Purification and characterization of 240-kDa cGMP-dependent protein kinase substrate of vascular smooth muscle. Close resemblance to inositol 1,4,5-trisphosphate receptor

• Published in: The Journal of biological chemistry 1994-04, Vol.269 (15), p.11640-11647
• Main Authors: KOGA, T, YOSHIDA, Y, JI-QUN CAI, MD OMEDUL ISLAM, IMAI, S
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Aorta - metabolism; Biological and medical sciences; Calcium Channels - isolation & purification; Calcium Channels - metabolism; Calmodulin - metabolism; Cell receptors; Cell structures and functions; Centrifugation, Density Gradient; Chromatography, Affinity; Cyclic GMP-Dependent Protein Kinases - metabolism; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Inositol 1,4,5-Trisphosphate - metabolism; Inositol 1,4,5-Trisphosphate Receptors; Kinetics; Microsomes - enzymology; Miscellaneous; Molecular and cellular biology; Molecular Sequence Data; Molecular Weight; Muscle, Smooth, Vascular - metabolism; Phosphorylation; Receptors, Cytoplasmic and Nuclear - isolation & purification; Receptors, Cytoplasmic and Nuclear - metabolism; Substrate Specificity; Swine; Transferases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)78173-3

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.