The active cross-bridge motions of isolated thick filaments from myosin-regulated muscles detected by quasi-elastic light scattering

Abstract

Intensity fluctuation spectroscopy has been used successfully as a probe that can detect an increase in high-frequency internal motions of isolated thick filaments of Limulus muscle upon the addition of calcium ions. We have attributed such motions to cross-bridge motion instead of to an increase in the flexibility of the filament backbone. Here we show that after cleavage of the S-1 and then the S-2 moieties with papain, cross-linking the myosin heads to the filament backbone, or heat denaturation (42 degrees C, 10 min), the increase in the high frequency internal motions in the thick filaments no longer occurs. Congo Red, which has been shown to induce shortening of isolated myofibrils, also increases the high-frequency motions of the isolated filaments. Furthermore, the increase is suppressed by treating the filaments with a myosin ATPase inhibitor such as vanadate ions (10 mM) or by replacing ATP with either an equimolar CrADP or the nonhydrolyzable ATP analogue beta, gamma-imido-adenine-5'-triphosphate (AMP-PNP). Calcium ions have a similar effect on isolated thick filaments from scallop muscle, where the myosin is known to be regulatory. Calcium ions have no such effect on thick filaments isolated from frog muscle, which is believed not to be regulated by calcium binding to myosin. These results confirm our earlier supposition that the additional high frequency internal motions of the thick filaments isolated from striated muscle of Limulus are related to the energy dependent, active cross-bridge motions.

Effect of ATP depletion on the isolated thick filament of limulus striated muscle

“…The r values are expressed in such a way that the r value of the same filament preparation suspended in relaxing solution is taken as 1. As previously demonstrated with ATP-containing relaxing solution (Fan et al, 1985a), the addition of calcium ions to the relaxing solution in which AMP-PNP was substituted for ATP did not affect the r values. Magnesium ion concentrations between 1 and 5 mM had no effect on the r values from filaments suspended in ATP-free solution (Table II).…”

“…2). Although both calcium ions and an ATP-free condition increased the F values of thick filament suspensions, the effect of the latter was different from that of the former (Fan et al, 1983a;Kubota et al, 1983;Fan et al, 1985a) in the following ways. (a) The increase of F values in the ATP-free condition did not require energy.…”

“…(c) The increase of r values in ATP-free solution was insensitive to phenylmethylsulfonyl fluoride (PMSF). The increase of r values resulting from calcium ions is sensitive both to heat treatment (Fan et al, 1985a) and to PMSF (Fan et al, 1985c). The results are presented in Table IV.…”

“…(a) The increase of F values in the ATP-free condition did not require energy. Energy from the hydrolysis of ATP is indispensable for the increase of F values induced by calcium ions (Fan et al, 1985a). (b) The increase of r values in ATP-free solution was not sensitive to heat treatment (420C, 10 min).…”

“…The light-scattering spectrometer used and the method of data treatment have been described previously (Kubota et al, 1983;Fan et al, 1985a). An argon ion laser operated at 488 nm and -100 mW was used.…”