THE C-TERMINUS OF CARDIAC TROPONIN-I IS ESSENTIAL FOR FULL INHIBITORYACTIVITY AND CA2+ SENSITIVITY OF RAT MYOFIBRILS

Although the C terminus of troponin I is known to be important in myof ilament Ca2+ regulation in skeletal muscle, the regulatory function of this region of cardiac troponin I (cTnI) has not been defined. To add ress this question, the following recombinant proteins were expressed in Escherichia coli and purified: mouse wildtype cTnI (WT cTnI; 211 re sidues), cTnI-(1-199) (missing 12 residues), cTnI-(1-188) (missing 23 residues), and cTnI-(1-151) (missing 60 residues), The inhibitory acti vity of cTnI and the mutants was tested in myofibrils, from which cTnI .cTnC was extracted by exchanging endogenous cardiac troponin with exo genous cTnT causing the Ca2+ sensitivity of the myofibrils to be lost, Addition of increasing amounts of exogenous WT cTnI or cTnI-(1-199) t o cTnT-treated myofibrils at pCa 8 caused a concentration-dependent in hibition of the maximum ATPase activity, However, cTnI-(1-188) and cTn I-(l-151) inhibited this activity to about 75% and 50% of that of the WT cTnI, respectively, We also formed a complex of either WT cTnI or e ach of the mutants with cTnC, reconstituted the complex into the cTnT- treated myofibrils, and measured the Mg2+-ATPase activity as a functio n of pCa, We found that the cTnI-(1-188).cTnC complex only partially r estored Ca2+ sensitivity, whereas the cTnI-(1-151).cTnC complex did no t restore any Ca2+ sensitivity, Each cTnI C terminal deletion mutant w as able to bind to cTnC, as shown by urea-polyacrylamide gel-shift ana lysis and size exclusion chromatography, Each mutant also co-sedimente d with actin, Our results indicate that residues 152-199 (C-terminal t o the inhibitory re gion) of cTnI are essential for full inhibitory ac tivity and Ca2+ sensitivity of myofibrillar ATPase activity in the hea rt.