EFFECT OF NUCLEOTIDES AND ACTIN ON THE INTRAMOLECULAR CROSS-LINKING OF MYOSIN SUBFRAGMENT-1

The heavy chain of myosin subfragment-1 (S1) is cleaved by limited try psinolysis into three fragments, 27, 50, and 20 kDa-aligned in this or der from the N-terminus. The tertiary structure of the molecule is ess entially not affected by trypsinolysis. The spatial relations between the various regions of the molecule and the nucleotide- and actin-indu ced intramolecular movements were studied by cross-linking tryptic S1 with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-(3-dim ethylaminopropyl)-3-ethylcarbodiimide (EDC), phenylenediglyoxal (PDG), and glutaraldehyde. The formation of crosslinked products was monitor ed by SDS-PAGE, using the fluorescent probes 9-anthronitrile and iodoa cetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS), which specifi cally label the 27- and 20-kDa fragments, respectively. The reaction w ith the cross-linkers leads to the formation of 50-kDa/20-kDa, 27-kDa/ 20-kDa, 27-kDa/50-kDa, and 20-kDa/light chain cross-linked products. O f these, the most intensive was the formation of the 50-kDa/20-kDa pro ducts, which appeared as a doublet on the SDS-PAGE with all the cross- linkers. This indicates that the interface between the two fragments i s rather extended. The presence of MgATP or MgADP promoted the formati on of the 20-kDa/50-kDa cross-linked products, especially with the low er electrophoretic mobility band, when EEDQ was used as a cross-linker . With PDG as a cross-linker, MgATP also affected the cross-link forma tion between the 20-kDa fragment and the light chains whereas it had n o influence on the formation of other products. On the other hand, the effect of actin on the cross-linking with the various cross-linkers w as quite extensive, and it was manifested in the reduction of cross-li nk formation between the various S1 domains. It is concluded that both nucleotides and actin induce intramolecular movements in S1 and that the nucleotide-induced movements are more restricted than those induce d by actin, which extend to larger regions of the molecule.