Ionic interaction of myosin loop 2 with residues located beyond the N-terminal part of actin probed by chemical cross-linking.
To probe ionic contacts of skeletal muscle myosin with negatively charged residues located beyond the N-terminal part of actin, myosin subfragment 1 (S1) and actin split by ECP32 protease (ECP-actin) were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). We have found that unmodified S1 can be cross-linked not only to the N-terminal part, but also to the C-terminal 36 kDa fragment of ECP-actin. Subsequent experiments performed on S1 cleaved by elastase or trypsin indicate that the cross-linking site in S1 is located within loop 2. This site is composed of Lys-636 and Lys-637 and can interact with negatively charged residues of the 36 kDa actin fragment, most probably with Glu-99 and Glu-100. Cross-links are formed both in the absence and presence of MgATP.P(i) analog, although the addition of nucleotide decreases the efficiency of the cross-linking reaction.
Pliszka
Barbara
B
Department of Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur Street, PL-02-093 Warsaw, Poland. b.pliszka@nencki.gov.pl
Martin
Brian M
BM
Karczewska
Emilia
E
D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D003432:Cross-Linking Reagents / Q000737:chemistry*; D007477:Ions / Q000737:chemistry; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism*; D010196:Pancreatic Elastase / Q000378:metabolism; D011817:Rabbits
D000199:Actins; D003432:Cross-Linking Reagents; D007477:Ions; D010196:Pancreatic Elastase; D009218:Myosins