Effects of calcium and magnesium on the actomyosin adenosine 5'-triphosphatase of stably phosphorylated gizzard myosin

Abstract

There are conflicting reports on the effect of Ca2+ on actin activation of myosin adenosine-triphosphatase (ATPase) once the light chain is fully phosphorylated by a calcium calmodulin dependent kinase. Using thiophosphorylated gizzard myosin, Sherry et al. [Sherry, J. M. F., Gorecka, A., Aksoy, M. O., Dabrowska, R., & Hartshorne, D. J. (1978) Biochemistry 17, 4417-4418] observed that the actin activation of ATPase was not inhibited by the removal of Ca2+. Hence, it was suggested that the regulation of actomyosin ATPase activity of gizzard myosin by calcium occurs only via phosphorylation. In the present study, phosphorylated and thiophosphorylated myosins were prepared free of kinase and phosphatase activity; hence, the ATPase activity could be measured at various concentrations of Ca2+ and Mg2+ without affecting the level of phosphorylation. The ATPase activity of myosin was activated either by skeletal muscle or by gizzard actin at various concentrations of Mg2+ and either at pCa 5 or at pCa 8. The activation was sensitive to Ca2+ at low Mg2+ concentrations with both actins. Tropomyosin potentiated the actin-activated ATPase activity at all Mg2+ and Ca2+ concentrations. The calcium sensitivity of phosphorylated and thiophosphorylated myosin reconstituted with actin and tropomyosin was most pronounced at a free Mg2+ concentration of about 3 mM. The binding of 125I-tropomyosin to actin showed that the calcium sensitivity of ATPase observed at low Mg2+ concentration is not due to a calcium-mediated binding of tropomyosin to F-actin. The actin activation of both myosins was insensitive to Ca2+ when the Mg2+ concentration was increased above 5 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of arterial myosin in experimental renal hypertension in the dog.

“…34 Purified myosin from arteries of both control and hypertensive animals revealed light chains with apparent molecular weights of 20 and 17 kd. A similar observation was made for myosin purified from chicken gizzard, 25 -36 bovine stomach, 37 guinea pig vas deferens, 23 and swine pulmonary artery. 24 The electrophoretic mobility of these light chains is not altered in response to hypertension either on SDS-PAGE (data not shown) or on isoelectric focusing (Figure 2).…”

“…Actin and tropomyosin were prepared from chicken gizzard. 25 The yields of myosin from arteries of normotensive and hypertensive animals were similar. Approximately 2 mg 35-70% ammonium sulfate fractions per gram arterial media and 0.2 mg pure myosin per milligram 35-70% ammonium sulfate fraction were obtained from both groups.…”

“…43 The Ca 2+ -activated ATPase activity of smooth muscle myosin isolated from a variety of smooth muscles is lower than K + -EDTAstimulated activity. 23 - 25 Modification of the sulf hydryl group of the myosin is associated with changes in high salt ATPase activities. 43 In the present study, oxidation of the sulf hydryl groups was prevented by using dithiothreitol.…”

Mutagenesis Analysis of Functionally Important Domains within the C-terminal End of Smooth Muscle Caldesmon

“…Smooth muscle actin, CaD, myosin (fully phosphorylated), and tropomyosin were purified from chicken gizzard (31)(32)(33). Bovine brain calmodulin was prepared according to Dedman and Kaetzel (34).…”

Do thin filaments of smooth muscle contain calponin?

“…Smooth muscle myosin was purified from chicken gizzard using the method of Persechini et al, [21] and it was thiophosphorylated according to the method of Heaslip and Chacko [22]. The extent of phosphorylation was confirmed by urea gel electrophoresis.…”