Location of the sites of reaction of N-ethylmaleimide in papain and chymotryptic fragments of the gizzard myosin heavy chain

Abstract

The thiol of the gizzard myosin heavy chain, which reacts most rapidly with N-ethylmaleimide (MalNEt), has been located in the subfragment 2 region of myosin rod by fragmentation of [14C]-MalNEt-labeled myosin with papain and chymotrypsin. MalNEt reacts more slowly with thiols present in the 70- and 25-kilodalton (kDa) papain fragments of subfragment 1. The reaction of MalNEt with thiols present in these regions is increased on addition of ATP by factors of 2 and 10, respectively, when myosin is modified in 0.45 M NaCl where it is present in the extended, 6S conformation. The rate of increase of Mg2+-activated adenosinetriphosphatase (ATPase) activity, which reflects the loss of ability of myosin to assume the folded, 10S conformation, and the rate of loss of K+-EDTA-activated activity produced by MalNEt are both accelerated 5- to 10-fold on addition of ATP. The rates at which ATPase activities change agree closely to the reaction rates of MalNEt with the 25-kDa region of subfragment 1; therefore, the changes in these activities can be attributed to modification of a thiol of the 25-kDa segment. An increase in actin-activated ATPase activity produced by reaction of myosin with MalNEt in 0.45 M NaCl is accelerated by ATP by a factor of at least 4. Reaction with [14C]MalNEt in the presence of MgATP and 0.2 M NaCl, where myosin is in the 10S form, inhibits the incorporation of radioactive MalNEt into the 25-kDa papain fragment of subfragment 1. It also prevents the increase in actin-activated ATPase activity and preserves the ability of myosin to assume the 10S form.

Formation and movement of myosin-containing structures in living fibroblasts.

“…Although the actin-activated Mg2+-ATPase was three times higher than that with unlabeled, unphosphorylated myosin, the degree of activation by actin filaments was reduced for the fluorescent myosin. These observations were again consistent with the labeling of the SH-C group (Onishi, 1985;Nath et al, 1986).…”

“…Proteolytic analyses indicated that the tetramethylrhodamine groups were associated primarily with the SH-B of the 17-kD light chain, and the SH-C group of the heavy chain near the COOH terminus of the S1 head (Nath et al, 1986). While important characteristics of myosin, including selfassembly and actin-activated ATPase activities, were preserved qualitatively after fluorescent labeling, there were quantitative changes similar to those induced by other sulfhydryl reacting agents (Onishi, 1985;Chandra et al, 1985;Nath et al, 1986).…”

H2O2-induced Kinetic and Chemical Modifications of Smooth Muscle Myosin

“…Constructs of SMM with altered regulation typically have abnormally high ATPase activities in the unphosphorylated state and an abnormally low ATPase in the phosphorylated state (36,(42)(43)(44)(45)(46)(47). Alkylation of SMM thiols causes a similar effect (41,48,49) most likely attributed to reaction of SH1(Cys 717 ). The effects of H 2 O 2 on pHMM and upS1 were very similar in our studies, suggesting that they are not mediated by modification of the most reactive thiol on SMM, which is in the S2 region of the rod (Cys 892 ) (49) and which is not present in S1.…”

Monoclonal antibodies detect and stabilize conformational states of smooth muscle myosin.

“…Four antibodies with epitopes in the central portion of therod ($2.1, $2.2, LMM.1, and LMM.2) bind tightly to synthetic filaments and block their disassembly to the folded monomer in the presence of MgATE The stabilization is probably due to a physical cross-linking between myosin molecules in the filament. Alternatively, antibody binding could induce a change in the myosin that mimics phosphorylation, although only modifications to the head have been observed to have this effect (Nath et al, 1986).…”