Antibodies directed against N-terminal residues on actin do not block acto-myosin binding.
Several studies using a variety of approaches have suggested a possible role for the amino-terminal residues of skeletal muscle actin in acto-myosin interaction. In order to assess the significance of acto-S-1 contacts involving the N-terminal segment of actin, we have prepared polyclonal antisera against a synthetic peptide corresponding to the seven amino-terminal residues of rabbit skeletal muscle actin (alpha-N-terminal peptide). Affinity-purified immunoglobulin (Ig) G (and Fab) prepared from these antisera reacts strongly and specifically with the amino-terminal segment of both G- and F-actin but not with myosin subfragment 1 (S-1). This specificity was determined by Western blot analysis of actin and its proteolytic fragments and the inhibition of the above reactivity by the alpha-N-terminal peptide. The alpha-N-terminal peptide did not interact with S-1 in solution, affect S-1 and actin-activated S-1 MgATPase, or cause dissociation of the acto-S-1 complex. In separate experiments F-actin could be cosedimented with S-1 and affinity-purified IgG or Fab by using an air-driven ultracentrifuge. Densitometric analysis of sodium dodecyl sulfate/polyacrylamide gels of pellet and supernatant fractions from such experiments demonstrated the binding of both S-1 and IgG or Fab to the same F-actin protomer. Our results suggest that, while the acidic N-terminal amino acids of actin may contact the myosin head, these residues cannot be the main determinants of acto-S-1 interaction.
Miller
L
L
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Kalnoski
M
M
Yunossi
Z
Z
Bulinski
J C
JC
Reisler
E
E
D000199:Actins / Q000276:immunology / Q000378:metabolism*; D000205:Actomyosin / Q000378:metabolism*; D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D003956:Dialysis; D004797:Enzyme-Linked Immunosorbent Assay; D007140:Immunoglobulin Fab Fragments; D009132:Muscles / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D011817:Rabbits
D000199:Actins; D000906:Antibodies; D000936:Antigen-Antibody Complex; D007140:Immunoglobulin Fab Fragments; D000205:Actomyosin; D009218:Myosins