Cysteine-374 of actin resides at the gelsolin contact site in the EGTA-resistant actin-gelsolin complex.
The interaction of pig plasma gelsolin (G) and actin (A) was examined by using photoreactive 4-maleimidobenzophenone-actin (BPM-actin) in which BPM was previously conjugated to Cys-374 of actin through the maleimide moiety. In the presence of micromolar [Ca2+], the major cross-linked product observed after irradiation of the mixture of gelsolin (82 kDa) and actin (42 kDa) had an apparent molecular mass of 130 kDa although gelsolin predominantly existed in the form of an A2G complex (170 kDa). No cross-linked product was detected in the absence of Ca2+. BPM-actin itself did not give any cross-linked product. By use of fluorescent-labeled gelsolin, the cross-linked 130 kDa was shown to be an AG complex. The cross-linked complex was also formed from the A2G complex after removal of Ca2+ by [ethylenebis-(oxyethylenenitrilo)]tetraacetic acid (EGTA) followed by irradiation, indicating that it was the EGTA-resistant AG complex that was cross-linked. The results show that Cys-374 at the C-terminal segment of actin in the EGTA-resistant AG complex is 9-10 A apart from gelsolin. Furthermore, it was shown that the EGTA-resistant actin molecule once incorporated in the A2G complex did not exchange with free actin in the presence of Ca2+. This was also supported by the effect of phosphatidylinositol 4,5-bisphosphate, which did not dissociate the EGTA-resistant actin molecule from the A2G complex in the presence of Ca2+, but did after removal of Ca2+.
Doi
Y K
YK
Department of Food Science, Kyoto Women's University, Japan.
Banba
M
M
Vertut-Doï
A
A
D000199:Actins / Q000097:blood* / Q000737:chemistry; D000345:Affinity Labels; D000818:Animals; D001577:Benzophenones / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000097:blood*; D003432:Cross-Linking Reagents / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry*; D004533:Egtazic Acid / Q000494:pharmacology; D005456:Fluorescent Dyes; D018260:Gelsolin; D008301:Maleimides; D008840:Microfilament Proteins / Q000097:blood*; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D013552:Swine
D000199:Actins; D000345:Affinity Labels; D001577:Benzophenones; D002135:Calcium-Binding Proteins; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D018260:Gelsolin; D008301:Maleimides; D008840:Microfilament Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D004533:Egtazic Acid; C046109:benzophenone-4-maleimide; D003545:Cysteine; D002118:Calcium