Temperature-induced change of thick filament and location of the functional sites of myosin.

The combination of the small-angle X-ray camera to use the synchrotron radiation (National Lab. of High Energy Physics, Tsukuba) and a sensitive X-ray detecting system (FCR System, Fuji Medical System Inc.) using the imaging plate enabled us to record a small angle X-ray diagram of liver rabbit muscle within few minutes. Live relaxed rabbit muscle kept at 25 degrees C gave clear relaxed pattern, in which myosin layer line can be observed up to 13th order. When it was cooled down to 5 degrees C, it gave the small-angle X-ray pattern which looks like that obtained from contracting frog muscle, whereas cooled muscle produced no tension. The 8th meridional reflection almost vanished. The pattern is different from that obtained from rigor muscle: actin layer line at 72 A spacing can not be observed. The "stiffness" and (1,1) reflection on the equator increased. This indicates that more crossbridges are at the vicinity of the thin filaments without developing tension at low temperature. This might be related to the marked decrease in the rate of the step from M*ADP to M.ADP at low temperature. The position of the ATP binding site, SH1, actin binding site(s) was determined by three-dimensional image reconstruction method. Actin-binding site was determined by comparing the three-dimensional image of actin-tropomyosin-S1 and that of actin-tropomyosin-troponin-Ca. The position of ATP binding site and SH1 was determined by three-dimensional reconstruction of the complexes of actin-tropomyosin and S1 of which the ATP binding site or SH1 was labelled with avidin. It was found that SH1 locates near the actin-binding site at the same side of S1. The distance from head-rod junction to ATP binding site and SH1 was similar. But they locate at the different side of S1 and the distance between two sites is about 5 nm, which is consistent with that obtained by energy transfer method.