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M. T. Walker, P. R. Robinson; Spectral Analysis Of Endogenous Mouse Melanopsin. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4642.
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Melanopsin is a protein that belongs to the opsin class of proteins in the G protein coupled receptor super family. Melanopsin is expressed in about 1-3% of retinal ganglion cells in the mammalian retina. With so few cells in the retina expressing melanopsin, much of the spectral analysis of the photopigment has used heterologously expression melanopsin. Like other vertebrate opsins, expressed melanopsin binds a vitamin A-based chromophore 11-cis-retinal in the dark, and the photopigment formed can be activated in a light dependent manner. Previous work from our lab has demonstrated that the absorbance maximum of purified expressed melanopsin in detergent solution is 420nm. The spectral absorbance of the purified expressed melanopsin can be red-shifted 60nm with the removal NaCl from the solution.Using an antibody against the N-terminal of mouse melanopsin, we are able to select melanopsin expressing cells from mouse retinas. This enriched sample of melanopsin expressing cells are then used to measure the absorbance endogenous melanopsin.
We use a molecular and biochemical approach to identify the spectral properties of endogenous mouse melanopsin. Melanopsin expressing cells are selected from mouse retinas by disassociating the retina, labeling melanopsin expressing cells with a melanopsin antibody, and finally sorting melanopsin expressing cells using an immuno-affinity magnetic bead procedure. Melanopsin sorted cells are solubilized in detergent and the photopigment absorbance is measured.
Similar to the heterologously expressed melanopsin, endogenous mouse melanopsin reconstituted with 11-cis-retinal has a maximum wavelength absorbance of 420nm in solution. Surprisingly, the absorbance maximum of melanopsin photopigment from dark adapted retinas is drastically red-shifted from that of the reconstituted photopigment.
These experiments demonstrate that the reconstitution of both heterologously and endogenously expressed mouse melanopsin with 11-cis-retinal results in spectra with a 420 nm absorbance maximum. The difference in absorbance maximum observed for pigment from dark adapted retinas suggests that reconstituted melanopsin might not represent the stable dark form of the photopigment.
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