F-actin-myosin-subfragment-1 (S1) interactions. Identification of the refractory state of S1 with the S1 dimer.
Several conflicting experiments have been carried out concerning the existence of a refractory state for myosin subfragment 1 (S1; i.e. unable to bind to F-actin), in the presence of Mg-nucleotide compounds, at low temperature. Contradictory experiments have been published on the existence of a S1 dimer, under the same conditions. By taking into account some elementary, but crucially important, precautions in the preparation of myosin and S1, it is possible to maintain intact the head-to-head sites of dimerisation on both myosin and S1. Moreover, it has been shown that Mg2+ alone, at low temperature, is able to induce a reversible S1-S1 dimerisation [Morel, J. E. & Garrigos, M. (1982) Biochemistry 21, 2679-2686). Here, we have studied the intrinsic viscosity of S1, in the presence of 2 mM MgCl2, versus temperature, between 0.5-20 degrees C. At approximately 0.5-2.0 degrees C, the intrinsic viscosity of S1 was found to be 19.8 cm3/g. Above 2.5-3.0 degrees C, a steep decrease in the intrinsic viscosity was observed. Between 7 degrees C and 20 degrees C, a constant intrinsic viscosity of 6.7 cm3/g was measured. Therefore, there is a dramatic temperature transition between approximately 2.5-7.0 degrees C (width, 4.5 degrees C; midpoint, 5 degrees C): below 2.5 degrees C we observed the presence of a S1 dimer (confirmed by analytical ultracentrifugation performed at 1 degree C) and above 7-8 degrees C we observed the presence of a S1 monomer (confirmed by analytical ultracentrifugation performed at 8 degrees C). We have also studied the interactions of F-actin with S1, under the same conditions of temperature, and in the presence of 2 mM MgCl2, by using preparative ultracentrifugation. At 1-2 degrees C, S1, which is in the dimeric form, is unable to bind to F-actin. At 6-20 degrees C, F-actin and S1 bind stoichiometrically (1S1/1actin), with an equilibrium constant of 0.5 microM-1, under our experimental conditions. We also performed binding experiments at 3-5 degrees C. Although we were unable to clearly reach the asymptote corresponding to stoichiometry, by assuming a 1:1 ratio, we found a temperature transition in the equilibrium constants, between 3-5 degrees C (width, 2 degrees C; midpoint, 4 degrees C). These phenomena are comparable to those observed for the dimer. Thus, we have shown that there is also a temperature transition in the F-actin-S1 binding process. We conclude that the dimeric and the refractory states of S1 are identical.
Grussaute
H
H
Laboratoire de Biologie, Ecole Centrale de Paris, Châtenay-Malabry, France.
Ollagnon
F
F
Morel
J E
JE
D000199:Actins / Q000737:chemistry* / Q000648:ultrastructure; D000818:Animals; D008274:Magnesium / Q000494:pharmacology; D008854:Microscopy, Electron; D015879:Myosin Subfragments / Q000737:chemistry* / Q000648:ultrastructure; D011817:Rabbits; D013696:Temperature; D014783:Viscosity
D000199:Actins; D015879:Myosin Subfragments; D008274:Magnesium