G-actin conformational change and polymerization induced by paraquat.
Paraquat (1,1'-dimethyl-4,4'-bipyridilium dichloride) is a broad-spectrum herbicide that is highly toxic to animals (including man), the major lesion being in the lung. In mammalian cells, paraquat causes deep alterations in the organization of the cytoskeleton, marked decreases in cytoskeletal protein synthesis, and alterations in cytoskeletal protein composition; therefore, the involvement of the cytoskeleton in cell injury by paraquat was suggested. We previously demonstrated that monomeric actin binds paraquat; moreover, prolonged actin exposure to paraquat, in depolymerizing medium, induces the formation of actin aggregates, which are built up by F-actin. In this work we have shown that the addition of paraquat to monomeric actin results in a strong quenching of Trp-79 and Trp-86 fluorescence. Trypsin digestion experiments demonstrated that the sequence 61-69 on actin subdomain 2 undergoes paraquat-dependent conformational changes. These paraquat-induced structural changes render actin unable to completely inhibit DNase I. By using intermolecular cross-linking to characterize oligomeric species formed during paraquat-induced actin assembly, we found that the herbicide causes the formation of actin oligomers characterized by subunit-subunit contacts like those occurring in oligomers induced by polymerizing salts (i.e., between subdomain 1 on one actin subunit and subdomain 4 on the adjacent subunit). Furthermore, the oligomerization of G-actin induced by paraquat is paralleled by ATP hydrolysis.
DalleDonne
I
I
University of Milan, Department of Biology, Italy.
Milzani
A
A
Colombo
R
R
D000199:Actins / Q000737:chemistry / Q000187:drug effects*; D000818:Animals; D002850:Chromatography, Gel; D003850:Deoxyribonuclease I / Q000378:metabolism; D015233:Models, Statistical; D018482:Muscle, Skeletal / Q000187:drug effects; D010269:Paraquat / Q000494:pharmacology*; D010710:Phosphates / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism; D014461:Ultracentrifugation
D000199:Actins; D010710:Phosphates; D014364:Tryptophan; D003850:Deoxyribonuclease I; D014357:Trypsin; D010269:Paraquat