In most published studies of actin polymerization, the identity of the tightly-bound divalent cation has been poorly characterized. This is significant because Ca++ is present in most actin preparations, but the tightly bound divalent cation in vivo is most likely Mg++. We have very recently published findings that attribute a portion of the lag phase in the polymerization of Ca-actin by added MgCl2 to actin bound divalent cation exchange. We have also recently reported that the ration of forward polymerization rate constants, K+Mg/K+Ca, has a value around 2 and that the critical actin concentration (CAC) for Ca-actin is 10-20 x the CAC for Mg-actin. Based on this work, we propose further research comparing Mg-actin with Ca-actin. The mk+, CAC, and mk-values for both types of actin will be determined at a variety of conditions using pyrene-labeled actin monomer. The average polymer lengths of Mg-actin and of Ca-actin will be measured by electron microscopy, thereby allowing the number concentration of polymer ends, m, to be calculated. Differences in the binding of tropomyosin to Mg-F-actin and Ca-F-actin will be sought. We will also study the ability of troponin to regulate the myosin subfragment 1 (S-1) ATPase activity activated by both types of actin. In preliminary work we have found that monomeric Ca-actin activates the S-1 ATPase activity significantly more than does Mg-actin. This interesting finding implies a structural difference between Ca-actin and Mg-actin; we will study this further by using DNase I inhibition by actin and SH reactivity as structural probes. The technique of applied hydrostatic pressure will be used to measure the volume change of activation DeltaV*, for the elongation reactions of Mg-actin and Ca-actin. The effect of actin-bound divalent actin on oligomer formation (nucleation) will also be investigated using a unique pyrene-labeled monomer actin assay. From these studies, a clearer understanding of the role of the actin-bound cation in the reactions of actin will be gained as well as a better characterization of the properties of actin.
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