Ca(2+)-dependent regulation of vascular smooth-muscle caldesmon by S.100 and related smooth-muscle proteins.
1. We have investigated the ability of bovine brain S.100, and of three related proteins from sheep aorta smooth muscle, to confer Ca(2+)-sensitivity on thin filaments reconstituted from smooth-muscle actin, tropomyosin and caldesmon. 2. At 37 degrees C in pH 7.0 buffer containing 120 mM-KCl, approximately stoichiometric amounts of S.100 reversed caldesmon's inhibition of the activation of myosin MgATPase by smooth-muscle actin-tropomyosin. The [S.100] which reversed by 50% the inhibition by caldesmon (the E.C.50) was 2.5 microM when [caldesmon] = 2-3 microM in the assay mixture. When [KCl] was decreased to 70 mM, E.C.50 = 11.5 microM; at 25 degrees C in 70 mM-KCl, up to 20 microM-S.100 had no effect. When skeletal-muscle actin rather than smooth-muscle actin was used to reconstitute thin filaments, 20 microM-S.100 did reverse inhibition by caldesmon, at 25 degrees C in buffer containing 70 mM-KCl. This dependence on conditions is also characteristic of the calmodulin-caldesmon interaction. 3. These results suggested that S.100 or a related protein might interact with caldesmon in smooth muscle. We therefore attempted to prepare such a protein from sheep aorta. Three proteins were purified: an Mr-17,000 protein (yield 16 mg/kg), an abundant Mr-11,000 protein (yield 48 mg/kg), and an Mr-9000 protein (yield 4 mg/kg). Neither of the last two low-Mr proteins had any effect on activation of myosin MgATPase by reconstituted thin filaments. The protein of Mr 17,000 had Ca(2+)-sensitizing activity, and behaved exactly like brain calmodulin in the assay system.
Pritchard
K
K
Cardiac Medicine Department, National Heart and Lung Institute, London, U.K.
Marston
S B
SB
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D001011:Aorta / Q000737:chemistry; D001921:Brain; D002118:Calcium / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D066298:In Vitro Techniques; D009130:Muscle, Smooth / Q000378:metabolism*; D009218:Myosins / Q000037:antagonists & inhibitors; D009418:S100 Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012756:Sheep
D002148:Calmodulin-Binding Proteins; D009418:S100 Proteins; D009218:Myosins; D002118:Calcium