Characterization and localization of adenylate cyclase during development of Dictyostelium discoideum

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Virginia Polytechnic Institute and State University

Cyclic AMP functions as the chemotactic signal during aggregation of single-celled amoebae of the cellular slime mold Dictyostelium discoideum. Evidence suggests that cyclic AMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. Biochemical characterization of adenylate cyclase, the cyclic AMP synthetic enzyme, was accomplished using a sensitive radioimmunoassay. The enzyme was found to be pellet-bound. The non-ionic detergents, Triton X-100 and Lubrol PX, were not effective for solubilizing this activity. Magnesium or manganese could serve as the required divalent cation, with the Mn-supported activity over 4-fold greater than the Mg-supported activity. Typical mammalian adenylate cyclase modulators such as guanyl nucleotides, fluoride, and cholera toxin did not activate the Dictyostelium enzyme. Calcium, in conjunction with its protein receptor calmodulin, did not appear to regulate the enzyme. An endogenous extracellular, heat-stable substance was found to inhibit Dictyostelium adenylate cyclase.

By use of ultramicrotechniques adenylate cyclase activity was localized in the pre-spore cells of the culminating individual with no activity detected in the pre-stalk region. Lack of detectable activity in the pre-stalk cells may be due to a masking by the endogenous inhibitor. An increasing gradient of activity was found in the pre-spore mass with activity increasing from the uppermost area to the base. No striking localization was seen prior to the pre-culmination stage of development. Two peaks in cyclic AMP levels, as measured in individuals were found during development. One coincided with aggregation, the other occurred at the culmination stage. A gradient of cyclic AMP within the culminating individual paralleled the gradient of adenylate cyclase activity. The tip of the individual had greater levels of cyclic AMP than the middle pre-spore region, and the upper stalks had higher levels than the lower stalks.

These data indicate an enzymatic potential for establishing a gradient of cyclic AMP. At the culmination stage of development this molecule could act to direct the chemotactic movements of the pre-stalk cells as well as provide positional information for the terminal differentiation of the pre-spore cells into mature spores.