Calcium‐Dependent Calmodulin Binding to Chromaffin Granule Membranes: Presence of a 65‐Kilodalton Calmodulin‐Binding Protein

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Abstract
The presence of calmodulin-binding sites on [cow] chromaffin granule membranes was investigated. Saturable, high-affinity 125I-calmodulin-binding sites (Kd = 9.8 nM; Bmax [maximum binding capacity] = 25 pmol/mg protein) were observed in the presence of 10-4 M free calcium. A second, nonsaturable, calmodulin-binding activity could also be detected at 10-7 M free calcium. No binding occurred at lower calcium levels. When chromaffin granule membranes were delipidated by solvent extraction, calmodulin binding was observed at 10-4 M free calcium. No binding was detected at lower calcium concentrations. A calcium concentration of 10-7 M promotes the binding of calmodulin to some solvent-soluble components of the chromaffin granule membrane. Calmodulin-binding proteins associated with the granule membrane were identified by photoaffinity cross-linking. A calmodulin-binding protein complex, of MW 82K [kilodalton], was formed in the presence of 104 M free calcium. This cross-linked product was specific because it was not detected either in the absence of calcium, in the presence of nonlabeled calmodulins or in the absence of cross-linker activation. When solvent-treated membranes were used, a second, specific, calmodulin-binding protein complex (70K) was formed. Since the apparent MW of calmodulin in the electrophoresis system was 17K, 2 calmodulin-binding proteins of MW 65K and 53K are present in the chromaffin granule membrane. This result was confirmed by the use of calmodulin-affinity chromatography. When detergent-solubilized membranes were applied on the column in the presence of calcium, 2 polypeptides of apparent MW of 65K and 53K were specifically eluted by EGTA [ethylene glycol bis (.beta.-amino ethyl ether) N,N,N'',N''-tetraacetic acid] buffers. Since detergent treatments or solvent extractions are necessary to detect the 53K calmodulin-binding protein, only the 65K calmodulin-binding polypeptide may play a role in the interaction between calmodulin and secretory granules in chromaffin cells.