Separation and characterization of the two functional regions of troponin involved in muscle thin filament regulation.
Mild proteolytic cleavage of the troponin complex yields TnT1, the N-terminal fragment of troponin T, and TnT2IC, a complex of the C-terminal fragment of troponin T (TnT2) with troponin I (TnI) and troponin C (TnC) [Morris, E. P., & Lehrer, S. S. (1984) Biochemistry 23, 2214-2220]. Both TnT1 and TnT2IC bind tightly to the tropomyosin.actin (Tm.actin) thin filament and influence the interaction of myosin subfragment 1 (S1) with Tm.actin. TnT1 does not affect the rate of S1 binding to Tm.actin but does increase the cooperativity with which S1 "turns on" Tm.actin, monitored by the excimer fluorescence of a pyrene label attached to Cys 190 of Tm [Geeves, M.A., & Lehrer, S. S. (1994) Biophys. J. 67, 273-282]. The apparent cooperative unit size of Tm.actin is increased from 6 to 9 by TnT1 and to 12 by whole troponin. In contrast, TnT2IC has no effect on the cooperativity of Tm.actin but does make the apparent S1-binding rate constant, kapp, Ca(2+)-sensitive; i.e., in the absence of Ca2+, kapp is reduced 2-3-fold by both TnT2IC and whole troponin. Thus, the N- and C-terminal regions of TnT appear to act independently in modulating effects of S1 binding to the Tm.actin thin filament that are important in regulation.
Max Planck Institute für Molekulare Physiologie, Dortmund, Germany.
S
S
Schaertl
S S
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Lehrer
M A
MA
Geeves
D000199:Actins / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D007700:Kinetics; D018482:Muscle, Skeletal / Q000378:metabolism*; D015879:Myosin Subfragments / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments; D011485:Protein Binding; D011817:Rabbits; D013997:Time Factors; D014336:Troponin / Q000302:isolation & purification / Q000378:metabolism*; D020107:Troponin T
D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D014336:Troponin; D020107:Troponin T; D000255:Adenosine Triphosphate; D002118:Calcium