Abstract
The cardiac troponin (Tn) complex, consisting of a Ca 2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT), has been reconstituted from purified troponin subunits isolated from bovine heart muscle. The Ca 2+ binding properties of cardiac Tn were determined by equilibrium dialysis using either EGTA or EDTA to regulate the free Ca 2+ concentration. Cardiac Tn binds 3 mol Ca 2+/mol and contains two Ca 2+-binding sites with a binding constant of 3 x 10 8 M -1 and one binding site with a binding constant of 2 x 10 6 M -1. In the presence of 4 mM MgCl 2, the binding constant of the sites of higher affinity is reduced to 2 x 10 7 M -1, while Ca 2+ binding to the site at the lower affinity is unaffected. The two high affinity Ca 2+ binding sites of cardiac Tn are analogous to the two Ca +-Mg 2+ sites of skeletal Tn, while the single low affinity site is similar to the two Ca 2+ specific sites of skeletal Tn. The Ca 2+ binding properties of the complex of TnC and TnI (1:1 molar ratio) were similar to those of Tn. Cardiac TnC also binds 3 mol of Ca 2+/mol and contains two sites with a binding constant of 1 x 10 7 M -1 and a single site with a binding constant of 2 x 10 5 M -1. Assuming competition between Mg 2+ and Ca 2+ for the high affinity sites of TnC and Tn, the binding constants for Mg 2+ were 0.7 and 3.0 x 10 3 M -1, respectively. The Ca 2+ dependence of cardiac myofibrillar ATPase activity was similar to that of an actomyosin preparation regulated by the reconstituted troponin complex. Comparison of the Ca 2+ binding properties of cardiac Tn and the cardiac myofibrillar ATPase activity as a function of [Ca 2+] and at millimolar [Mg 2+] suggests that activation of the ATPase occurs over the same range of [Ca 2+] where the Ca 2+-specific site of cardiac Tn binds Ca 2+.
Original language | English (US) |
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Pages (from-to) | 11688-11693 |
Number of pages | 6 |
Journal | Journal of Biological Chemistry |
Volume | 255 |
Issue number | 24 |
State | Published - Dec 1 1980 |
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology