A similar calmodulin-binding protein expressed in chromaffin, synaptic, and neurohypophyseal secretory vesicles

The presence of calmodulin-binding proteins in three neurosecretory vesicles (bovine adrenal chromaffin granules, bovine posterior pituitary secretory granules, and rat brain synaptic vesicles) was investigated. When detergent-solubilized membrane proteins from each type of secretory organelle were applied to calmodulin-affinity columns in the presence of calcium, several calmodulin-binding proteins were retained and these were eluted by EGTA from the columns. In all three membranes, a 65-kilodalton (63 kilodaltons in rat brain synaptic vesicles) and a 53-kilodalton protein were found consistently in the EGTA eluate. 125I-Calmodulin overlay tests on nitrocellulose sheets containing transferred chromaffin and posterior pituitary secretory granule membrane proteins showed a similarity in the protein bands labeled with radioactive calmodulin. In the presence of 10(-4) M calcium, eight major protein bands (240, 180, 145, 125, 65, 60, 53, and 49 kilodaltons) were labeled with 125I-calmodulin. The presence of 10 microM trifluoperazine (a calmodulin antagonist) significantly reduced this labeling, while no labeling was seen in the presence of 1 mM EGTA. Two monoclonal antibodies (mAb 30, mAb 48), previously shown to react with a cholinergic synaptic vesicle membrane protein of approximate molecular mass of 65 kilodaltons, were tested on total membrane proteins from the three different secretory vesicles and on calmodulin-binding proteins isolated from these membranes using calmodulin-affinity chromatography. Both monoclonal antibodies reacted with a 65-kilodalton protein present in membranes from chromaffin and posterior pituitary secretory granules and with a 63-kilodalton protein present in rat brain synaptic vesicle membranes. When the immunoblotting was repeated on secretory vesicle membrane calmodulin-binding proteins isolated by calmodulin-affinity chromatography, an identical staining pattern was obtained. These results clearly indicate that an immunologically identical calmodulin-binding protein is expressed in at least three different neurosecretory vesicle types, thus suggesting a common role for this protein in secretory vesicle function.