 72, 2572-2576 Flores, J., and Sharp, G. W. G. (1975) J. Clin. Iwest. 56, 1345

1349 Beckman, B., Flares, J., Witkum, P. A., and Sharp, G. W. G.

(1974) J. CZin. Inuest. 53, 1202-1205 Ganguly, U., and Greenough, W. B. (1975)Proc. Natl.Acad. Sci.

U. S. A. ‘72. 3561-3564 Field, M. (1974) Proc. N&Z. Acad. Sci. U. S. A. 71, 3299-3303 Bennett, V.. and Cuatrecasas, P. (1975)J. Membrane A&. 22, l-

28 Bennett, V., Mong, L., and Cuatrecasas, P. (1975) J. Membrane

BioZ. 24, 107-129 Blume, A. J., Dalton, C., and Sheppard, H. (1973) Proc. N&Z.

Acad. Sci. U. S. A. 70, 3099-3102 Blume, A. J., and Foster, C. J. (1975) J. BioZ. Chem. 250, 5003-

5008 Blume, A. J., and Foster, C. J. (1976) J. Neurochem. 26,305-311 Blume, A. J., and Foster, C. J. (1976) J. BioZ. Chem. 251, 3399-

3404 Amano, T., Richelson, E., and Nirenberg, M. (1972) Proc. N&Z.

Acad. Sci. U. S. A. 69, 258-263 Salomon, Y., Londos, C., and Rodbell, M. (1974)AnaZ. B&hem.

58, 541-548 Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.

(1951) J. BioZ. Chem. 193, 265-275 Vaughan, M., Pierce, N. F., and Greenough, W. B., III (1970)

Nature 226, 658-659 Kimberg, D. V., Field, M., Johnson, J., Henderson, A., and

Gershon, E. (1971) CZin. Inuest. 50, 1218-1232 Hewlett. E. L.. Guerrant. R. L.. Evans. D. J.. Jr.. and Green-

ough, ‘W. B.,‘III (1974) hature’249, 371-373 Rodbell, M.. Birnbaumer, L., Pohl. S. L.. and Krans. H. M. J.

(1971j J. BioZ. Chem. 246, i877-1882 Lefkowitz, R. J. (1974) J. Biol. Chem. 249, 6119-6124 Londos, C., Salomon, Y., Lin, M. C., Harwood, J. P., Schramm,

M., Wolff, J., and Rodbell, M. (1974) Proc. N&Z. Acad. Sci. U. S. A. 71, 3087-3090

Glossman, H., and Gips, H. (1974) Naunyn-Schmiedeberg’s Arch. Pharmakol. Exp. Pathol. 286, 239-249

Yount, R. G., Babcock, D., Ballantyne, W., and Ojala, D. (1971) Biochemistry 10, 2484-2489

Perkins. J. P. (1973) in Advances in Cvclic Nucleotide Research (Greengard, P., ed) Vol. 3, pp. l-64, Raven Press, New York

Salomon, Y., Lin, M. C., Londos, C., Rendell, M., and Rodbell, M. (1975) J. BioZ. Chem. 250, 4239-4245

by guest on October 24, 2020

http://ww

w.jbc.org/

Dow

nloaded from

3774 Cholera Toxin Modification of Adenylate Cyclase

42. Schramm, M., and Rodbell, M. (1975) J. Biol. Chem. 250, 2232- 48. Wheeler, M. A., Solomon, R. A., Cooper, C., Hertzberg, L., 2237 Mehta, H., Miki, N., and Bitensky, M. W. (1976) J. Infect. Dis.

43. Harwood, J. P., LBw, H., and Rodbell, M. (1973) J. Biol. Chem. 133, 589-595 248, 6239-6245 49. Gill, M. D. (1976) J. Infect. Dis. 133, S55-S63

44. Cuatrecasas, P., Jacobs, S., and Bennett, V. (1975) Proc. Natl. 50. Gill, M. D. (1976) Biochemistry 15. 1242-1248 Acad. Sci. U. S. A. 72, 1739-1743 51. Wodnar-Filipowicz, A., and iai, C. Y. (1976) Arch. Biochem.

45. Lefkowitz, R. J., and Caron, M. G. (1975) J. Biol. Chem. 250, Biophys. 176, 465-471 4418-4422 52. Donta, S. T. (1976) J. Infect. Es. 133, S115&S119

46. Rodbell, M. (1975) J. Biol. Chem. 250, 5826-5834 47. Gill, M. D. (1975)Proc. N&Z. Acad. Sci. U.S. A. 72,2064-2068

53. Kimura, N., Nakane, K., and Nagata, N. (1976) Biochem. Bio- phys. Res. Commun. 70, 1250-1256

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S L Levinson and A J Blumeactivity after cholera toxin treatment.

Altered guanine nucleotide hydrolysis as basis for increased adenylate cyclase

1977, 252:3766-3774.J. Biol. Chem. 

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