Direct and Enzyme-mediated Photoaffkity Labeling of Membrane- associated Actin in Dictyostelium discoideum *

A single specifically labeled protein (Mr = 42,000) is found when Dictyostelium discoideum crude membrane (ghost) preparations are photoaffinity-labeled with either [32P]8N3-cAMP or [32P]8N3-AMP. With the CAMP reagent, there is a close correlation between the labeling of this protein and the membrane phosphodiesterase activity. During cell differentiation the developmental time courkes of the photoaffinity labeling of the M, = 42,000 protein and the membrane phosphodiesterase are identical. In addition, cyclic nucleotide substrates of the membrane phosphodiesterase also inhibit the photoaffinity labeling of the M, = 42,000 protein. AMP, which is a poor inhibitor of the membrane phosphodiesterase, is a very effective inhibitor of the photoaffinity labeling. These results suggest that the M, = 42,000 protein is labeled by [32P]8N+UVIP produced by the action of membrane phosphodiesterase on [32P]8N3CAMP. The membrane phosphodiesterase of ghosts does in fact mediate this conversion. When ghosts are photoaffinity-labeled with [32P]8N3-AMP, the M, = 42,000 protein is again specifically labeled. In contrast to the results with the CAMP photoaffinity reagent, labeling with the AMP ligand is inhibited only by the noncyclic adenine nucleotides AMP, ADP, and ATP. The protein containing this AMP binding site is quantitatively extracted from the membrane with 0.6 M KI (but not detergents) and quantitatively binds to DNase I. Tryptic peptide maps of the photoaffinity-labeled protein indicate a relatively discrete localization of the attached radioactivity. Photoaffinity-labeled peptides co-migrate on sodium dodecyl sulfate gels with peptides derived from D. discoideum membrane-associated actin. These properties identify the labeled protein as membrane-associated actin. The photoaffinity-labeled AMP binding site may be distinct from the ATP binding site of actin.