Adrenal Chromaffin Cell Calmodulin: Its Subcellular Distribution and Binding to Chromaffin Granule Membrane Proteins

Bovine adrenal medullae were homogenized in the presence or in the absence of EGTA and different subcellular fractions were prepared by differential and density gradient centrifugations. In the presence of the chelating agent, 69% of the total calmodulin, measured by radioimmunoassay, was present in the cytosol; the rest was bound to different membrane-containing fractions (nuclei, microsomal, and crude granule fraction). When the chelating agent was omitted, 43% of the calmodulin was present in the cytosol, the remaining calmodulin being membrane-bound. Further resolution of the crude granule fraction by sucrose density centrifugation demonstrated that the distribution of calmodulin in the density gradient was similar to the distribution of chromaffin granules rather than to that of mitochondria, Golgi elements, and lysosomes. In this case, there was also more calmodulin bound to chromaffin granules when EGTA was omitted from the density gradient. Experiments with 125I-calmodulin indicated the presence of high-affinity binding sites (KD = 1.3 X 10(-8) M; Bmax = 30 pmol/mg protein) for calmodulin in chromaffin granule membranes. Further, photoaffinity crosslinking experiments with 125I-calmodulin followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography indicated the presence of three calmodulin-binding polypeptide complexes (84,000; 41,000; and 38,000 daltons) in chromaffin granule membranes. These polypeptides were not labelled when either Ca2+ was omitted or an excess of nonradioactive calmodulin was present in the photolysis buffer, indicating the Ca2+ dependency and the specificity of the interaction. On the basis of the results described, it is suggested that the cellular levels of Ca2+ control the cellular distribution of calmodulin and its binding to specific chromaffin granule membrane proteins. Further, it is also suggested that the interactions between calmodulin and granule proteins might play a role in stimulus-secretion coupling.