Proc. Nat. Acad. Sci. USA

Vol. 72, No. 7, pp. 2572-2576, July 1975

Biochemistry

Cholera toxin activation of adenylate cyclase in cancer cell membrane fragments ("A fragment" of cholera toxin/plasma cell membranes)

MARK W. BITENSKY, MARCIA A. WHEELER, HEMLATA MEHTA, AND NAOMASA MIKI Department of Pathology, Yale University School of Medicine, 310 Cedar St., New Haven, Connecticut 06510

Communicated by Lewis Thomas, April 18,1975

ABSTRACT Activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by cholera toxin (84,000 daltons, 5.5 S) is demonstrated in plasma membrane fragments of mouse ascites cancer cells. The activation of adenylate cyclase is mediated by a macromolecular cyclase activating factor (MCAF), which has a sedimentation constant of 2.7 S and a molecular weight of about 26,000. MCAF is derived from; and may be identical to the "A fragment" of cholera toxin. Generation of MCAF depends on prior interaction of cholera toxin with either dithiothreitol, NADH, NAD, or a low-molecular-weight component (less than 700 daltons) present in cytoplasm. Subsequent exposure of this pretreated cholera toxin to cell membranes from a variety of mouse ascites cancer cells is followed rapidly by the appearance of MCAF, which no longer requires dithiothreitol, NADH, or NAD for the activation of adenylate cyclase. Activation of adenylate cyclase by MCAF in ascites cancer cell membrane fragments is not reversed by repeated washing of these membrane fragments. Adenylate cyclase in normal cell membrane fragments fails to respond either to cholera toxin or MCAF in the presence of dithiothreitol. In striking contrast, the adenylate cyclase in membrane fragments from five ascites cancer cells responds to either MCAF or native cholera toxin preincubated with dithiothreitol, NADH, or NAD.

Cholera toxin has a molecular weight of 84,000 (1), and is composed of an A fragment with a molecular weight of 25,000 and four smaller fragments each with a molecular weight of 15,000 (2). The latter appear capable of binding monosialoganglioside (GM1) on the cell surface (3, 4). The mechanism of action of cholera toxin was suggested by the finding that secretion of water and chloride by jejunal mucosa was increased by cyclic adenosine 3':5'-monophosphate (cAMP), epinephrine, theophylline, and prostaglandin Ei (5, 6). It was found that exposure to cholera toxin markedly increased the content of cAMP (7) and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity (8) in mucosal cells. Subsequently, it was demonstrated that cholera toxin could activate adenylate cyclase in a variety of other tissues (9-12). Certain features of the interaction of cholera toxin with adenylate cyclase have appeared consistently in several cell types. These include (i) a time lag between exposure to cholera toxin and the appearance of increased adenylate cyclase activity (9-12), (ii) a requirement for intact cells (9-12), and (Mii) adenylate cyclase activation which is independent of protein synthesis (13). We report here the activation of adenylate cyclase by cholera toxin in suspensions of membrane fragments prepared for sarcoma 180 and other mouse ascites cancer cells. This activation depends on both a low-molecular-weight (less then 700 daltons) cytoplasmic component and a macroAbbreviations: cAMP, cyclic adenosine 3':5'-monophosphate; MCAF, macromolecular adenylate cyclase activation factor.

molecular (about 26,000 daltons) adenylate cyclase activating factor (MCAF) which derives from cholera toxin. We initially selected sarcoma 180 because of excellent growth characteristics, homogeneity, availability as a free suspension, and a substantial adenylate cyclase activity which responds vigorously to cholera toxin. We describe some physical and chemical properties of MCAF, some molecules which substitute for the low-molecular-weight cytoplasmic component, and the capacity of different cell membrane fragments to either generate or respond to MCAF. We also discuss some properties of normal and cancer cells which may explain differences in their ability to respond to cholera toxin in broken cell systems.

MATERIALS AND METHODS Purified cholera toxin (5 mg/ml) was provided by the National Institute of Allergy and Infectious Diseases and was dialyzed against 100 mM Tris.HCl buffer, pH 7.5. Carrierfree 125I (5 mCi) in 0.1 N NaOH was purchased from New England Nuclear Corp. "Insoluble" enzymes were purchased from Sigma Chemical Co. Ascites cancer cells were kindly provided by Dr. Alan Sartorelli of Yale Medical School, and were maintained in the ascites form and transplanted as previously described (14). Ascites cancer cells were harvested (no. 18 needle) into a citrate (0.45%)-normal saline solution and washed by centrifugation at 1000 rpm for 2 min at 40 in a Sorvall HB-4 rotor. The pellet was homogenized in 20 mM glycylglycine buffer, pH 7.5, containing 5 mM MgCl2 (Buffer A) at a ratio of 2 volumes of packed cells to 1 volume of Buffer A, with a motor driven Teflon on glass homogenizer (12 strokes at 1000 rpm at ice temperature). The homogenate (2-4 ml) was layered on a discontinuous sucrose density gradient consisting of 10 ml each of solutions with densities of 1,23, 1.19, and 1.16 in a 40 ml tube. The tubes were centrifuged at 23,000 X g for 30 min in a Sorvall HB-4 rotor. Material at the interface between densities 1.23 and 1.19, which contained over 90% of the adenylate cyclase, was harvested, suspended in Buffer A, and rehomogenized with a Teflon on glass homogenizer. Succinic dehydrogenase measurements (15) indicate that the gradient-prepared membranes contain small amounts of mitochondrial contamination. This homogenate was diluted 20 times with Buffer A and centrifuged at 36,400 X g for 10 min. Three volumes of pellet (gradientprepared membranes) were then suspended in 1 volume of Buffer A. Adenylate cyclase activity was assayed at 300 in a reaction volume of 20 Al which contained 50 mM Tris-HCl buffer, pH 7.5, 5 mM MgCl2, 1 mM [3H]ATP (diluted to 100 Ci/ mol, New England Nuclear Corp.), a regenerating system (2 2572

