Specific interactions of the alkali light chain 1 in skeletal myosin heads probed by chemical cross-linking

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Abstract
We have investigated the enzymatic properties of the 120K cross-linked heavy-chain-light-chain derivative formed upon reaction of chymotryptic myosin subfragment 1 (S-1) isoenzymes with the bis(imido esters) dimethyl 3,3''-dithiobis(propionimidate) and dimethyl suberimidate. The formation of the 120K product was accompanied for S-1(A1) but not for S-1(A2) by a loss of the actin-activated ATPase without alteration of the Ca2+-ATPase whereas the Mg2+-ATPase was increased 2-fold. Up to 70%, the inhibition of the acto-S-1(A1) ATPase activity was closely correlated with the extent of cross-linking of the A1 light chain; this activity could be largely restored upon cleavage of the cross-link using the reversible cross-linker dimethyl 3,3''-dithiobis(propionimidate). The covalent link affected the acto-S-1(A1) Mg2+-ATPase activity by reducing 3-fold the Mmax and increasing 2-fold the Kapp. On reacting for the first time the hydrophobic, carboxyl group directed cross-linker N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) with the acto-S-1(A1 + A2) complex, we found that the N-terminal tail of the A1 light chain was cross-linked to actin to an extent much larger than observed earlier with the water-soluble 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; like the latter agent, EEDQ elicited the covalent union of the A1 subunit to the COOH-terminal part of actin. This cross-linker appears to be a valuable chemical probe of the F-actin-Al light-chain interaction. Finally, no cross-linking of actin to the isolated A1 light chain was observed in spite of the reported binding of F-actin to this light chain in the isolated state; in contrast, the cross-linking occurred when the Aq subunit was complexed to the isolated COOH-terminal 20K fragment of the S-1 heavy chain. These results suggest that the heavy chain changes the conformation of the light subunit and thereby determines its cross-linking ability to actin.