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 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.
Koga
T
T
Department of Pharmacology, Niigata University School of Medicine, Japan.
Yoshida
Y
Y
Cai
J Q
JQ
Islam
M O
MO
Imai
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000378:metabolism; D015220:Calcium Channels / Q000302:isolation & purification* / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010766:Phosphorylation; D018160:Receptors, Cytoplasmic and Nuclear / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine
D015220:Calcium Channels; D002147:Calmodulin; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate; D017869:Cyclic GMP-Dependent Protein Kinases