Conformational transitions within the head and at the head-rod junction in smooth muscle myosin studied with a limited proteolysis method.
It was previously shown that tryptic digestion of subfragment 1 (S1) of skeletal muscle myosins at 0 degree C results in cleavage of the heavy chain at a specific site located 5 kDa from the NH2-terminus. This cleavage is enhanced by nucleotides and suppressed by actin and does not occur at 25 degrees C, except in the presence of nucleotide. Here we show a similar temperature sensitivity and protection by actin of an analogous chymotryptic cleavage site in the heavy chain of gizzard S1. The results support the view that the myosin head, in general, can exist in two different conformational states even in the absence of nucleotides and actin, and indicate that the heavy chain region 5 kDa from the NH2-terminus is involved in the communication between the sites of nucleotide and actin binding. We also show here for the first time that the S1-S2 junction in gizzard myosin can be cleaved by chymotrypsin and that this cleavage (observed in papain-produced S1 devoid of the regulatory light chain) is also temperature-dependent but insensitive to nucleotides and actin. It is suggested that the temperature-dependent alteration in the flexibility of the head-rod junction, which is apparent from these and similar observations on skeletal muscle myosin [Miller, L. & Reisler, E. (1985) J. Mol. Biol. 182, 271-279; Redowicz, M.J. & Strzelecka-Gołaszewska, H. (1988) Eur. J. Biochem. 177, 615-624], may contribute to the temperature dependence of some steps in the cross-bridge cycle.
Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, Warszawa, Poland.
M J
MJ
Redowicz
A
A
Sobieszek
H
H
Strzelecka-Gołaszewska
D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D005895:Gizzard, Avian; D006868:Hydrolysis / Q000187:drug effects; D009130:Muscle, Smooth / Q000737:chemistry*; D015879:Myosin Subfragments / Q000032:analysis* / Q000378:metabolism; D010206:Papain; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism; D013696:Temperature; D014422:Turkeys
D000199:Actins; D015879:Myosin Subfragments; D011506:Proteins; D002918:Chymotrypsin; D010206:Papain