N-Ethylmaleimide-modified subfragment-1 and heavy meromyosin inhibit reactivated contraction in motile models of retinal cones

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Abstract
The mechanism of contraction in motile models of teleost retinal cones was examined by using N-ethylmaleimide (NEM)-modified myosin fragments (NEM-S-1 and NEM-heavy meromyosin [HMM]) to prevent access of native myosin to actin filaments during reactivation of contraction. In the diurnal light/dark cycle, retinal cones of green sunfish (Lepomis cyanellus) and bluegill (L. macrochirus) exhibit length changes of > 90 .mu.m. The motile myoid region of the cone contracts from 100 .mu.m in the dark to 6 .mu.m in the light. Motile models for cone contraction have been obtained by lysis of dark-adapted retinas with the nonionic detergent, Brij-58. These cone motile models undergo Ca2+- and ATP-dependent reactivated contraction, with morphology and rate comparable to those observed in vivo (Burnside et al., 1982). The cone myoids contain longitudinally oriented actin filaments which bind myosin subfragment-1 (S-1) to form characteristic arrowhead complexes which dissociate in the presence of MgATP (Burnside, 1978). Modification of S-1 or HMM with the SH reagent, NEM, produces new species, NEM-S-1 and NEM-HMM, which still bind actin but fail to detach in the presence of MgATP (Meeusen, et al., 1979). NEM-S-1 and NEM-HMM were used to test whether cone contraction depends on an actomyosin force-generating system. Reactivated contraction of cone models is inhibited by NEM-S-1 and NEM-HMM, but not by the unmodified species, S-1 and HMM. Reactivated cone contraction exhibits NEM-S-1 and NEM-HMM sensitivity as well as Ca2+- and ATP-dependence. These observations are consistent with an actomyosin-mediated mechanism for force production during cone contraction.