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Two smooth muscle myosin heavy chain isoforms differ in their amino terminus by the presence [(+)insert] or absence [(-)insert] of a seven-amino acid insert. Animal studies show that the (+)insert isoform is predominantly expressed in rapidly contracting phasic muscle and the (-)insert isoform is mostly found in slowly contracting tonic muscle. The expression of the (+)insert isoform has never been demonstrated in human smooth muscle. We hypothesized that the (+)insert isoform is present in humans and that its expression is commensurate with the organ's functional requirements. We report, for the first time, the sequence of the human (+)insert isoform and quantification of its expression by real-time PCR and Western blot analysis in a panel of human organs. The (+)insert isoform mRNA and protein expression levels are significantly greater in small intestine compared with all organs studied except for trachea and are significantly greater in trachea compared with uterus and aorta. To assess the functional significance of this differential myosin isoform expression between organs, we measured the rate of actin filament movement ([v.sub.max]) when propelled by myosin purified from rat organs, because the rat and human inserts are identical and their remaining sequences show 93% identity. [v.sub.max] exhibits a rank correlation from the most tonic to the most phasic organ. The selective expression of the (+)insert isoform observed among human organs suggests that it is an important determinant of tissue shortening velocity. A differential expression of the (+)insert isoform could also account for altered contractile properties observed in human pathology. phasic and tonic smooth muscle; real-time polymerase chain reaction; in vitro motility assay

Autophosphorylation of smooth muscle myosin light chain kinase (MLCKase) occurs in the presence of Ca2+ and calmodulin (CM), and shows that dimerization of the kinase is involved in the autocatalytic reaction. The main autophosphorylation site is identified as Thr-863 or Thr-865 and is located on the 24-kDa C-terminal fragment of the kinase. The initial phosphorylation rates and maximal incorporation levels are highest at a molar ratio of 2 MLCKase to 1 CM.

Studies conducted on turkey gizzard myosin light chain kinase (MLCKase) show that MLCKase activity and the affinity for calmodulin decreases in the presence of Ca2+/calmodulin (CM), but calmodulin increases the activity. Cross-linking experiments show that the kinase undergoes oligomerization, and the oligomers affinity for CM is more than the affinity shown by the dimers. The kinase forms dimers readily, which are in equilibrium with the monomeres and oligomeres, and the activity of MLCKase is also controlled by the interactions of the monomeres, even in the absence of CM.

Ammonium sulfate fractionation and divalent cation precipitation were applied to purify major contractile proteins from relaxed actomyosin extracted from molluscan catch muscle myofibrils, as a supramolecular complex composed of twitchin (TW), myosin (MY), and myorod (MR). The phosphorylation of twitchin by PKA-like kinase and the phosphorylation of myosin and myorod by myosin-associated kinase were found to play crucial role in the development of the catch state.

The effects of changes in Mg2+ concentration on the kinetics of stretch activation were investigated on skinned rat heart preparations under maximal Ca2+ activation. Muscle strips of hyper- and hypothyroid rat hearts were investigated at 0.5 and 1 mM free Mg2+; the total ATP concentration was 8 mM which resulted in saturating MgATP2- concentrations above 5 mM. Preparations containing exclusively the cardiac α-myosin heavy chain (hyper- and hypothyroid atria, hyperthyroid ventricles) showed an acceleration of the kinetics of stretch activation by a factor of about 1.5 (P<0.01, paired t-test) when free Mg2+ was decreased from 1 to 0.5 mM. Conversely, preparations containing exclusively the β-myosin heavy chain isoform showed only a small acceleration by a factor of 1.05 (P<0.05, paired t-test) under the same conditions. The fact that the Mg2+ sensitivity was dependent on the myosin heavy chain isoform excludes the possibility that Mg2+ exhibits only unspecific effects on contractile proteins. Several hypotheses for explaining the observed Mg2+ effects are discussed. The conditions used in our experiments might be close to the physiological situation and, thus, changes of Mg2+ concentration must be considered as possible factors modulating the contractile kinetics especially of atrial muscle tissue. [ABSTRACT FROM AUTHOR]