Equilibrium linkage analysis of cardiac thin filament assembly. Implications for the regulation of muscle contraction.
A major focus in studies of muscle contraction has been the effect of Ca2+ on the interactions among the thin filament's five constituent polypeptides: actin, tropomyosin, troponin C (TnC), troponin T (TnT), and troponin I (TnI). We have investigated these interactions by analyzing thin filament assembly as a linear lattice binding problem with linkage relationships in the associations of tropomyosin, actin, and troponin. Binding of TnT, the binary TnT.TnI complex, or the ternary troponin complex (+/- Ca2+) to tropomyosin was measured spectrofluorimetrically after labeling cardiac tropomyosin with N-(1-pyrene)iodoacetamide. The affinity constants ranged between 0.2 and 0.6 microM-1 in the presence of 300 mM KCl. Also, the affinities of tropomyosin, tropomyosin-troponin, tropomyosin.TnT, and tropomyosin.TnT.TnI for an isolated site on F-actin were determined. The actin association constants were 0.0006 microM-1 for tropomyosin, 1 microM-1 for tropomyosin.TnT, 2 microM-1 for tropomyosin.TnT.TnI, 0.5 microM-1 for tropomyosin.troponin, and 0.5 microM-1 for tropomyosin-troponin.Ca2+. Linked equilibrium analysis permitted calculation of the affinities for actin.tropomyosin of TnT (400 microM-1), TnT.TnI (1600 microM-1), troponin (500 microM-1), and troponin.Ca2+ (300 microM-1). Therefore, both troponin and tropomyosin.troponin retain high actin-affinity even when Ca2+ is present or when TnI is removed, and even in the absence of cooperative contributions. The results are discussed in consideration of increasing evidence for a Ca(2+)-regulated azimuthal movement of tropomyosin on F-actin (Lehman, W., Craig, R., and Vibert, P. (1994) Nature 368, 65-67). It is proposed that tropomyosin movement may be due to switching between TnI-mediated and TnT-mediated binding of troponin-tropomyosin to distinct sites on F-actin.
Department of Internal Medicine, University of Iowa, Iowa City 52242.
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D000199:Actins / Q000378:metabolism; D000818:Animals; D002417:Cattle; D008840:Microfilament Proteins / Q000737:chemistry / Q000502:physiology*; D009200:Myocardial Contraction; D011485:Protein Binding; D011817:Rabbits; D014335:Tropomyosin / Q000378:metabolism; D014336:Troponin / Q000378:metabolism
D000199:Actins; D008840:Microfilament Proteins; D014335:Tropomyosin; D014336:Troponin