Localization of adenylate cyclase in Dictyostelium discoideum. I. Preparation and biochemical characterizations of cell fractions and isolated plasma membrane vesicles.

A difference in the organization of adenylate cyclase and 3′5′-cyclic phosphodiesterase in isolated plasma membranes was observed. Observation of this difference was made possible by the development of a new technique for the lysis of Dictyostelium discoideum using the polyene antibiotic amphotericin B. A particulate fraction prepared from the cell lysate contains adenylate cyclase, 3′5′-cyclic phosphodiesterase and 5′-nucleotidase. The yield of adenylate cyclase is 40% higher than in paniculate fractions prepared from cells lysed by sonication or with Triton X-100. Purification of the particulate fraction on discontinuous sucrose gradient completely separates membranes from mitochondria and other cellular material as shown by electron microscopic analysis of different fractions. Biochemical characterization of the purified membrane fraction shows it contains adenylate cyclase, 3′5′-cyclic phosphodiesterase and 5′-nucleotidase activities while electron microscopic analysis shows a vesicular morphology. Additional studies on the purified membranes used Triton X-100, trypsin and phospholipase C to probe the relationship between membrane structural elements and enzymatic activities. The results of these studies show distinct differences in the organization of each enzyme molecule within the membrane.