No abstract

SUMMARY In a search for additional Ca 2+ regulatory components in vascular smooth muscle, a novel troponin T-like protein was purified from bovine aorta smooth muscle. The isolated protein was separated into several isoforms on isoelectric focusing. The major isoelectric variants were focused in the pH region of 8.4 to 9.1. The protein had slightly different molecular masses in the M r range of 35,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its molar ratio relative to tropomyosin in the muscle extract was estimated to be 0.9 = 1.0. The novel protein bound to the immobilized calmodulin and exhibited a number of common physicochemical properties with gizzard (Af r = 34,000) calmodulin-binding and F-actin-binding protein. The aorta and gizzard proteins were immunologically cross-reactive. Both proteins shared a common antigenic determinant with COOHterminal segments of rabbit skeletal and bovine cardiac troponin T and bound to the immobilized smooth muscle tropomyosin. Both proteins interacted with rabbit skeletal troponin C in the presence and absence of Ca 2 + , but they did not interact with troponin I. These results suggest that the novel protein, which is designated calponin, may be a specialized component of smooth muscle thin filament involved in the regulation of contractile apparatus. (Hypertension 11: 620-626, 1988) KEYWORDS • vascular smooth muscle • troponin T • tropomyosin • calmodulin-binding protein * calcium T HE contractile process of the muscle is regulated by changes in the sarcoplasmic Ca 2+ concentration. In cardiac and skeletal muscles, actin-myosin interaction is regulated by a specific actin-linked mechanism, the troponin-tropomyosin system.'" 3 Recent studies have suggested that the contraction of vascular smooth muscle is regulated by a Ca 2+ -dependent mechanism in addition to myosin light chain phosphorylation.4 "*The maintenance of vascular smooth muscle tone characterized by a prolonged force production with low energy cost (latch contraction) 7 " 9 could be a major contributor to peripheral vascular resistance. To understand potential myogenic factors responsible for the hypertensive state, the regulatory protein (or proteins) in the second Ca 2+ -dependent mechanism must be identified.We have reported novel proteins with M, values of 33,000 to 35,000 that are present in large amounts in a variety of vertebrate smooth muscles.

Calponin inhibited the actin-activated myosin MgATPase activity in a dose-dependent manner without affecting the phosphorylation level of myosin light chain. This inhibition was Ca2(+)-independent. The decrease in enzymatic activity of myosin was correlated with binding of calponin to actin-tropomyosin filaments. Caldesmon showed a further inhibition of the calponin-induced inhibition of MgATPase activity of the thiophosphorylated myosin. Calponin-induced inhibition of the myosin MgATPase activity was reversed by the addition of calmodulin only in the presence of Ca2+. These results suggest that calponin acts as an inhibitory component of smooth muscle thin filaments.

Background: Calponin is a calmodulin-and actinbinding protein expressed in smooth muscle. It promotes actin polymerization and inhibits actinactivated myosin ATPase activity. Despite the molecular and functional characterization of calponin in vitro, the physiological role of calponin in vivo has not been clarified.