Purification and characterization of novel calmodulin-binding protein from cardiac muscle.

A novel protein which represents the most abundant calmodulin-binding protein in bovine heart cytosolic fraction was purified to apparent homogeneity. The purification procedure involved DEAE-Sepharose CL-6B (to remove calmodulin), calmodulin-Sepharose 4B affinity, and Sepharose 6B column chromatographies. This purified calmodulin-binding protein is a highly asymmetric protein with a sedimentation coefficient of approximately 5.0 S and a Stokes radius of about 83.0 A. The molecular weight of the calmodulin-binding protein was determined to be 175,000 from the sedimentation constant and Stokes radius of the protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein showed a single protein band with an apparent molecular weight of 140,000. The result suggests that the protein is monomeric. Although this molecular weight is similar to that of caldesmon, a known ubiquitous calmodulin-binding protein, the protein did not react with caldesmon-specific antibodies, nor did it display a proteolytic fragmentation pattern similar to that of the former. In addition, caldesmon was found almost exclusively in the particulate fraction in low ionic strength cardiac muscle extract, whereas this protein is purified the soluble fraction.