The calcium and magnesium binding sites on cardiac troponin and their role in the regulation of myofibrillar adenosine triphosphatase

M. J. Holroyde, S. P. Robertson, J. D. Johnson, R. J. Solaro, J. D. Potter

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209 Scopus citations

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 languageEnglish (US)
Pages (from-to)11688-11693
Number of pages6
JournalJournal of Biological Chemistry
Volume255
Issue number24
StatePublished - 1980

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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