The characterization of vanadate-trapped nucleotide complexes with spin-labelled myosins.

The properties of spin-labelled myosin, prepared from rabbit skeletal and scallop adductor muscle, on forming a long-lived complex with ADP and vanadate (M.ADP.Vi), have been investigated. In the case of an iodoacetamide-based label attached to rabbit myosin or subfragment 1, M.ADP.Vi formation is characterized by a marked increase in the mobility of the probe, similar to that seen during steady-state ATPase activity. Hence, this complex appears to be a good analogue of the M**ADP.Pi state. The kinetics of M.ADP.Vi formation were determined by following the electron paramagnetic resonance (e.p.r.) signal with time and were analysed according to the scheme: (formula; see text) After correction for Vi polymerization, K'4 = 3.2 X 10(-4)M, k'-3 = 8.7 X 10(-3) s-1 and k'3 = 1.5 X 10(-4) s-1. The major effect of spin-labelling the reactive SH1 thiol is to increase k'3, so that M.ADP.Vi dissociates over a period of hours rather than days. In contrast, a maleimide-based spin-label attached to rabbit myosin does not exhibit a large change in mobility, on formation of the M.ADP.Vi complex. However, the small change observed in both the conventional and saturation transfer spectra questions the assumption that this probe is completely insensitive to librational motion during ATPase activity. The immobilized spectrum of the iodoacetamide-based spin label attached to scallop myosin is insensitive to M.ADP.Vi formation in the presence or absence of Ca2+. Under these conditions, the label appears to reflect gross head motion and hence this observation lends no support to the idea that, in the myosin-linked regulatory system, Ca2+ operates by controlling the flexibility of the subfragment 1-subfragment 2 joint.