Definition of an N-terminal actin-binding domain and a C-terminal Ca2+ regulatory domain in human brevin.
Brevin is a Ca2+-modulated actin-associated protein that will sever F-actin and cap barbed filament ends. Limited proteolysis with chymotrypsin or subtilisin cleaves the molecule approximately in half. Cleavage is approximately 10-fold more rapid in Ca2+ than in EGTA. The two fragments are readily separated from each other and from undigested brevin by high pressure liquid chromatography on a DEAE resin. A 40,000-mol-wt fragment from the N-terminal is not retained by DEAE, while a 45,000-mol-wt C-terminal fragment binds more tightly than brevin. The N-terminal fragment retains approximately 10% of the nucleation activity, caps barbed ends, and retains 50% of the total severing activity defined by dilution induced depolymerization of pyrenyl actin, but, in contrast to brevin, none of these functions are affected by Ca2+. Fluorescent actin binding studies and gel-filtration demonstrate that the 40,000-mol-wt fragment binds two actin monomers. The 45,000-mol-wt C-terminal fragment has no severing, nucleating, or capping activity. Cross-reaction with two monoclonal antibodies against two specific Ca2+-induced conformations of human platelet gelsolin suggest that both Ca2+ binding sites are located on the carboxyl half of the brevin molecule. One epitope, defined as the rapidly exchanging Ca2+ binding site in the gelsolin-actin complex, is lost when a 20,000-mol-wt fragment is cleaved from the carboxyl terminal. The second epitope, related to the poorly exchanging Ca2+ binding site in the complex, is nearer the middle of the brevin molecule.
Bryan
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Hwo
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D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology*; D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification; D004533:Egtazic Acid / Q000494:pharmacology; D018260:Gelsolin; D006801:Humans; D007700:Kinetics; D008840:Microfilament Proteins; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding
D000199:Actins; D002352:Carrier Proteins; D018260:Gelsolin; D008840:Microfilament Proteins; D010446:Peptide Fragments; C016953:brevin; D004533:Egtazic Acid; D002118:Calcium