Inhibition of Aggregation and Differentiation of Dictyostelium

Proc. Nat. Acad. Sci. USA Vol. 69, No. 5, pp. 1128-1130, May 1972

Inhibition of Aggregation and Differentiation of Dictyostelium discoideum by Antibodies Against Adenosine 3':5'-Cyclic Monophosphate Diesterase (amoeboid slime mold/chemotaxis/gel diffusion) EDMOND A. GOIDL*, BRUCE M. CHASSY, LESLIE L. LOVE, AND MICAH I. KRICHEVSKY * Department of Chemistry, American University, Washington, D.C. 20016; and Environmental Mechanisms Section, National Institute of Dental Research, National Institute of Health, Bethesda, Maryland 20014 Communicated by Marshall Nirenberg, February 25, 1972

ABSTRACT A specific antibody directed against cle pyruvate kinase (EC2.7.1.40), 0.25mM NADH (13-DPNH), adenosine 3': S'-cyclie monophosphate diesterase (ci. and 10 mM cAMP. Hydrolysis of cAMP was monitored con- AMP diesterase; EC 3.1.4.c), which is secreted by the NADH amoeboid slime mold, Dictyostelium discoldeum, was tinuously by observation of the resultant oxidation of piepared in rabbits. Purified gammaglobulin fractions at 340 nm in a 1 ml, 1 cm cuvette at 300, in a Gilford model that contain the antibodies inhibited the activity of 2400 recording spectrophotometer. cAMP diesterase preparations in vitro and interfered with aggregation and development in vivo. While cells that Photography. Photomicrographs were taken with a 10.1 X were treated with antibody were unable to aggregate be- 12.7 cm (4 X 5 in.) Polaroid camera mounted on a Zeiss- cause of the inability to destroy cAMP, they aggregated Tessovar. Polaroid Film Type 55 P/N was used. normally when washed free of antibody. Chemicals. 3':5'-cycic AMP, and adenosine triphosphate Amoebae of the class Acrasiales are capable of aggregation and were obtained from Schwarz Biochemical (Orangeburg, N.Y.). differentiation when challenged by changes in their environ- Myokinase, phosphoenol pyruvate, lactic acid dehydrogenase, ment, such as a shortage of food or a sudden drop in humidity NADH, and puruvate kinase were purchased from CalBio- (1). Adenosine 3': 5'-cyclic monophosphate (cAMP) (2) and chem (Los Angeles, Calif.). Rabbit 7S gammaglobulins were other 3': 5'-cyclic nucleotides (3) have a chemotactic effect on obtained from Pentex (Kankakee, Ill.). Freund's adjuvant the amoebae. The chemotaxis is followed by formation of was kindly provided by Dr. I. Green from the Laboratory of aggregates because cyclic nucleotides, or their metabolites, Immunology, NIAID, NIH. Mycobacterium tuberculosis make the amoebae mutually adhesive (1, 3). Both cAMP and HaRv was also provided by Dr. I. Green. AMP can trigger and stimulate other events associated with differentiation (4). The presence of a cAMP diesterase (EC Experimental Animals. White New Zealand Rabbits, 6 3.1.4b) (5, 6) makes it difficult to distinguish which effects are months old (2.5-4.0 kg) were obtained from the Animal Pro- mediated by cAMP per se. This cAMP diesterase probably duction Section, National Institutes of Health, Bethesda, Md. information passed between amoe- enhances the chemotactic Rabbits were inoculated with 500 1g of 95% bae; once a molecule of cAMP has exerted its attraction, it Immunizations. homogeneous 3':5'-cyclic cAMP diesterase (8) emulsified in might be metabolized to maintain the signal-to-noise ratio withM. tuberculosis HvRv in four here was performed to ascertain if the complete Freund's adjuvant (3). The study reported and were bled 30 after immuniza- action of cAMP diesterase is an obligate part of the chemo- sites, subcutaneously, days tactic process. The experimental course was to prepare an tion. antibody specific for cAMP diesterase, determine its anti- Gammaglobulin Fractionation. 7S Gammaglobulins were enzyme activity in vitro, and then observe its effect on amoe- precipitated at 40 by slow addition, with constant stirring, of boid aggregation. cold saturated ammonium sulfate to a final concentration of The MATERIALS AND METHODS 50%. The preparation was left to stir overnight. precipi- tate was collected by centrifugation at 10,000 X g, washed Growth and Harvesting. Dictyostelium discoideum, strain twice with cold 50% ammonium sulfate, and dissolved in NC-4j grown with Escherichia coli B as the food source in 0.01 M phosphate buffered saline (pH 7.4). The solution was complete medium, was used in these experiments (7). Amoe- dialyzed against 0.01 M phosphate buffered saline (4 changes bae, free of bacteria, were prepared by repeated differential of 100 volumes each). centrifugation (7). Washed amoebae were suspended in a small volume of distilled water. RESULTS Enzyme Assay. Enzyme activity was measured in a mixture Characterization of specificity of the antibody-containing containing purified enzyme, 0.1 M Tris-buffer (pH 7.4), 5 IU of fractions rabbit muscle myokinase (EC 2.7.4.3) (1 IU, International The center well of Ouchterlony plates was filled with 12 al of Unit, = 1 Mmol/min at 300), 1 mM adenosine triphosphate the immunizing antigen (cAMP diesterase). The peripheral (ATP), 5 mM phosphoenolpyruvate (PEP), 2 IU of lactic acid wells contained comparable volumes of the purified gamma- dehydrogenase (EC 1.1.2.7) (from rabbit), 2 IU of rabbit mus- globulin fractions from specific antisera to the diesterase. The 1128 Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Specific Antibodies to cAMIP Diesterase 1129

