Using antibodies against Dictyostelium membranes to identify an actin-binding membrane protein.
Polyclonal antibodies made against Dictyostelium discoideum membranes were used to block the interaction of those membranes with actin. As expected, actin interacted mostly with the internal surface of the membrane, demonstrated by the fact that whole cells could only absorb out a minor fraction of the blocking antibody. The antibody was used to show that the membrane component(s) which interacted with actin were probably integral; they could be extracted with detergent but not with solutions designed to extract peripheral membrane proteins. To identify the responsible protein(s), Western transfers of membranes were cut into fractions which were tested for their ability to absorb out the blocking activity of the antibody. We observed a single peak at a molecular weight of approximately 20,000, and thus conclude that a 20,000-mol-wt protein is a major integral membrane actin-binding protein in Dictyostelium. This approach to the identification of proteins involved in actin-membrane interaction has allowed us to make the first identification of an actin-binding membrane protein which is based on its activity in native membranes.
D000199:Actins / Q000378:metabolism*; D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004023:Dictyostelium / Q000378:metabolism*; D018260:Gelsolin; D007140:Immunoglobulin Fab Fragments; D007700:Kinetics; D008840:Microfilament Proteins; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits
D000199:Actins; D000906:Antibodies; D000936:Antigen-Antibody Complex; D002352:Carrier Proteins; D018260:Gelsolin; D007140:Immunoglobulin Fab Fragments; D008840:Microfilament Proteins; C016953:brevin