Proc. Nat. Acad. Sci. USA 72 (1975)

Biochemistry: Bitensky et al. Table 1. Effects of cholera toxin on adenylate cyclase activity in homogenates and gradient-prepared membranes from sarcoma 180

Adenylate cyclase activity

Membrane Additions

preparation Homogenates Homogenates Homogenates Homogenates Gradient Gradient None Gradient Gradient

Gradient Gradient

36 147 126 156 50 78 8 430 422

None Cholera toxin L-Epinephrine L -Epinephrine and cholera toxin None Cholera toxin Cytoplasm Cytoplasm and cholera toxin Cytoplasm and cholera toxin and then washing (2 times) Dithiothreitol Dithiothreitol and cholera toxin

51 451

Cholera toxin was used at a concentration of 100 Ag/ml, dithiothreitol at 2 mM, and L-epinephrine 0.01 mM. Cytoplasm represents the 100,000 x g supernatant (1 hr, 40) obtained by the centrifugation of sarcoma 180 homogenates. Gradient-prepared membranes ("gradient") were washed by dilution in 20 volumes of buffer A and collected by centrifugation at 34,000 x g for 10 min. Cytoplasm or dithiothreitol was preincubated with cholera toxin for 20 min at 300. Adenylate cyclase is expressed as pmol of cAMP synthesized per min/mg of protein. The values are the averages of six determinations and replicate values agreed within 5%.

mg/ml of pyruvate kinase and 40 mM phosphoenolpyruvate) and 0.5 mM isobutylmethylxanthine (Aldrich Chemical). The reaction was initiated by the addition of 30-50 Aig of membrane protein and after 15 min was terminated by boiling (3 min). Labeled cAMP was isolated by polyethyleneimine cellulose thin-layer chromatography as previously described (16). Phosphodiesterase (3':5'-cyclic adenosine monophosphate 5'-nucleotidohydrolase, EC 3.1.4.17) activity was assayed at 300 by incubating 10 ,uM [3H]cAMP (diluted to 1000 Ci/ mole, New England Nuclear) with enzyme for 5 min at 300 as previously described (16, 17). Cyclic AMP was measured A

500 ,5 :oo

RESULTS Effects of Cholera Toxin on Broken Cells. When cholera toxin was incubated with homogenates of sarcoma 180, basal activity of adenylate cyclase was increased 4- to 5-fold. Stimulation of adenylate cyclase activity by both epinephrine and cholera toxin was less than additive (Table 1). The concentration of cholera toxin required for maximal activation of adenylate cyclase in the homogenates was 100,ug/ml (Fig. IB) while smaller amounts of cholera toxin (1 ,.g/ml) are required for maximal activation of adenylate cyclase in the intact cell suspensions (Fig. IA). The content of cAMP in intact cells incubated in Tyrode's solution with cholera toxin (5 jig/ml) for 60 min at 370 was B

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by a modification of the method of Gilman (18) in that binding protein was prepared from rabbit muscle by the method of Reiman et al. (19), and samples were prepurified with a basic aluminum oxide column (0.5 X 1.0 cm) (20). Protein concentrations were determined by the method of Lowry et al. (21) with bovine serum albumin as standard. Ultracentrifugation in a sucrose density gradient (5-20%) was done by the method of Martin and Ames (22). The gradients were centrifuged for 16 hr at 50,000 rpm in a Beckman SW-50 rotor at 40. Tube contents (3.5 ml) were harvested from the bottom in 25 equal fractions. Hemoglobin (4.5 S, 64,500 daltons) and cytochrome c (1.7 S, 12,600 daltons) were used as standards. MACF activity was measured by incubation with gradient-purified membranes and the adenylate cyclase reaction mixture for 15 min at 300. Iodinated (125I-labeled) cholera toxin (100 jig) was prepared using carrier-free 125I and the chloramine-T method (4, 23). We achieved a specific activity of 0.68 MCi of 125I/ ,ug of cholera toxin. Iodinated cholera toxin was examined on 7.5% polyacrylamide gel electrophoresis containing sodium dodecyl sulfate (24). Fragment A (25,000 daltons) had 83% and fragment B (15,000 daltons) had 17% of the total radioactivity: Nonspecific background contamination in the entire gel accounted for less than 0.5% of the total radioactivity. Gel filtration was done with a Sephadex G-100 column (1.5 X 80 cm) equilibrated with 100 mM sodium phosphate buffer, pH 7.5.

400

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Cholera toxin activation of adenylate cyclase in cancer cell membrane fragments.

Activation of adenylate [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by cholera toxin (84,000 daltons, 5.5 S) is demonstrated in plasma membrane ...
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