Binding of actin to liver cell membranes: the state of membrane-bound actin.
Previous work has shown that actin binds specifically and saturably to liver membranes stripped of endogenous actin (Tranter, M. P., S. P. Sugrue, and M. A. Schwartz. 1989. J. Cell Biol. 109:2833-2840). Scatchard plots of equilibrium binding data were linear, indicating that binding is not cooperative, as would be expected for F- or G-actin. To determine the state of membrane-bound actin, we have analyzed the binding of F- and G-actin to liver cell membranes. G-actin in low salt depolymerization buffer and EF-actin, a derivative that polymerizes very poorly in solution, bind to liver cell membranes as well as untreated actin in polymerization buffer. Phalloidin-stabilized F-actin binds, but to a lesser extent. The binding of F- and G-actins are mutually competitive and are inhibited by ATP, suggesting that both forms of actin bind to the same sites. For untreated actin in polymerization buffer, the time course of binding is biphasic, with an initial rapid component which is followed by a plateau phase, then a second, slower component. The binding kinetics of pure F-actin and pure G-actin are both monophasic and match the fast and slower components, respectively, of untreated actin. In the reconstituted system, membrane-bound actin does not stain with rhodamine-phalloidin, nor are actin filaments detected by EM. Distinct regions of amorphous material, however, are visible, which stain with an anti-actin antibody. The exact nature of this material has yet to be determined. A model of actin binding is presented.
Tranter
M P
MP
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 02115.
Sugrue
S P
SP
Schwartz
M A
MA
D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D008954:Models, Biological; D010590:Phalloidine; D011108:Polymers; D011817:Rabbits; D012235:Rhodamines; D013194:Staining and Labeling; D013552:Swine
D000199:Actins; D011108:Polymers; D012235:Rhodamines; D010590:Phalloidine; D000255:Adenosine Triphosphate