Characterization of an actin-myosin head interface in the 40-113 region of actin using specific antibodies as probes.
Evidence for the participation of the 1-7 and 18-28 N-terminal sequences of actin at different steps of actin-myosin interaction process is well documented in the literature. Cross-linking of the rigor complex between filamentous actin and skeletal-muscle myosin subfragment 1 was accomplished by the carboxy-group-directed zero-length protein cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodi-imide. After chaotropic depolymerization and thrombin digestion, which cleaves only actin, the covalent complex with Mr 100,000 was characterized by PAGE. The linkage was identified as being between myosin subfragment 1 (S-1) heavy chain and actin-(1-28)-peptide. The purified complex retained in toto its ability to combine reversibly with fresh filamentous actin, but showed a decrease in the Vmax. of actin-dependent Mg2(+)-ATPase. By using e.l.i.s.a., S-1 was observed to bind to coated monomeric actin or its 1-226 N-terminal peptide. This interaction strongly interfered with the binding of antibodies directed against the 95-113 actin sequence. Moreover, S-1 was able to bind with coated purified actin-(40-113)-peptide. Finally, antibodies directed against the 18-28 and 95-113 actin sequence, which strongly interfered with S1 binding, were unable to compete with each other. These results suggest that two topologically independent regions are involved in the actin-myosin interface: one located in the conserved 18-28 sequence and the other near residues 95-113, including the variable residue at position 89. Other experiments support the 'multisite interface model', where the two actin sites could modulate each other during S-1 interaction.
Labbé
J P
JP
UPR 8402, Centre de Recherches de Biochimie Macromoléculaire (CNRS), U249 (INSERM), Université de Montpellier 1, France.
Méjean
C
C
Benyamin
Y
Y
Roustan
C
C
D000199:Actins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001665:Binding Sites; D015153:Blotting, Western; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005022:Ethyldimethylaminopropyl Carbodiimide; D008970:Molecular Weight; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D013917:Thrombin / Q000378:metabolism
D000199:Actins; D000906:Antibodies; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; D010446:Peptide Fragments; D013917:Thrombin; D017301:Ca(2+) Mg(2+)-ATPase; D005022:Ethyldimethylaminopropyl Carbodiimide