Functional importance of Ca2+-deficient N-terminal lobe of molluscan troponin C in troponin regulation.
Ca(2+)-binding sites I and II in the N-terminal lobe of molluscan troponin C (TnC) have lost the ability to bind Ca(2+) due to substitutions of the amino acid residues responsible for Ca(2+) liganding. To evaluate the functional importance of the Ca(2+)-deficient N-terminal lobe in the Ca(2+)-regulatory function of molluscan troponin, we constructed chimeric TnCs comprising the N-terminal lobes from rabbit fast muscle and squid mantle muscle TnCs and the C-terminal lobe from akazara scallop TnC, TnC(RA), and TnC(SA), respectively. We characterized their biochemical properties as compared with those of akazara scallop wild-type TnC (TnC(AA)). According to equilibrium dialysis using (45)Ca(2+), TnC(RA), and TnC(SA) bound stoichiometrically 3 mol Ca(2+)/mol and 1 mol Ca(2+)/mol, respectively, as expected from their primary structures. All the chimeric TnCs exhibited difference-UV-absorption spectra at around 280-290 nm upon Ca(2+) binding and formed stable complexes with akazara scallop troponin I, even in the presence of 6M urea, if Ca(2+) was present. However, when the troponin complexes were constructed from chimeric TnCs and akazara scallop troponin T and troponin I, they showed different Ca(2+)-regulation abilities from each other depending on the TnC species. Thus, the troponin containing TnC(SA) conferred as high a Ca(2+) sensitivity to Mg-ATPase activity of rabbit actomyosin-akazara scallop tropomyosin as did the troponin containing TnC(AA), whereas the troponin containing TnC(RA) conferred virtually no Ca(2+) sensitivity. Our findings indicate that the N-terminal lobe of molluscan TnC plays important roles in molluscan troponin regulation, despite its inability to bind Ca(2+).
Doi
Teppei
T
Laboratory of Biochemistry and Biotechnology, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan.
Satoh
Ai
A
Tanaka
Hiroyuki
H
Inoue
Akira
A
Yumoto
Fumiaki
F
Tanokura
Masaru
M
Ohtsuki
Iwao
I
Nishita
Kiyoyoshi
K
Ojima
Takao
T
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D002678:Chimera / Q000235:genetics; D008969:Molecular Sequence Data; D008974:Mollusca / Q000737:chemistry*; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D014335:Tropomyosin / Q000378:metabolism; D019209:Troponin C / Q000737:chemistry / Q000378:metabolism*; D019210:Troponin I / Q000737:chemistry / Q000378:metabolism; D020107:Troponin T / Q000737:chemistry / Q000378:metabolism
D002413:Cations, Divalent; D010455:Peptides; D014335:Tropomyosin; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium