Ca2+-dependent regulation of vascular smooth-muscle caldesmon by S.100 and related smooth-muscle proteins

Abstract

1. We have investigated the ability of bovine brain S.100, and of three related proteins from sheep aorta smooth muscle, to confer Ca2+-sensitivity on thin filaments reconstituted from smooth-muscle actin, tropomyosin and caldesmon. 2. At 37°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 μM when [caldesmon] = 2-3 μM in the assay mixture. When [KCl] was decreased to 70 mM, E.C.50= 11.5 μM; at 25°C in 70 mM-KCl, up to 20 μM-S.100 had no effect. When skeletal muscle actin rather than smooth-muscle actin was used to reconstitute thin filaments, 20 μM-S.100 did reverse inhibition by caldesmon, at 25°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 M(r)-17000 protein (yield 16 mg/kg), an abundant M(r)-11000 protein (yield 48 mg/kg), and an M(r)-9000 protein (yield 4 mg/kg). Neither of the last two low-M(r) proteins had any effect on activation of myosin MgATPase by reconstituted thin filaments. The protein of M(r)-17000 had Ca2+-sensitizing activity, and behaved exactly like brain calmodulin in the assay system.