ATPase In Smooth Muscle Research Articles

Myosin I from mammalian smooth muscle is regulated by caldesmon-calmodulin.

A single-headed monomeric myosin (myosin I) was isolated from pig urinary bladder smooth muscles and purified to homogeneity. Myosin I from smooth muscle is composed of a 110-kDa heavy chain and three 17-kDa light chains. The heavy chain from smooth muscle myosin I does not cross-react with the antibody against conventional myosin (myosin II) from smooth muscle, but it does show antigenic similarity to adrenal medulla myosin I heavy chain. The light chain from smooth muscle myosin I is similar to calmodulin in molecular weight, amino acid composition, and migration on SDS-polyacrylamide gel electrophoresis in the presence of Ca2+. The high salt ATPase activity of myosin I in the presence of CaCl2 is higher than that in K(+)-EDTA. Smooth muscle actin causes a 5-10-fold activation of the Mg-ATPase activity of myosin I. In the presence of Ca2+, exogenous calmodulin enhances the actin-activated ATPase activity of myosin I, and the increased activity is associated with the binding of exogenous calmodulin to myosin I heavy chain. A maximum of 4 mol of light chains/mol of myosin I heavy chain is observed in the presence of exogenous calmodulin. Caldesmon, a calmodulin/actin-binding protein, inhibits the actin-activated ATPase activity of myosin I. This inhibition is reversed by exogenous calmodulin in the presence of Ca2+. The actin activation of myosin I ATPase exhibits around 50% Ca2+ sensitivity in the presence of exogenous calmodulin. When caldesmon is bound to actin, Ca2+ sensitivity is increased to 80% in the presence of calmodulin. Therefore, smooth muscle caldesmon, which is thought to play a role in the regulation of actin activation of myosin II, also regulates the actin activation of myosin I ATPase in smooth muscle.

Inhibition of actin-activated myosin Mg 2+-ATPase in smooth muscle by Ruthenium red

Ruthenium red was found to inhibit actin-activated myosin Mg 2+-ATPase in smooth muscle and to bind to myosin heavy chain, but not to F-actin. The inhibition by Ruthenium red of actin-activated Mg 2+-ATPase was of the competitive type with respect to actin ( K i 4.4μM) and of the non-competitive type with respect to ATP ( K i 6.6μM). However, Ruthenium red scarcely dissociated the acto-heavy meromyosin complex during the ATPase reaction. These results suggest that Ruthenium red interacts directly with the binding site for F-actin on the myosin heavy chain. This site is considered to be necessary not for maintaining the binding affinity of myosin for F-actin, but for activation of the Mg 2+-ATPase.

Activation of actin-activated ATPase in smooth muscle by phosphorylation of myosin light chain with protease-activated kinase I.

The 20,000-dalton light chain of myosin from chicken gizzard has been shown to be phosphorylated in a Ca2+ and calmodulin-independent manner by the activated form of a protease-activated kinase from rabbit reticulocytes. Protease-activated kinase I incorporates phosphate stoichiometrically into the phosphorylatable light chain (P-light chain) in isolated myosin light chains and in actomyosin. The same serine residue appears to be phosphorylated by the protease-activated kinase and the Ca2+-dependent myosin light chain kinase. This conclusion is based on results from two-dimensional peptide maps of chymotryptic and tryptic digests of the phosphorylated P-light chain and from phosphoamino acid analysis of acid hydrolysates. Phosphorylation of the P-light chain by the proteolytically activated protein kinase stimulates the actin-activated Mg-ATPase activity of myosin in the absence of Ca2+. The extent of stimulation of the ATPase activity is similar to that observed upon phosphorylation of actomyosin by the Ca2+-dependent myosin light chain kinase. A proteolytically activated protein kinase with chromatographic properties and substrate specificity similar to protease-activated kinase I from reticulocytes has also been identified in gizzard. Protease-activated kinase I has been shown to be distinct from the Ca2+-dependent myosin light chain kinase by the mode of activation and specificity with other substrates, including phosphorylation of a unique site on myosin P-light chain from skeletal muscle (Tuazon, P. T., Stull, J. T., and Traugh, J. A. (1982) Biochem. Biophys. Res. Commun. 108, 910-917).