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Two major F\u2010actin\u2010binding proteins from <jats:italic>Dictyostelium discoideum<\/jats:italic>, a cytoplasmic 30 kDa protein and a 17 kDa integral membrane protein, and two minor membrane polypeptides of 19 kDa and 15 kDa were detected by this method. Using F\u2010actin affinity and immunoaffinity chromatography, the 17 kDa polypeptide was identified as ponticulin, a previously described actin\u2010binding glycoprotein from <jats:italic>D. discoideum<\/jats:italic> plasma membranes (Wuestehube, L.J., and Luna, E.J. [1987]: <jats:italic>J. Cell Biol.<\/jats:italic> 105:1741\u20131751). The binding of F\u2010actin to ponticulin on blots is specific because unlabeled F\u2010actin competes with <jats:sup>125<\/jats:sup>I\u2010labeled F\u2010actin and because G\u2010actin does not bind. Nitrocellulose\u2010bound ponticulin displays binding characteristics similar to those of purified plasma membranes in solution, e.g., F\u2010actin binding is sensitive to high salt and to elevated temperatures. Under optimal conditions, <jats:sup>125<\/jats:sup>I\u2010labeled F\u2010actin blot overlays are at least as sensitive as are immunoblots with an antibody specific for ponticulin. When blotted onto nitrocellulose after 2\u2010D gel electrophoresis, all isoforms of ponticulin and of the 19 kDa and 15 kDa polypeptides appear to bind F\u2010actin in proportion to their abundance. Thus the actin\u2010binding activities of these proteins do not appear to be regulated by modifications that affect isoelectric point. However, the actin\u2010binding activity of nitrocellulose\u2010bound ponticulin is diminished when the protein is exposed to reducing agents, suggesting an involvement of disulfide bond(s) in ponticulin function. The <jats:sup>125<\/jats:sup>I\u2010labeled F\u2010actin blot overlay assay also may enable us to identify F\u2010actin\u2010binding proteins in other cell types and should provide a convenient method for monitoring the purification of these proteins.<\/jats:p>","DOI":"10.1002\/cm.970180303","type":"journal-article","created":{"date-parts":[[2005,2,24]],"date-time":"2005-02-24T03:02:55Z","timestamp":1109214175000},"page":"164-179","source":"Crossref","is-referenced-by-count":25,"title":["Direct binding of F\u2010actin to ponticulin, an Integral plasma membrane glycoprotein"],"prefix":"10.1002","volume":"18","author":[{"given":"Catherine P.","family":"Chia","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anne L.","family":"Hitt","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elizabeth J.","family":"Luna","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2005,2,4]]},"reference":[{"key":"e_1_2_1_2_1","doi-asserted-by":"publisher","DOI":"10.1002\/cm.970140203"},{"key":"e_1_2_1_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/315410a0"},{"key":"e_1_2_1_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/0005-2736(86)90324-X"},{"key":"e_1_2_1_5_1","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.99.4.1434"},{"key":"e_1_2_1_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/0304-4157(89)90006-3"},{"key":"e_1_2_1_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(83)90382-3"},{"key":"e_1_2_1_8_1","doi-asserted-by":"publisher","DOI":"10.1002\/cm.970050608"},{"key":"e_1_2_1_9_1","doi-asserted-by":"publisher","DOI":"10.1002\/bies.950030206"},{"key":"e_1_2_1_10_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.80.19.5927"},{"key":"e_1_2_1_11_1","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.104.3.513"},{"key":"e_1_2_1_12_1","doi-asserted-by":"publisher","DOI":"10.1038\/335649a0"},{"key":"e_1_2_1_13_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.cb.04.110188.002415"},{"key":"e_1_2_1_14_1","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.100.6.1884"},{"key":"e_1_2_1_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/0304-4157(89)90017-8"},{"key":"e_1_2_1_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/0014-4827(89)90178-X"},{"key":"e_1_2_1_17_1","first-page":"267a","article-title":"Purification of ponticulin, an actin\u2010binding integral membrane protein, by Triton X\u2010114 extraction and its detection by [125]I\u2010labeled F\u2010actin on blot overlays","volume":"109","author":"Chia C. 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