Smooth muscle caldesmon controls the strong binding interaction between actin-tropomyosin and myosin.
We have demonstrated that caldesmon does not alter the affinity of weak binding actomyosin complexes when it inhibits actin-tropomyosin activation at physiological ratios (1 per 14 actins), and we proposed that it acts upon the strong binding complexes in the same way that troponin-tropomyosin does. We therefore compared the effect of caldesmon, caldesmon fragments, and troponin upon the interaction of the strongly bound complexes S-1.ADP, S-1.adenylyl imidodiphosphate (AMP.PNP), and N-ethylmaleimide-treated myosin subfragment-1 (NEM-S-1) with actin-tropomyosin. In 0.17 M ionic strength buffer [14C]iodoacetamide-labeled S1.ADP bound to actin-smooth muscle tropomyosin with no evidence of cooperativity; Kd = 0.8 +/- 0.3 microM (n = 5). Inhibitory concentrations of sheep aorta caldesmon or rabbit skeletal muscle troponin made the binding highly cooperative. At low levels of saturation the apparent Kd was 10-40 microM with 10 microM caldesmon and 8-20 microM with 6 microM troponin; at > 50% saturation the binding was indistinguishable from actin-tropomyosin alone. A similar result was obtained for the binding of [14C]iodoacetamide-labeled S-1.AMP.PNP to actin-smooth muscle tropomyosin at 0.03 M ionic strength (Kd = 0.47 +/- 0.05 microM). Binding was slightly cooperative and became highly cooperative in the presence of inhibitory concentrations of troponin, caldesmon, and the human caldesmon fragments H7 (amino acids 622-767) and H9 (amino acids 726-793). We conclude that caldesmon and troponin both act as allosteric effectors of the "on"/"off" equilibrium of actin-tropomyosin. 0.1 NEM-S-1/actin potentiated actin-smooth muscle tropomyosin activation of myosin MgATPase 7-fold at 0.03 M ionic strength. Caldesmon inhibited the ATPase in the presence and absence of 0.5 microM NEM-S-1. NEM-S-1 reactivated actin-tropomyosin, which had been inhibited by troponin, caldesmon, H7, or H9. This is compatible with opposing effects of NEM-S-1 and caldesmon or troponin upon the actin-tropomyosin on/off equilibrium.
Marston
S B
SB
Department of Cardiac Medicine, National Heart and Lung Institute, London, United Kingdom.
Fraser
I D
ID
Huber
P A
PA
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000502:physiology*; D005033:Ethylmaleimide / Q000494:pharmacology; D007460:Iodoacetamide / Q000737:chemistry; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism*; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism*; D009994:Osmolar Concentration; D011485:Protein Binding; D011817:Rabbits; D012756:Sheep; D014335:Tropomyosin / Q000378:metabolism*
D000199:Actins; D002148:Calmodulin-Binding Proteins; D015879:Myosin Subfragments; D014335:Tropomyosin; D005033:Ethylmaleimide; D007460:Iodoacetamide