Symplectin

The luminescent substance of the squid Symplectoteuthis oualaniensis was first obtained in the form of insoluble particles by Tsuji et al. (1981). The suspension of the particles emitted light in the presence of monovalent cations such as K+, Rb+, Na+, Cs+, NH+, and Li+ (in decreasing order of effect). Molecular oxygen was needed for the luminescence. Divalent ions such as Ca + and Mg + did not trigger light emission. The light emission (Amax 470 nm) was optimal in the presence of 0.6 M KCl or NaCl and at a pH of 7.8.

The soluble form of the Symplectoteuthis photoprotein was isolated and purified from the granular light organs of the squid and was named "symplectin" (Takahashi and Isobe 1994; Fujii et al 2002). The light organs were first extracted with a pH 6 buffer containing 0.4 M KCl to remove impurities, and then symplectin was extracted from the residue with a pH 6 buffer containing 0.6 M KCl. All solutions used in the experiments contained 0.25 M sucrose, 1 mM dithiothreitol, and 1 mM EDTA. The 0.6 M KCl extract was chromatographed by size-exclusion HPLC on a TSK G3000SW column. Symplectin was eluted as two major components of oligomers, having molecular masses of200 kDa or more, and a minor component of monomer (60 kDa). All processes of extraction and purification were carried out at 4 °C. Warming up a solution of symplectin, adjusted to pH 8, to room temperature causes the luminescence reaction to begin, and the light emission lasts for hours.

A tryptic digestion of the KCl extract increased the content of the 60-kDa species at the expense of the two high-molecular-weight species, accompanied by the formation of 40-kDa and 16-kDa species. SDS-PAGE analysis of the two high-molecular-weight oligomers revealed that they consist mainly of the 60-kDa protein. The 60-kDa protein and the 40-kDa protein were fluorescent in the SDS-PAGE analysis. The spent protein of symplectin after luminescence (aposymplectin) could be reconstituted into original symplectin by treatment with dehydrocoelenterazine (Isobe et al. 2002).

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