Abstract
The binding of the NH2-terminal region of troponin T (TnT) to the COOH-terminal region of tropomyosin (Tm) and the head-to-tail overlap between Tm molecules is thought to provide a pivotal link between troponin (Tn) and Tm (White, S.P., Cohen, C., and Phillips, G.N., Jr. (1987) Nature 325, 826-828). To further explore the structure-function relationship of the NH2-terminal region of TnT, we studied the binding of a 26,000-dalton TnT fragment (26K-TnT, Ohtsuki, I., Shiraishi, F., Suenaga, N., Miyata, T., and Tanokura, M.J. (1984) J. Biochem. (Tokyo) 95, 1337-1342) which corresponds to residues 46-259 of TnT2f, the major isoform of TnT in rabbit fast twitch muscle, to immobilized alpha-Tm. Both 26K-TnT and TnT2f were retained by the alpha-Tm affinity column in the presence of 150 mM NaCl. However, upon increasing the NaCl concentration 26K-TnT was eluted from the column at a higher ionic strength than was TnT. When applied alone, the binary complex of TnI and TnC (TnC.TnI) was not retained by the alpha-Tm affinity column. When applied subsequently to prebound TnT2f or 26K-TnT, TnI.TnC was retained by the alpha-Tm affinity column and eluted together with TnT2f or 26K-TnT as ternary troponin complexes. Whether Ca2+ was present or not, Tn containing 26K-TnT was eluted at a higher ionic strength than was Tn containing TnT2f, indicating that removal of the first 45 residues of TnT2f strengthens the binding of Tn to Tm. In the presence of Tm, reconstituted Tn containing 26K-TnT conferred Ca2+ sensitivity on actomyosin-S1 MgATPase, and the steepness of the pCa-ATPase relation was unchanged with respect to the actoS1 ATPase regulated by TnT2f. It is concluded that the first 45 residues of TnT2f are not essential for anchoring the troponin complex to the thin filament and do not play a crucial role in the cooperative response of regulated actoS1 ATPase to Ca2+.
Highlights
From the $Departmentof Physiology and Biophysics, University of Washington, Seattle, Washington 98195 and the §Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101
NHz-terminal regionof tail overlap between Tm mole- (TnT), we studied the bindionfg identifiedtworegions as the sites of interaction with Tm
TnI.TnC was retained by the a-Tm affinity column head-to-tail overlap of Tm, where T1 interacts with the NHe and eluted together with TnTozrf26K-TnT as ternary terminus of the adjoining T m [11,12,13]
Summary
Purification of 26K-TnT-We have developed a new procedure for the purification of 26K-TnT, which is described under “Materials and Methods.” Using crude Tn as starting material, the procedure first separates the 26K-TnfrTagment from T n T by anion-exchangechromatographyonDEAEcellulose. Purification of 26K-TnT-We have developed a new procedure for the purification of 26K-TnT, which is described under “Materials and Methods.”. Using crude Tn as starting material, the procedure first separates the 26K-TnfrTagment from T n T by anion-exchangechromatographyonDEAEcellulose. When crude T n was passed through aDE52 column, the 26K-TnTfragment and TnIemerged in theflow-through fraction, whereas T n T was retained by the column. The differentchromatographic behavior of the 26,000-Da frag-. Aminoacidcompositionwas determined witha Waters Picotag system [35]
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