Structure-function studies of the amino-terminal region of bovine cardiac troponin T.
The length and amino acid sequence of the amino-terminal region of troponin T (TnT) is regulated by alternative mRNA processing in both mammals and birds. To study the function of this region, three forms of bovine cardiac TnT were compared: isoforms TnT1 and TnT2, which differ by the presence or absence of residues 15-19 and TnT 39-284. TnT 39-284 was prepared by chemical cleavage of TnT1 at Cys-39. All three forms of TnT successfully reconstituted with troponin I and troponin C, resulting in troponins designated Tn1, Tn2, and TnCN. Three properties of the reconstituted troponins were compared. 1) Tn1 and TnCN had indistinguishable effects on tropomyosin polymerization. Addition of either 8 microM Tn1 or 8 microM TnCN increased the viscosity (eta rel) of 5 microM tropomyosin from 1.0 to 1.63 at 10 degrees C. 2) All of the three troponins conferred Ca2+ dependence to the MgATPase rate of myosin S-1-actin-tropomyosin. In the presence of saturating concentrations of Tn2, Tn1, or TnCN, 50% MgATPase activation occurred at pCa 6.0, 5.9, or 5.75, respectively. 3) The affinity of the Ca2+-specific binding site of reconstituted Tn1 was 50% stronger than the affinity of the same site on TnCN. These results suggest that the amino-terminal region of cardiac TnT is not a completely Ca2+-insensitive domain, but rather modulates the interaction of Ca2+ with troponin and with the thin filament. Furthermore, the effects of TnT on tropomyosin-tropomyosin binding are predominantly due to portions of TnT carboxyl-terminal to residue 38.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D007700:Kinetics; D009206:Myocardium / Q000032:analysis*; D009994:Osmolar Concentration; D011108:Polymers / Q000378:metabolism; D011817:Rabbits; D013329:Structure-Activity Relationship; D014335:Tropomyosin / Q000378:metabolism; D014336:Troponin / Q000032:analysis*; D020107:Troponin T
D011108:Polymers; D014335:Tropomyosin; D014336:Troponin; D020107:Troponin T; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium