Isolation and characterization of the inositol trisphosphate receptor from smooth muscle.
The release of Ca2+ from internal stores is requisite to muscle contraction. In skeletal muscle and heart, the Ca2+ release channels (ryanodine receptor) of sarcoplasmic reticulum, involved in excitation-contraction coupling, have recently been isolated and characterized. In smooth muscle, inositol 1,4,5-trisphosphate (IP3) is believed to mobilize Ca2+ from internal stores and thereby modulate contraction. We describe the isolation of an IP3 receptor from smooth muscle. Bovine aorta smooth muscle microsomes were solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and the IP3 receptor was purified by sucrose gradient centrifugation and column chromatography with heparin-agarose and wheat germ agglutinin-agarose. The purified receptor bound 2.7 +/- 0.18 nmol of IP3 per mg of protein with a Kd of 2.4 +/- 0.24 nM. That is, the purified receptor has been enriched about 1000-fold compared with the original microsomes, whereas the Kd for IP3 remains unchanged. The receptor is an oligomer of a single polypeptide with a Mr of 224,000 as determined by SDS/PAGE. Negative-staining electron microscopy reveals that the receptor is a large pinwheel-like structure having surface dimensions of approximately 250 X 250 A with fourfold symmetry. The IP3 receptor from smooth muscle is similar to the ryanodine receptor with regard to its large size and fourfold symmetry, albeit distinct with regard to appearance, protomer size, and ligand binding.
Chadwick
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CC
Department of Molecular Biology, Vanderbilt University, Nashville, TN 37235.
Saito
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A
Fleischer
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S
D000818:Animals; D001011:Aorta / Q000378:metabolism; D015220:Calcium Channels; D002417:Cattle; D002846:Chromatography, Affinity; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007700:Kinetics; D008854:Microscopy, Electron; D008861:Microsomes / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism / Q000648:ultrastructure; D018160:Receptors, Cytoplasmic and Nuclear; D012995:Solubility
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate