Aequorea

Direct Metal Analyses. Calcium ion can be detected to a lower limit of 10 M hy Aequorea bioluminescence. Strontium interferes to a minor extent (270,271). 

Harvey (1952) noted R. S. Anderson has tested Aequorea at Friday Harbor and found that his hydromedusan will luminesce under strict anaerobic conditions. It was an extremely important observation retrospectively. The ability to luminesce in the absence of oxygen had been also observed with Medusa hemisphaerica by Macartney (1810) and with radiolarians, ctenophores, and Pelagia by Harvey (1926b). 

Shimomura and Johnson, 1969). The blue fluorescent substance showed an ultraviolet absorption maximum at 350 nm (in ethanol) thus, it was named AF-350 (renamed coelenteramine later). We obtained about 1 mg of AF-350 from 125 mg of pure aequorin that had been extracted and purified from 2.5 tons of Aequorea, and then determined its structure as shown in Fig. 4.1.9 (Shimomura and Johnson, 1972). 

Properties of GFP. Aequorea GFP is a relatively stable protein with a molecular weight of about 27,000 (Shimomura, 1979). The... 

Fig. 4.1.16 Luminescence spectrum of aequorin triggered by Ca2+ (solid line /.max 465 nm), and the fluorescence spectra of Aequorea GFP excitation (dashed line A.max 400 nm and 477 nm) and emission (dash-dot line 7max 509 nm). The dotted line is the fluorescence excitation spectrum of GFP in the light organs, showing that 480 nm excitation peak is almost missing — an evidence showing that GFP in light organs exists in an aggregated form having a very low E value at 480 nm.

The gene of Aequorea GFP was cloned by Prasher et al. (1992), and expressed in E. coli and Caenorhabditis elegans by Chalfie et al. (1994) and in E. coli by Inouye and Tsuji (1994a). The X-ray structure of recombinant GFP was solved by Ormo et al. (1996) and Yang et al. (1996,1997). The protein is in the shape of a cylinder consisting of 11 strands of (3-sheets and an a-helix inside (which contains the chromophore), with short helical segments on the ends of the cylinder. Thus the chromophore is sealed and protected from the outside medium. 

Fig. 4.1.17 Graphic illustration of Forster-type resonance energy transfer from aequorin to Aequorea GFP. In the vessel at left, a solution contains the molecules of aequorin and GFP randomly distributed in a low ionic strength buffer. The vessel at right contains a solution identical with the left, except that it contains some particles of DEAE cellulose. In the solution at right, the molecules of aequorin and GFP are coadsorbed on the surface of DEAE particles. Upon an addition of Ca2+, the solution at left emits blue light from aequorin (Xmax 465 nm), and the solution at right emits green light from GFP (Xmax 509 nm).

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