Effect of α-actinin on actin viscosity

Abstract

As determined by analytical ultracentrifugation, purified α-actinin does not form stable complexes with G-actin, myosin, tropomyosin, or the tropomyosintroponin complex. However, α-actinin forms a stable complex with F-actin polymerized either in 100 mM KC1 or in 2mM MgCl 2 without KCl. Viscosity studies confirm that α-actinin interacts as strongly with Mg 2+-polymerized actin as it does with KCl-polymerized actin. When measured at o°, addition of 0.02–0.05 parts of purified α-actinin to 1 part of actin, by weight, causes a 4–5-fold increase in specific viscosity of F-actin. At 37″, 0.10–0.20 parts of purified α-actinin to 1 part of actin are required to cause a 2–3-fold increase in specific viscosity of F-actin. Addition of α-actinin above this level at 37″, results in precipitation of F-actin. Tropomyosin (0.25 parts to 1 part actin, by weight) has no effect on the α-actinin-induced increase in F-actin viscosity at o° but almost completely abolishes the effect of α-actinin on F-actin viscosity at 37°. The effects of temperature on the α-actinin-induced increase in F-actin viscosity are completely reversible. Trypsin treatment of α-actinin-F-actin mixtures for 5 min at trypsin to actin ratios of 1:50, by weight, completely destroys the α-actinin-induced increase in F-actin viscosity at 37°. Subsequent incubation of the trypsin-treated mixture at o°, however, shows the ability of α-actinin to increase F-actin viscosity at o° is only partially destroyed by 15 min of trypsin digestion. Similar results are obtained by treating purified α-actinin with trypsin and then mixing the treated α-actinin with untreated actin. Neither temperature nor trypsin has any effect on viscosity of F-actin in the absence of α-actinin; hence, trypsin and temperature affect α-actinin and the α-actinin-F-actin interaction. The results are consistent with the hypothesis that at o° α-actinin cross-links F-actin by binding strongly both along the length and at one end of the F-actin strand. At 37°, however, strong binding of α-actinin is restricted primarily to one end of the F-actin strand, and any weak binding that may occur along the length of the F-actin strand is abolished by tropomyosin.