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P 9~~~ I FIG. 1. The effect of normal gammaglobulin on aggregation and differentiation. A and B represent amoebae 24 and 48 hr, respectively, after exposure to the gammaglobulin. (X40).

plates were left in a humid chamber at room temperature results were observed with controls containing water, buff- (230) for 8 hr and then kept at 40 overnight. A control dif- ered saline, or normal immunoglobulin. fusion plate with normal gammaglobulin and cAMP di- In order to test the effect of specific antibody on differentia- esterase under comparable experimental conditions gave no tion, 0.2 ml of purified gammaglobulins (8.0 mg/ml) obtained precipitin lines. The antibody-containing fractions gave single from the antiserum to cAMP diesterase was spread uniformly clear, sharp microprecipitin lines, indicating a high specificity on the agar 2 hr before placing the droplets that contained the toward the antigenic determinants of the cAMP. amoebae on the agar. At-the time the amoebae were placed on the agar no difference from the control with phosphate buffer In vitro effect of purified gammaglobulins obtained from specific antisera on cAMP diesterase activity was observed. There was no observable aggregation and no evidence of any differentiation after 12 hr. A few preaggre- Equal volumes of solutions of specific gammaglobulin (800 gates comparable in size to those seen in controls after 6 hr on ug/ml) or commercial rabbit 7S gammaglobulin (800 ug/ml) agar with phosphate buffer or normal gammaglobulin were and purified cAMP diesterase (100 pg/ml) were mixed and apparent after 24 hr (Fig. 2A). A few preaggregates were visi- allowed to stand at room temperature. At the end of 24-hr ble at 48 hr (Fig. 2B). This pattern is distinctly different from incubation, the reaction mixtures were centrifuged for 30 min that seen in the controls that fruited completely within 30 hr. at 4000 X g at 40; a measured aliquot of supernatant was mixed The amoebae did not differentiate further than the preag- with the substrate, and the oxidation of NADH was moni- gregate stage. tored at 340 nm. In 24 hr, the purified gammaglobulin preparation from the antiserum to cAMP diesterase lowered the Reversibility of the effect of antibody on differentiation enzyme activity by 84%, compared to the effect of gamma- and aggregation of D. discoideum globulin from normal rabbit serum. Normal serum or gamma- Amoebae were allowed to aggregate on Millipore filter pads globulin alone caused a transient stimulation in enzyme overlaid on fiberglass discs. The amoebae on discs containing activity. This stimulation was not observed with antibody- water or phosphate buffer fruited within 24 hr, while those on containing serum or antibody-containing gammaglobulin. discs containing 0.2 ml antibody (gammaglobulin) failed to aggregate after 48 hr. All filters were then removed from Effect of purified gammaglobulins obtained from a their specific antiserum on the development of D. discoideum petri dishes and placed on discs containing distilled water. At the end of 5 days, the amoebae that had been A solution (0.2 ml) of normal gammaglobulin (8 mg/ml) was inhibited by antibody began to differentiate, while those left spread over the surface of an entire agar plate and allowed to on antibody-containing discs remained unchanged. diffuse through the agar. After 4 hr, droplets (50 ,1) of amoebae were placed on the plate. Grex (preaggregates) and the DISCUSSION evidence of streaming left by migrating amoebae are clearly The basic hypothesis examined in this study is that cAMP visible at 24 hr (Fig. 1A). Final differentiation is observed by diesterase plays an obligate role in aggregation and differentia- 48 hr (Fig. 1B). The outcome is clearly "typical." Similar tion. Shaffer made this suggestion years before the identifica- Downloaded by guest on September 28, 2021 1130 Immunology: Goidl et al. Proc. Nat. Acad. Sci. USA 69 (1972) A I B

