Effect of actomyosin on heat-induced gelation of myosin.

Abstract

Myosin and reconstituted actomyosin free of regulatory proteins were mixed in 0.6M KCl and 20 HIM phosphate buffer at pH 6.0 and were tested quantitatively for thermally induced gelation properties by measuring the rigidity of the system at 65°C. Full enhancement of gelation was attained when the weight ratio of myosin-to-actomyosin was about 4. The addition of regulatory proteins to actomyosin could restore calcium sensitivity of the contractile system, but did not affect the heat-induced gelation of myosin in the presence of actomyosin, suggesting that regulatory proteins play no role in the heat-induced gelation of the system. Neither the single and double headed subfragments, both capable of interacting with F-actin, nor the helical tail subfragments, devoid of the intracting site with F-actin, exhibited changes in thermogelling properties when mixed with F-actin. However, upon addition to F-actomyosin, the tail subfragments revealed a significant effect on the gelation of actomyosin, whereas the headed subfragments exerted no influence over gelation of the system. These results indicate that the enhancing effect of F-actin on the heat-induced gelation of myosin was brought about solely by the limited amount of F-actomyosin formed in the system, which acts as a cross-linker between the tail portion of bound and free myosin molecules.