Purification and subunit structure of bovine brain modulator binding protein.

A protein which can associate with the Ca”-dependent modulator protein resulting in the inhibition of the Ca’+-activatable cyclic nucleotide phosphodiesterase has been discovered recently and designated as modulator binding protein (Wang, J. H., and Desai, R. (1977) J. Biol. Chem. 252,4175-4183; Klee, C. B., and Krinks, M. H. (1978) Biochemistry 17, 120-126). This protein is purified from bovine brain to near homogeneity. The purification procedure is based mainly on the Ca’+dependent association of this protein with the highly acidic modulator protein. The purified modulator binding protein is shown to be a globular protein of molecular weight 85,000. It contains two distinct subunits, subunits A and B, of molecular weights about 60,000 and 14,500, respectively. The mass ratio of subunits A/ B is determined to be about 2.3 : 1. From these results, the subunit structure of modulator binding protein is suggested to be AB2. In the presence of 6 M urea, modulator binding protein dissociates into its constituent subunits, and subunits A and B can be separated on a G-100 Sephadex column. The isolated subunit A exhibits inhibitory activity against the Ca”-activatable cyclic nucleotide phosphodiesterase. Subunit B has no phosphodiesterase inhibiting activity, nor does it affect the activity of subunit A in the enzyme reaction. These results suggest that subunit A is responsible for the association of modulator binding protein to the modulator protein. The function of subunit B is not known at present.