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. 2012 Jun 20;287(33):27930–27940. doi: 10.1074/jbc.M111.337295

FIGURE 3.

FIGURE 3.

Effect of ATP on rabbit ventricular myofibrils and reconstituted thin filaments + S1. Panel A shows the time course of IANBD fluorescence as Ca2+ was removed from TnCIANBDT53C and D65A TnCIANBDT53C rabbit myofibrils in the presence of ATP. TnCIANBDT53C rabbit myofibrils (Ca2+ Saturated Control versus EGTA + ATP trace, no observed rate) and D65A TnCIANBDT53C rabbit myofibrils (D65A versus EGTA + ATP trace, 203 ± 11/s) in Buffer A + 200 μm Ca2+ were mixed with an equal volume of the Buffer A + 10 mm EGTA and 2 mm ATP at 15 °C. The D65A TnCIANBDT53C Ca2+-free myofibril base line was acquired by rapidly mixing the D65A myofibrils in Buffer A + 5 mm EGTA against equal volumes of Buffer A + 5 mm EGTA (D65A Rigor). Panel B shows the time course of IANBD fluorescence decay from Ca2+-free TnCIANBDT53C rabbit myofibrils by mixing with ATP in the presence or absence of ADP. TnCIANBDT53C myofibrils in Buffer A + 5 mm EGTA ± 2 mm ADP were rapidly mixed with equal volumes of the Buffer A + 5 mm EGTA + 2 mm ATP (Ca2+-free Myofibrils versus ATP trace, 163 ± 8/s, and Ca2+-free Myofibrils + ADP versus ATP trace, 6.9 ± 0.6/s, respectively). To examine the effect of ATP on the Ca2+-saturated state, TnCIANBDT53C myofibrils in Buffer A + 200 μm Ca2+ were mixed with an equal volume of Buffer A + 200 μm Ca2+ + 2 mm ATP (Ca2+ Saturated versus Ca2+ + ATP trace, no observed rate). The Ca2+-free base line (Ca2+-free Control trace) from Fig. 1B was included as a reference point for the fluorescence decay induced by ATP. Panel C shows the time course of IAANS fluorescence decay from Ca2+-free reconstituted thin filaments containing TnCIAANST53C Tn upon dissociation of myosin-S1. TnCIAANST53C reconstituted thin filaments with a myosin S1 to actin subunit ratio of 1:7, 2:7 and 4:7 in Buffer A (without Tween 20) + 5 mm EGTA were rapidly mixed with equal volumes of the Buffer A (without Tween 20) + 5 mm EGTA + 2 mm ATP. The rates of S1 detachment reconstituted at the ratios of 1:7, 2:7, and 4:7 concentrations were ∼300/s, 268 ± 11/s, and 265 ± 10/s, respectively. All data were collected at 15 °C.