Regulation of actomyosin interactions in Limulus muscle proteins.
Contraction of striated muscle from Limulus polyphemus, the horseshoe crab, is regulated by both calcium binding to a troponin-tropomyosin-dependent thin filament array and a myosin light chain kinase-dependent phosphorylation of myosin. We have isolated myosin from Limulus striated muscle and examined how these two regulatory systems affect the sliding velocity of actin filaments over myosin, using an in vitro motility assay. Our results show that in the presence of ATP, Limulus myosin must be phosphorylated in order to move actin filaments. No movement was observed for actin filaments interacting with dephosphorylated Limulus myosin. Calcium was not required for actin movement. In contrast, when both troponin and tropomyosin are bound to actin filaments, calcium is required for the movement of actin filaments over phosphorylated myosin. These results demonstrate that the "off" state of either the thin filament or thick filament regulatory system is dominant and that for the movement to occur, both phosphorylated Limulus myosin and an activated troponin-tropomyosin system are required. Tropomyosin by itself increases the sliding velocity of actin filaments over phosphorylated Limulus myosin about 10-fold in a calcium-independent manner. Tropomyosins from turkey gizzard smooth muscle, bovine cardiac muscle, and Limulus muscle all have a profound effect in increasing the velocity. Troponin alone does not change the velocity. Partial sequences of the tryptic phosphopeptides of Limulus myosin regulatory light chains generated following the phosphorylation by gizzard myosin light chain kinase yield ATS(PO4)NVFAMFEQNQIA for 21 kDa and SGS(PO4)NVFSMFT for 31-kDa light chain.
Wang
F
F
Laboratory of Molecular Cardiology National Heart, Lung and Blood Institute, Bethesda, Maryland 20892.
Martin
B M
BM
Sellers
J R
JR
D000199:Actins / Q000302:isolation & purification / Q000378:metabolism; D000205:Actomyosin / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D005895:Gizzard, Avian; D006737:Horseshoe Crabs; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009124:Muscle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D014335:Tropomyosin / Q000302:isolation & purification / Q000378:metabolism; D014336:Troponin / Q000302:isolation & purification / Q000378:metabolism; D014422:Turkeys
D000199:Actins; D009124:Muscle Proteins; D010748:Phosphopeptides; D014335:Tropomyosin; D014336:Troponin; D000205:Actomyosin; D009219:Myosin-Light-Chain Kinase