BISPHOSPHORYLATION OF CARDIAC TROPONIN-I MODULATES THE CA2-DEPENDENT BINDING OF MYOSIN SUBFRAGMENT S1 TO RECONSTITUTED THIN-FILAMENTS()

We have reconstituted thin filaments comprising pyrene-labelled actin (pyr-actin), tropomyosin (Tm) and cardiac troponin (cTn), cTn was isol ated in two defined phosphorylation states; completely dephosphorylate d on all subunits and with only the cTnI subunit bisphosphorylated, Th e thin filament was saturated with cTn at a pyr-actin/Tm/cTn ratio of 7:1:1, The calcium-dependent binding of S1 to thin filaments was measu red in a stopped-flow spectrophotometer and the dependence of the obse rved rate constant on [Ca2+] fitted to the Hill equation, The only sig nificant difference between the two phosphorylation states of the fila ments was a 0.36 decrease in the pCa(50) on bisphosphorylation.