N-terminal modification and its effect on the biochemical characteristics of Akazara scallop tropomyosins expressed in Escherichia coli.
Akazara scallop striated muscle tropomyosin mutants without a fused amino acid (nf-Tm), and with Ala- (A-Tm) or Asp-Ala- (DA-Tm) fused at the N-terminus were expressed in Escherichia coli cells. Among them, nf-Tm alone has an initial methionine. The native Akazara scallop tropomyosin and DA-Tm showed similar alpha-helix contents and intrinsic viscosity, but nf-Tm and A-Tm exhibited lower values than those of the native tropomyosin. According to the relative viscosity, all the expressed tropomyosins appear to have lost head-to-tail polymerization ability. Though nf-Tm has extremely low actin-binding ability, the ability was almost completely recovered with a two amino acid fusion but incompletely with a one amino acid fusion. On the other hand, an amino acid fusion, irrespective of the number, seemed to inhibit the Mg-ATPase activity of actomyosin. However, the bacterially expressed tropomyosins together with Akazara scallop troponin do not confer the full Ca(2+)-regulation ability of Mg-ATPase activity of actomyosin. These results support that N-terminal blocking probably by an acetyl group of Akazara scallop tropomyosin plays an important role not only in head-to-tail polymerization and actin-binding, as known for vertebrate tropomyosin, but also in maintaining the secondary or higher structure and Ca(2+)-regulation together with troponin.
Laboratory of Biochemistry and Biotechnolgy, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate 041-8611.
Akira
A
Inoue
Takao
T
Ojima
Kiyoyoshi
K
Nishita
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008974:Mollusca / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D013696:Temperature; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000199:Actins; D014335:Tropomyosin