Abstract
We investigated the effects of a novel marine toxin, mycalolide B, on actin polymerization and actin-activated myosin Mg(2+)-ATPase activity using purified actin and myosin from rabbit skeletal muscle. The results were compared with cytochalasin D which inhibits actin polymerization by binding to the barbed end of F-actin. By monitoring fluorescent intensity of pyrenyl-actin, mycalolide B did not accelerate actin polymerization but quickly depolymerized F-actin, whereas cytochalasin D accelerated actin nucleation and depolymerized F-actin at slower rate. The kinetics of depolymerization suggest that mycalolide B severs F-actin. The relationship between the concentration of total actin and F-actin at different concentration of mycalolide B suggests that mycalolide B forms 1:1 complex with G-actin. Viscometry and electron microscopic observation further suggest that actin filament was depolymerized by mycalolide B. Unlike cytochalasin D, furthermore, mycalolide B suppressed actin-activated myosin Mg(2+)-ATPase activity. We concluded that mycalolide B severs F-actin and sequesters G-actin and may serve as a novel pharmacological tool for analyzing actin-mediated cell functions.
Highlights
Chemicals-Mycalolide B sp. by the method described was isolated from marine previously(Fusetani et al, sponge Mycale 1989)
The results lasin D and N-(1-pyrene)iodoacetamidewere obtained from Sigma and were compared with cytochalasDinwhich inhibits actin MolecularProbes (Eugene,OR), respectively.[Y-~~PIAwTaPs purchased polymerization by binding to the barbed end of F-actfirno.m Amersham (Backinghamshire,UK)
By monitoring fluorescent intensity of pyrenyl-actin, Preparation of Proteins-Actin was purified from rabbit skeletal mycalolide B did not accelerate actin polymerization muscle (Spudich and Watt, 1971) using buffer G containing 0.2 m but quickly depolymerized F-actin, whereas cytochala- CaCl, 0.2 m~ ATP, 0.5 m P-mercaptoethanol,and 2 mM Tris
Summary
Hartshorne, 1985).Measurements of acto-S1ATPase activity were performed by method described by Fiske and Subbarow (1925). The decay of fluorescence after thedilution of F-actin is olide B (Fig. 4B) This figurealsoshows that mycalolide B considered to be due to the net actin depolymerization from binds to G-actin in a 1:l molar ratio. Pretreatment the rateof depolymerization causedby cytochalasin D of F-actin withmycalolide B for 30 minbefore the dilution This may be rescent intensity wasdecreased by approximately 50% before because passage through the capillary of viscometer showed dilution) accelerated the subsequent depolymerization by similar effect topipettingin Fig. 2 and increased the dilution. In the absence of mycalolide B, long filaments of F-actin
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