Abstract

The 240-kDa, cGMP-dependent protein kinase substrate protein obtained from porcine aortic smooth muscle, whose phosphorylation was closely associated with stimulation of plasma membrane Ca(2+)-pump ATPase (Yoshida, Y., Sun, H.-T., Cai, J.-Q., and Imai, S. (1991) J. Biol. Chem. 266, 19819-19825), was purified to near homogeneity by three successive chromatographic runs with calmodulin-, concanavalin A-, and heparin-Sepharose columns from microsomes solubilized with Triton X-100. The purified protein was found to bind inositol 1,4,5-trisphosphate (InsP3) in a specific, heparin-inhibitable manner with a Kd of 2.0 nM and Bmax of 450 pmol/mg protein (the binding of inositol 1,3,4,5-tetrakisphosphate was much weaker). In sedimentation experiments on a linear sucrose density gradient the InsP3 binding activity was always with the 240-kDa protein. Protein kinase G phosphorylated the InsP3 receptor purified from the rat cerebellum as well as the 240-kDa protein. Sialic acid content of the protein measured with Limulus polyphemus agglutinin was not significantly different from that of the cerebellar InsP3 receptor. Thus, 240-kDa protein closely resembles InsP3 receptor and may be a type of InsP3 receptor. The only difference was the behavior on SDS-polyacrylamide gel electrophoresis. The 240-kDa protein presented itself as two polypeptides with similar but slightly differing M(r) values, both of which were phosphorylated by protein kinase G.