CYTOCHALASIN-B MAY SHORTEN ACTIN-FILAMENTS BY A MECHANISM INDEPENDENTOF BARBED END CAPPING

It is generally accepted that cytochalasin B (CB), as well as other cy tochalasins, shorten actin filaments by blocking monomer addition at t he fast-growing (''barbed'') end of these polymers. Despite the predom inance of this mechanism, recent evidence suggests that other interact ions may also occur between CB and F-actin. To investigate this possib ility further we have employed an actin derivative, prepared by substi tution at Cys(374) by a glutathionyl residue. We demonstrate here that CB did not significantly bind to glutathionyl F-actin under several i onic conditions. We further show that in the presence of CB the glutat hionyl-F-actin exhibits a significantly higher ATPase activity than th e non-modified F-actin. These data argue that the incorporation of glu tathionyl groups prevents the high-affinity binding of CB to the barbe d end of actin filaments, probably due to a decreased hydrophobicity o f the CB binding site by the introduction of the hydrophylic glutathio nyl residue. Despite the lack of substantial binding at equilibrium, w e have found that the addition of CB to glutathionyl-F-actin results i n extensive fragmentation of the filaments, as demonstrated by electro n microscopy and by a significant reduction of the relative viscosity of actin solutions. These results are consistent with the idea that CB shortens glutathionyl-actin filaments by a mechanism distinct from ba rbed end capping. Glutathionyl F-actin offers an interesting model to study the complex mechanism of interaction of actin filaments with cyt ochalasins and with the physiologically important actin capping/severi ng proteins.