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FIG. 2. The effect of antiserum to cAMP diesterase containing gammaglobulins on D. discoideum. A and B represent amoebae 24 and 48 hr after exposure to the antiserum,. respectively. (X40). tion of the enzyme (9). Recent mathematical~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-models of Presented as a thesis by E. A. G. in partial fulfillment of the chemotaxis of amoeboid slime mold treat enzyme activity as requirements for the Master of Science degree granted by Amer- one of a set of controlling constants (10, 11), though recent ican University. evidence suggests that the activity is a variable throughout 1. Bonner, J. T. (1967) in The Cellular Slime Molds (Princeton differentiation and is not a constant (8, 12). The underlying University Press), 2nd ed., p. 11. concept is that, while cAMP presents the amoebae with 2. Konijn, T. M., van de Meene, J. G. C., Bonner, J.T. & directional information, the cAMP must be destroyed after it Barkley, D. S. (1967) Proc. Nat. Acad. Sci. USA 58, 1152- is sensed. The hydrolysis of cAMP enhances concentration 1154. gradients and eliminates multidirectional information. This 3. Chassy, B. M., Love, L. L. & Krichevsky, M. I. (1969) does not preclude the possibility that cAMP has other pro- Proc. Nat. Acad. Sci. USA 64, 296-303. 4. Bonner, J. T. (1970) Proc. Nat. Acad. Sci. USA 65, 110- found influences at the cell surface or within the cell. It is 113. possible that the response to cAMP, per se, requires a hy- 5. Chang, Y. Y. (1969) Science 161, 57-59. drolysis. If the foregoing argument is correct, a direct re- 6. Chassy, B. M., Love, L. L. & Krichevsky, M. I. (1969) moval of enzyme activity would inhibit the chemotactic Fed. Proc. 28, 842. process. 7. Krichevsky, M. I. & Wright, B. E. (1963) J. Gen. Microbiol. The experimental path chosen in this work was to prepare a 32, 195-207. highly specific antibody against cAMP diesterase. The 8. Chassy, B. M. (1972) Science 175, 1016-1018. was of inhibiting 9. Shaffer, B. M. (1956) Science 123, 1172-1173. purified antibody-containing fraction capable 10. Keller, E. F. & Segel, L. A. (1970) Nature 227, 1365-1366. the enzyme. The effect of the antibody on amoebae was di- 11. Keller, E. F. & Segel, L. A. (1970) J. Theor. Biol. 26, 399- rectly tested. Not only did the presence of antibody interfere 415. with aggregation (Fig. 2A and B), but its removal allowed 12. Pannbacker, R. & Bravard, L. (1972) Science 175, 1014- aggregation to proceed. 1015. Downloaded by guest on September 28, 2021