Abstract
A wide range of values has been reported for the subunit and molecular weights of smooth muscle caldesmon. There have also been conflicting reports concerning whether caldesmon is a monomer or dimer. We attempted to resolve these uncertainties by determining the molecular weight of chicken gizzard smooth muscle caldesmon using the technique of sedimentation equilibrium in the analytical ultracentrifuge. Unlike previous methods that have been used to estimate the molecular weight of caldesmon, the molecular weight determined by equilibrium sedimentation does not depend upon assumptions about the shape of the molecule. We concluded that caldesmon in solution is monomeric with a molecular mass of 93 +/- 4 kDa, a value that is much less than those previously reported in the literature. This new value, in conjunction with sedimentation velocity experiments, led to the conclusion that caldesmon is a highly asymmetric molecule with an apparent length of 740 A in solution. The mass of a cyanogen bromide fragment, with an apparent mass of 37 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was determined to be 25.1 +/- 0.6 kDa using sedimentation equilibrium. These results imply that the reported molecular weights of other fragment(s) of caldesmon have also been overestimated. We have determined an optical extinction coefficient for caldesmon (E1%(280 nm) = 3.3) by determining its concentration from its refractive index which was measured in the analytical ultracentrifuge. From the above values of the molecular weight and the extinction coefficient, we redetermined that the caldesmon molecule has two cysteines and recalculated the stoichiometric molar ratio of actin/tropomyosin/caldesmon in the smooth muscle thin filament to be 28:4:1.
Highlights
It has been suggested that the actin thin filament is switched on by the binding of Ca2+to calmodulin which in turn binds to actin-bound caldesmon, resulting in the release of caldesmon’s inhibition of the actomyosin ATPase activity [3,4,5,6,7,8]
Molecular Weight andSubunit Composition-In equilibrium centrifugation runs, caldesmon in 0.1 M NaCl, 0.5 mM EDTA, 1 mM DTT, 5 mM Mops, pH 7, behaved as a single, monodisperse, nonideal component with a molecular mass of 93 & 4 kDa (Fig. 1).Fig. 2A shows sodium dodecyl sulfate (SDS) gels on a sample before and after a run indicating that no significant proteolytic degradation had occurred during the run
CyanogenBromideFragment-The molecular weight which we have determined for the intactcaldesmon molecule implies that themolecular weight of some or all of its fragments [47, 48] have been overestimated by using gel electrophoresis. In support of this idea, we found that a cyanogen bromide fragment, which migrates with an apparent molecular mass of about 37 kDa on SDS gel electrophoresis (Fig. 4A) and which retains the ability to bind calmodulin and F-actin [27]
Summary
A wide range of values has been reported for the subunit and molecular weights of smooth muscle caldesmon. In order to understand the molecular mechanisms underlying thin filament regulation, it is necessary to determine the binding parameters of caldesmon’s interaction with these various proteins and to correlate themwith caldesmon’s effect on actomyosin ATPase activity For these and other studies, it is necessary to know caldesmon’s subunit composition, moleccyanogen bromide fragment, with an apparemntass of ular weight, and concentration. We set out to determine the native molecular weight and subunit composition of caldesmon by means of sedimentation equilibrium in theanalytical ultracentrifuge, a method which depends neither on the shape of the molecule nor on the molecule’s ability to bind SDS.
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