Interaction of actin with N-ethylmaleimide modified heavy meromyosin in the presence and absence of adenosine triphosphate

Abstract

N-Ethylmaleimide modified heavy meromyosin in only 3-fold activated by actin rather than 200-fold as is normal heavy meromyosin (Silverman, R., Eisenberg, E., and Kielley, W. W. (1972), Nature (London) 240, 207). Ultracentrifuge studies demonstrated that in the absence of ATP the N-ethylmaleimide modified heavy meromyosin binds to actin at a ratio of 2 actins to 1 N-ethylmaleimide modified heavy meromyosin. However, it was found that most of the N-ethylmaleimide modified heavy meromyosin was not bound to actin during ATP hydrolysis. Ultracentrifuge studies demonstrated that in the presence of 25 or 50 mM KCl under conditions where the ATPase is maximally activated by actin, less than 5% of the N-ethylmaleimide modified heavy meromyosin was bound to actin. In the absence of KCl there was limited binding but even this binding did not appear to correlate with the N-ethylmaleimide modified heavy meromyosin ATPase rate. Turbidity and viscosity studies also indicated that in the presence of ATP under conditions of maximal actin activation the N-ethylmaleimide modified heavy meromyosin and actin are almost completely dissociated, whereas there is a marked increase in turbidity and viscosity after all of the ATP is hydrolyzed. These results suggest that in the presence of ATP and actin N-ethylmaleimide modified heavy meromyosin exists most of the time in a refractory state unable to bind to actin and only a small part of the time in a nonrefractory state which can interact with actin. It follows that the major rate-limiting step during actin activation is the transition from the refractory to the nonrefractory state. Since the actin activation of N-ethylmaleimide modified heavy meromyosin is lower than that of normal heavy meromyosin this transition may be slower for N-ethylmaleimide modified heavy meromyosin than for normal heavy meromyosin.

Association of microinjected myosin and its subfragments with myofibrils in living muscle cells.

“…A similar decrease (90-95%) was observed at other actin concentrations. Consistent results were also obtained in previous studies of sulfhydryl-modified myosins (Mulhern and Eisenberg, 1978;Mulhern et al, 1975;Takashi et al, 1976).…”

The binding of mutant actins to profilin, ATP and DNase I

“…Following translation, excess ATP was removed from re-RESULTS ticulocyte lysate using Bio-Rad AG 1-X8 anion-exchange resin chloridc form, 200-400 mesh (Mulhern et al, 1975 actin, and incubated at 2 0 T for 2 min with occasional mixing. The resin was pelleted by centrifuging at 5000 g for 20 s, the supernatant was removed and recentrifuged for a further 20 s. An aliquot of supernatant was removed and immediately mixed with an equal volume of rehydrated ATP-affinity gel.…”

“…Following translation, excess ATP was removed from re-RESULTS ticulocyte lysate using Bio-Rad AG 1-X8 anion-exchange resin chloridc form, 200-400 mesh (Mulhern et al, 1975). The resin was washed three times with 2 vol.…”

“…Actin binds to DNase I from several tissues (Lazarides and Lindberg, 1974;Nadano et al, 1991), inhibiting DNase I activity and the ability of the actin to polymerise (Lazarides and Lindberg, 1974). Not all types of DNase 1 bind actin (Lacks, 1981) and the biological function of this interaction remains unclear.…”

A quantitative model of actin-myosin interaction in skeletal muscle

“…the laboratories of Taylor Taylor, 1970, 1971) and Eisenberg (Eisenberg and Kielley, 1973;Mulhern et al, 1975) as well as others have shown that hydrolysis of ATP by actomyosin involves a complex sequence of reactions. The rate-limiting step in this sequence appears to be the transformation of the myosin-ATP complex from a "refractory" state (M*Pr) to a state (M**Pr) where binding of actin (AMPr) may occur (Eisenberg and Kielley, 1973).…”