The effect of troponin I phosphorylation on the Ca2+-binding properties of the Ca2+-regulatory site of bovine cardiac troponin.

The effect troponin I phosphorylation has on the Ca2+-binding properties of troponin C (TnC) in the whole troponin complex reconstituted from cardiac troponin I (TnI), troponin T, and TnC has been measured. To selectively follow Ca2+ binding at the Ca2+-specific sites of TnC, this subunit was labeled with the fluorescent probe 2-(4’-iodo-acetamidoanilino)naphthalene-6sulfonic acid before reconstituting the whole cardiac troponin complex, TnlA (Johnson, J. D., Collins, J. H., Robertson, S . P., and Potter, J. D. (1980) J. Biol. Chem 255, 9635-9640). Two different methods were used to obtain phosphorylated T ~ M . The first was to form TnlA using nonphosphorylated TnI (0.1 mol of P/mol) which was then phosphorylated with 3’:5’-cyclic AMP-dependent protein kinase and ATP. Control samples were treated similarly but without added ATP. This procedure catalyzed the incorporation of 0.9 f 0.3 mol of P/ mol of rlhlA. Autoradiograms of 12.5% sodium dodecyl sulfate-polyacrylamide slab gels indicated that more than 98% of the 32P incorporated into T ~ I A was associated with TnI. The second method was first to phosphorylate the TnI subunit with the enzyme described above (1.7 f 0.3 mol of P/mol) before reconstituting T n I A . Control, nonphosphorylated, TnI samples were treated the same but without added ATP and contained 0.1 mol of P/mol. These two samples of TnI were then used to form phosphorylated and nonphosphorylated TnlA preparations. Independent of the method used to obtain the phosphorylated T n r * complexes, a decrease in the pCa required to achieve 50% of the maximal Ca2+induced fluorescence change in T ~ I A was observed. The rate of removal of CaZ+ from T n l A was also found to increase from 14.5 s-l to 21 s” upon TnI phosphorylation. The measured rate constants for Ca2+ removal and the pCasoa values for nonphosphorylated and phosphorylated Tnw were used to model the time course of Ca2+ binding to Tnu in response to a Ca2+ transient similar to an in vivo cardiac Ca2+ transient. The physiological implications of these measurements and calculations are discussed.