PURIFICATION AND CHARACTERIZATION OF 240-KDA CGMP-DEPENDENT PROTEIN-KINASE SUBSTRATE OF VASCULAR SMOOTH-MUSCLE - CLOSE RESEMBLANCE TO INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR

The 240-kDa, cGMP-dependent protein kinase substrate protein obtained from porcine aortic smooth muscle, whose phosphorylation was closely a ssociated with stimulation of plasma membrane Ca2+-pump ATPase (Yoshid a, 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 ch romatographic runs with calmodulin-, concanavalin A-, and heparin-Seph arose columns from microsomes solubilized with Triton X-100. The purif ied protein was found to bind inositol 1,4,5-trisphosphate (InsP3) in a specific, heparin-inhibitable manner with a K(d) of 2.0 nM and B(max ) 450 pmol/mg protein (the binding of inositol 1,3,4,5-tetrakisphospha te was much weaker). In sedimentation experiments on a linear sucrose density gradient the InsP3 binding activity was always with the 240-kD a protein. Protein kinase G phosphorylated the InsP3 receptor purified from the rat cerebellum as well as the 240-kDa protein. Sialic acid c ontent 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-po lyacrylamide 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.