Improved method for mapping the binding site of an actin-binding protein in the actin sequence. Use of a site-directed antibody against the N-terminal region of actin as a probe of its N-terminus.
An antibody was raised against the N-terminal 18 residues of rabbit skeletal muscle actin. By the use of this antibody as the N-terminal probe of actin and the fluorescent label at Cys-374 as its C-terminal probe, binding sites of depactin (an actin-depolymerizing protein from starfish oocytes) were identified in the actin sequence according to the method of Sutoh [Sutoh, K. (1982) Biochemistry 21, 3654-3661]. Cross-linking of the one-to-one complex of actin and depactin with 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC) generated two types of cross-linked products with slightly different apparent molecular weights, denoted as 60KU and 60KL. By the use of the N-terminal probe, it was unequivocally revealed that the C-terminal actin segment of residues 357-375 participated in cross-linking with depactin to form 60KL. On the other hand, by the use of the C-terminal probe it was revealed that the N-terminal actin segment of residues 1-12 participated in cross-linking with depactin to form 60KU. Since EDC cross-links Lys residue with Asp or Glu residue only when they are in direct contact, the result indicates that some of the N-terminal residues 1-12 and the C-terminal residues 357-375 of actin participate in binding depactin. The introduction of the N-terminal probe (the antibody recognizing the actin N-terminus) has increased the flexibility of the mapping method for locating binding sites of actin-binding proteins in the actin sequence.
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism*; D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D051345:Destrin; D005022:Ethyldimethylaminopropyl Carbodiimide; D005260:Female; D018260:Gelsolin; D008840:Microfilament Proteins; D009132:Muscles / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D011817:Rabbits; D013215:Starfish
D051339:Actin Depolymerizing Factors; D000199:Actins; D000906:Antibodies; D000936:Antigen-Antibody Complex; D002352:Carrier Proteins; D051345:Destrin; D018260:Gelsolin; D008840:Microfilament Proteins; D011506:Proteins; C016953:brevin; D005022:Ethyldimethylaminopropyl Carbodiimide