Antibody and peptide probes of interactions between the SH1-SH2 region of myosin subfragment 1 and actin's N-terminus.
The negatively charged residues in the N-terminus of actin and the 697-707 region on myosin subfragment 1 (S-1), containing the reactive cysteines SH1 and SH2, are known to be important for actin-activated myosin ATPase activity. The relationship between these two sites was first examined by monitoring the rates of SH1 and SH2 modification with N-ethylmaleimide in the presence of actin and, secondly, by testing for direct binding of SH1 peptides to the N-terminal segment on actin. While actin alone protected SH1 from N-ethylmaleimide modification, this effect was abolished by an antibody against the seven N-terminal amino acids on actin, F(ab)(1-7), and was greatly reduced when the charge of acidic residues at actin's N-terminus was altered by carbodiimide coupling of ethylenediamine. Neither F(ab)(1-7) nor ethylenediamine treatment reversed the effect of F-actin on SH2 reactivity in SH1-modified S-1. These results show a communication between the SH1 region on S-1 and actin's N-terminus in the acto-S-1 complex. To test whether such a communication involves the binding of the SH1 site on S-1 to the N-terminal segment of actin, the SH1 peptide IRICRKG-NH2(4+) was used. Cosedimentation experiments revealed the binding of three to six peptides per actin monomer. Peptide binding to actin was affected slightly, if at all, by F(ab)(1-7). The antibody also did not change the polymerization of G-actin by the peptides. The peptides caused a small reduction in the binding of S-1 to actin and did not change the binding of F(ab)(1-7).(ABSTRACT TRUNCATED AT 250 WORDS)
Cartoux
L
L
Department of Chemistry, University of California, Los Angeles 90024.
Chen
T
T
DasGupta
G
G
Chase
P B
PB
Kushmerick
M J
MJ
Reisler
E
E
D000199:Actins / Q000737:chemistry* / Q000276:immunology / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D027682:Cation Transport Proteins; D003545:Cysteine / Q000737:chemistry*; D004563:Electrochemistry; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology; D011108:Polymers; D011817:Rabbits
D000199:Actins; D000906:Antibodies; D027682:Cation Transport Proteins; D015879:Myosin Subfragments; D009842:Oligopeptides; D010446:Peptide Fragments; D011108:Polymers; D000251:Adenosine Triphosphatases; C022761:potassium transporting ATPase; D000252:Calcium-Transporting ATPases; D003545:Cysteine; D005033:Ethylmaleimide