| Research Abstract |
The 240 kDa, cGMP-dependent protein kinase (G-kinase) substrate obtained from porcine aortic smooth muscle as a protein, whose phosphorylation was closely associated with stimulation of plasma membrance Ca^<2+>-pump ATPase(J.Biol.Chem., 266,19819-19825,1990), was purified to near homogeneity by three successive chromatographic runs with calmodulin -, concanavalin A - and heparin-Sepharose columns from Triton X-100 solubilzed microsomes. The purified protein was found to bind inositol 1,4,5-trisphosphate (IP^3)in a specific, heparin-inhibitable manner (Kd : 2.0nM ; Bmax : 450 pmol/mg protein). The protein also bound inositol 1,3,4,5, -tetrakisphosphate, though weakly. In sedimentation experiments on a linear sucrose density gradient the IP^3 binding activity was always with the 240-kDa protein. G-kinase phosphorylated the IP^3 receptor purified from 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 IP^3 receptor. Threr were, however, some differences : On SDS-PAGE the 240-kDa protein presented itself as two polypeptides with similar, but slightly differing Mr's both of which could be phosphlrylated by G-kinase, while cerebellar IP^3 receptor presented itself as a single band, and only the 240 kDa protein was found to bind with calmodulin. A certain immunological differences were also found. Thus, the 240 kDa protein may be a new member of IP^3 receptor superfamily.
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