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Titolo:
Functional role of Ca2+-binding site IV of scallop troponin C
Autore:
Ojima, T; Koizumi, N; Ueyama, K; Inoue, A; Nishita, K;
Indirizzi:
Hokkaido Univ, Fac Fisheries, Dept Marine Bioresources Chem, Hakodate, Hokkaido 041861, Japan Hokkaido Univ Hakodate Hokkaido Japan 041861 date, Hokkaido 041861, Japan
Titolo Testata:
JOURNAL OF BIOCHEMISTRY
fascicolo: 5, volume: 128, anno: 2000,
pagine: 803 - 809
SICI:
0021-924X(200011)128:5<803:FROCSI>2.0.ZU;2-X
Fonte:
ISI
Lingua:
ENG
Soggetto:
AMINO-ACID-SEQUENCE; STRIATED ADDUCTOR MUSCLE; AKAZARA SCALLOP; SKELETAL-MUSCLE; ATPASE ACTIVITY; LIGHT-CHAINS; MYOSIN; TROPOMYOSIN; CONTRACTION; MYOFIBRILS;
Keywords:
scallop troponin C; Ca2+-regulatory function; recombinants; Mg-ATPase activity;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
44
Recensione:
Indirizzi per estratti:
Indirizzo: Nishita, K Hokkaido Univ, Fac Fisheries, Dept Marine Bioresources Chem, Hakodate, Hokkaido 041861, Japan Hokkaido Univ Hakodate Hokkaido Japan 041861 ido 041861, Japan
Citazione:
T. Ojima et al., "Functional role of Ca2+-binding site IV of scallop troponin C", J BIOCHEM, 128(5), 2000, pp. 803-809

Abstract

Scallop troponin C (TnC) binds only one Ca2+/mol and the single Ca2+-binding site has been suggested to be site IV on the basis of the primary structure [K. Nishita, H. Tanaka, and T. Ojima (1994) J. Biol. Chem. 269, 3464-3468; T. Ojima, Ii. Tanaka, and K. Nishita (1994) Arch. Biochem. Biophys. 311, 272-276]. In the present study, the functional role of Ca2+-binding site IV of akazara scallop (Chlamys nipponensis akazara) TnC in Ca2+-regulation was investigated using a site-directed mutant with an inactivated site TV (TnC-ZEQ), N- and C-terminal half molecule mutants (TnC(N) and TnC(C)), and wild-type TnC (TnC(C)). Equilibrium dialysis using Ca-45(2+) demonstrated that TnC(W) and TnC(C) bind 0.6-0.8 mol of Ca2+/mol, but that TnC-ZEQ and TnC(N) bind virtually no Ca2+. The UV difference spectra of TnC(W) and TnC(C)showed bands at around 280-290 nm due to the perturbation of Tyr and Trp upon Ca2+-binding, while TnC-ZEQ and TnC(N) did not show these bands. In addition, TnC(W) and TnC(C) showed retardation of elution from Sephacryl S-200upon the addition of 1 mM CaCl2, unlike TnC-ZEQ and TnC(N). These results indicate that Ca2+ binds only to site IV and that Ca2+-binding causes structural changes in both the whole TnC molecule and the C-terminal half molecule. In addition, TnC(W), TnC-ZEQ, and TnC(C), but not TnC(N) were shown to form soluble complexes with scallop TnI at physiological ionic strength. Onthe other hand, the Mg-ATPase activity of reconstituted rabbit actomyosin in the presence of scallop tropomyosin was inhibited by scallop TnI and recovered by the addition of an equimolar amount of TnC(W), TnC-ZEQ, or TnC(C), but not TnC(N). These results imply that the site responsible for the association with TnI is located in the C-terminal half domain of TnC. Ternary complex constructed from scallop TnT, TnI, and Tnc, conferred Ca2+-sensitivity to the Mg-ATPase of rabbit actomyosin to the same extent as native troponin, but the TnC(N)-TnT-TnI and TnC-ZEQ-TnT-TnI complexes conferred no Ca2-sensitivity, while the TnC(C)-TnT-TnI complex conferred weak Ca2+-sensitivity. Thus, the major functions of scallop TnC, such as Ca2+-binding and interaction with TnI, are located in the C-terminal domain, however, the fullCa2+-regulatory function requires the presence of the N-terminal domain.

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Documento generato il 02/04/20 alle ore 00:10:52