Abstracts

The marine firefly “Cypridina hilgendorfii Müller” was discovered by F. Hilgendorf in around 1874 of Japan coast and introduced by W. Müller in ZOOLOGISCHE JAHRBÜCHER in 1891. The luminous Ostracod Cypridina, “Umihotaru called in Japanese”, is a typical luciferin-luciferase reaction without any cofactors. Before the discover of the green fluorescent protein (GFP) by O. Shimomura et al., he achieved to isolate and crystallize the Cypridina luciferin. N. Kishi et al. determined the chemical structure of this luciferin as a second success example of determined its chemical structure and Shimomura proposed the chemical reaction mechanism on the luciferin-luciferase. (S)-Cypridina luciferin has an imidazopyrazinone compound having three functional groups at the C2, C6, and C8 positions, which probably derives biogenetically from three amino acids: L-arginine, L-isoleucine, and L-tryptophan (or tryptamine). Cypridina luciferase is both glycoprotein and secretory protein. In the proposal mechanism of luciferin-luciferase reaction, when the luciferin bound to luciferase, the C7 of imidazopyrazinone is negatively charged and reacts easily with oxygen in C2 position to forming a dioxetaone. The excited state of oxyluciferin falls to its ground state, resulted in producing a blue light (lmax = 460 nm). During this reaction, the quantum yield was measured about 0.3 at 4 C, which is higher than coelenterazine-type bioluminescence system based on similar imidazopyrazinone compound. Two genes of Cypridina luciferases were cloned by F. I. Tsuji and our group. Cypridina luciferase has a higher molecular weight and have up to 17 potential disulfide bonds between the 34 cysteine residues in its 553-555 amino acids, resulting in stable protein with higher thermal stability. After transfection of these genes, the luciferase protein can express and secret to the out of cell in the mammalian, plant, and yeast cell but not express in E.coli. Until now, our group crystalized the recombinant Cypridina luciferase and determined its three-dimensional structure. The chemical synthesis procedure of Cypridina luciferin had already established as a commercial purpose by C. Wu. On the other hand, the application of Cypridina system was available in the biotechnology field for the in vitro cell functional analysis, ex vivo and in vivo imaging for tumor tissue etc. For instance, dual reporter assay using two secreted Cypridina and Gaussia luciferases can evaluate two gene expressions in the medium of living cell using Cypridina luciferin