The role of ATP, ADP and divalent cations in the formation of binary and ternary complexes of actin, cofilin and DNase I.
Actin is the major cytoskeletal protein of virtually all eukaryotic cells. Actin assembly/disassembly is involved in a variety of cellular processes and actin-binding proteins are essential in regulation of the pool of actin monomers. Cofilin and DNase I are actin-binding proteins, which form both binary (actin-DNase 1, cofilin-actin) and ternary (cofilin-actin-DNase I) complexes with actin. Here we use native gel electrophoresis to examine the roles of ATP, ADP, Ca2+ and Mg2+ in the formation of these complexes as well as on the ability of actin to self-assemble. Conditions which favour actin polymerisation are: ATP (no Me2+) > or = ADP (no Me2+) > ADP-Ca2+ = ADP-Mg2+ > ATP-Mg2+ > ATP-Ca2+. Preferential conditions for the formation of the binary actin-cofilin complex are: ADP-Mg2+ > or = ADP-Ca2+ >> ATP-Ca2+ approximately equals ATP-Mg2+ approximately equals ADP-No Me2+ approximately equals ATP-No Me2+. Actin forms a very tight complex with DNase I in the order: ATP-Ca2+ > or = ATP-Mg2+ approximately equals ADP-Mg2+ approximately equals ADP-Ca2+ > or = ADP-(no Me2+) > ATP-(no Me2+). Effectively, the complex does not form in the presence of ATP and the absence of free Me2+. Finally, the conditions which favour the formation of a ternary complex of cofilin-actin-DNase I resemble the actin-DNase I, namely: ATP-Ca2+ approximately equals ADP-Ca2+ approximately equals ADP-Mg2+ approximately equals ATPMg2+  ADP (no Me2+) > ATP-(no Me2+).
Chhabra
D
D
Department of Anatomy and Histology, Institute for Biomedical Research, The University of Sydney, Australia. dchhabra@anatomy.usyd.edu.au
Nosworthy
N J
NJ
dos Remedios
C G
CG
D000180:Acrylic Resins; D051339:Actin Depolymerizing Factors; D000199:Actins / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D002118:Calcium / Q000737:chemistry*; D002413:Cations, Divalent; D002645:Chickens; D003850:Deoxyribonuclease I / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D008274:Magnesium / Q000737:chemistry*; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics; D011817:Rabbits
D000180:Acrylic Resins; D051339:Actin Depolymerizing Factors; D000199:Actins; D002413:Cations, Divalent; D008840:Microfilament Proteins; C016680:polyacrylamide gels; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D003850:Deoxyribonuclease I; D008274:Magnesium; D002118:Calcium