. Author manuscript; available in PMC: 2008 Dec 16.

Published in final edited form as: Biochemistry. 2008 Dec 16;47(50):13383–13393. doi: 10.1021/bi801492x

Table 1.

Effects of modified cTnI mutants on reconstituted actomyosin ATPase activity

Troponin Complex	ATPase Activity		Ca²⁺ Sensitivity
Troponin Complex	EGTA	Ca²⁺	Ca²⁺ Sensitivity
cTnC(wt)-cTnT(wt)-cTnI(wt)	0.017	0.079	0.785
cTnC(wt)-cTnT(wt)-cTnI(Q131C)	0.015	0.074	0.797
cTnC(wt)-cTnT(wt)-cTnI(S151C)	0.019	0.084	0.773
cTnC(wt)-cTnT(wt)-cTnI(L160C)	0.016	0.079	0.797
cTnC(wt)-cTnT(wt)-cTnI(S167C)	0.018	0.085	0.788
cTnC(wt)-cTnT(wt)-cTnI(S188C)	0.017	0.077	0.779
cTnC(wt)-cTnT(wt)-cTnI(S210C)	0.016	0.072	0.778

Ca²⁺-dependent acto-S1 ATPase activity was measured at 30 °C in 60 mM KCl, 5.6 mM MgCl₂, 2 mM ATP, 30 mM imidazole (pH 7.0), 1 mM DTT and either 500 μM CaCl₂ for the Ca²⁺ state or 1 mM EGTA for the EGTA state. The protein concentrations used were 4.2 μM F-actin, 0.6 μM cTm, 0.6 μM cTn and 0.5 μM S1. The amounts of inorganic phosphate released were determined colorimetrically and expressed in μmol Pi/sec/g ⁴². Ca²⁺ sensitivity was defined as {1-(Activity_EGTA/ Activity_Ca)}×100⁴². cTnC(wt), cTnI(wt) and cTnT(wt) are wild-type cTnC, cTnI, and cTnT, respectively. The cTnI mutants were labeled at the single cysteine with AEDANS.