Mapping the domain of troponin T responsible for the activation of actomyosin ATPase activity. Identification of residues involved in binding to actin.
The in vitro Ca(2+) regulation of the actomyosin Mg(2+)-ATPase at physiological ratios of actin, tropomyosin, and troponin occurs only in the presence of troponin T. We have previously demonstrated that a polypeptide corresponding to the first 191 amino acids of troponin T (TnT-(1-191)) activates the actomyosin Mg(2+)-ATPase in the presence of tropomyosin. In order to further characterize this activation domain, we constructed troponin T fragments corresponding to residues 1-157 (TnT-(1-157)), 1-76 (TnT-(1-76)), 77-157 (TnT-(77-157)), 77-191 (TnT-(77-191)), and 158-191 (TnT-(158-191)). Assays using these fragments demonstrated the following: (a) residues 1-76 do not bind to tropomyosin or actin; (b) residues 158-191 bind to actin cooperatively but not to tropomyosin; (c) the sequence 77-157 is necessary for troponin interaction with residue 263 of tropomyosin; (d) TnT-(77-191) on its own activates the actomyosin ATPase activity as described previously for TnT-(1-191). TnT-(1-157), TnT-(1-76), TnT-(77-157), TnT-(158-191), and combinations of TnT-(158-191) with TnT-(1-157) or TnT-(77-157) showed no effect on the ATPase activity. We conclude that the activation of actomyosin ATPase activity is mediated by a direct interaction between amino acids 77 and 191 of troponin T, tropomyosin, and actin.
Oliveira
D M
DM
Departamento de Bioquimica, Instituto de Quimica, Universidade de São Paulo CP 26.077, CEP 05599-970 São Paulo, Brazil.
Nakaie
C R
CR
Sousa
A D
AD
Farah
C S
CS
Reinach
F C
FC
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D020013:Calcium Signaling; D002645:Chickens; D002942:Circular Dichroism; D004789:Enzyme Activation; D007700:Kinetics; D018482:Muscle, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014335:Tropomyosin / Q000378:metabolism; D020107:Troponin T / Q000737:chemistry* / Q000378:metabolism*
D000199:Actins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014335:Tropomyosin; D020107:Troponin T; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins