Bleaching, rhodopsin

Bleached rhodopsin must be regenerated to maintain normal vision 809... 

Bleached rhodopsin must be regenerated to maintain normal vision. Regeneration occurs by several mechanisms. The major pathway occurs in the retinal pigmented... 

Rhodopsin kinase (RK) phosphorylates bleached rhodopsin low [Ca2+] and recoverin (Recov) stimulate this reaction. Arrestin (Arr) binds phosphorylated carboxyl terminus, inactivating rhodopsin. 

Strissel, K.J., Sokolov, M., Trieu, L.H. and Arshavsky, V.Y. (2006) Arrestin translocation is induced at a critical threshold of visual signaling and is superstoichiometric to bleached rhodopsin. J. Neurosci. 26,... 

Bleaching is reversed in the dark and the red-purple color of rhodopsin returns. This is thought to occur by the reduction of all-Pms-retinal to vitamin Ai (retinal), which diffuses from the rod into the pigment epithelium, where it is converted enzymatically to the 1 l-c isomer of vitamin At. The enzymatic isomerization is followed by diffusion back into the rod, oxidation to 11 -rfr-retinal, and combination with opsin to form rhodopsin. This process is shown schematically in Figure 12.5.[Pg.289]

Table 12.3. Spectral Data for Rhodopsin and Its Bleaching Intermediates<18>...

The bleaching of rhodopsin has been found to lead to the all-trans form of retinal through several intermediate steps.(llb,45) These steps are temperature dependent consequently low temperatures must be used to observe the intermediate products. A solution of rhodopsin is subjected to a flash of light at liquid nitrogen temperature and intermediates are detected by changes in the absorption spectrum. The first intermediate, with an absorption maximum at 543 nm, believed to be an all-fra/w-retinal bound to opsin, has been termed prelumirhodopsin. Warming the sample to a temperature greater than... 

Figure 12.5. Cycle of reactions involved in the bleaching of rhodopsin by light and subsequent dark restoration.

FIGURE 49-4 Intermediates formed after photic bleaching of vertebrate rhodopsin. Numbers in parentheses are wavelengths of light (in nanometers) absorbed maximally by the individual intermediates. 

FIGURE 28. 283-MHz 19F NMR spectra of isomers of 8-F-rhodopsin in CFIAPS before (lower) and after photoirradiation (upper) (a) 11-cis (pulse delay, D5 = 5.0 s, number of acquisitions, NA = 5200, line broadening, LB = 80 Hz) (b) 9-cis (D5 = 50 ms, NA = 160000, LB = 80 Hz). Disappearance of the excess 9-cis aldehyde was due to repeated formation and bleaching of pigment during the irradiation process. Reprinted with permission from Reference 48. Copyright (1996) American Chemical Society... 

This enzyme [EC 2.7.1.125] catalyzes the reaction of ATP with rhodopsin to produce ADP and phosphorylated rhodopsin. The kinase acts on the bleached or activated form of rhodopsin. 

In the case of rhodopsin membrane under calcium gradient, the technique shows a nonlinearity of fluxes with respect to imposed gradients. We also notice some permeability variations just after rhodopsin bleaching and at last a calcium adsorption consequent to lighting performed in situ. 

A stack of about 1 000 disks in each rod cell contains the lightsensing protein rhodopsin,10 in which the chromophore 11-o i-retinal (from vitamin A) is attached to the protein opsin. When light is absorbed by rhodopsin, a series of rapid transformations releases all-frans-retinal. At this stage, the pigment is bleached (loses all color) and cannot respond to more light until retinal isomerizes back to the 11 -cis form and recombines with the protein. 

The visual chromophores. Rhodopsin has been an object of scientific interest for over 100 years.462 Wald and associates469 470 established that rhodopsin contains 11-ds-retinal bound to the opsin in Schiff base linkage (Eq. 23-36). When native rhodopsin is treated with sodium borohydride, little reduction is observed. However, after the protein is bleached by light, reduction of the Schiff base linkage becomes rapid, and the retinal is incorporated into a secondary amine, which was identified as arising from Lys 296. 

The eye of higher animals is remarkably sensitive to low light intensities, but saturation can occur relatively easily at high light intensities and the recovery is then slow. After the absorption of light rhodopsin (which has a red colour) is bleached when the retinal leaves the opsin, and recovery goes through a sequence of enzyme-catalysed reactions which takes several seconds. 

They did not start from reagent grade chemicals. The experiments involved the in-vitro bleaching of native rhodopsin in solution. They proposed that they caused the disassociation of rhodopsin into a protein and retinaldehyde in accordance with the conventional wisdom of the time. This wisdom conflicted somewhat with the earlier ideas of Kuhne but became the basis of the current conventional wisdom. They claimed to have removed the native retinene, and then recombined colorless rhodopsin-protein, free of all native retinene, with synthetic retinene, in high concentration. This was accomplished by letting the mixture set in the dark. The pH of these solutions was not described as a function of time. They demonstrated that the material after setting in the dark for 60 minutes exhibited a rise in extinction coefficient of 2 1 over a similar sample without the added retinene,. Both samples exhibited a peak absorption near 500 nm following the experiments. It is not clear why the reference sample showed any absorption at 500 nm if it was truly free of all native retinene,. 

Figure 5.5.10-5 CR Absorption spectrum of putative frog rhodopsin as a function of bleaching level. Curve 1 is unbleached. The material was prepared as a red powder and then placed in dilute solution. From Wolken, 1966. (A) Intrinsic ultraviolet peak of a retinoid. (B) intrinsic isotropic absorption peak of a retinol (al) complexed with opsin. From wolken, 1966.

Figure 7. Laser-induced absorbance changes at 561 nm as a function of time in detergent solubilised bovine rhodopsin (X) and isorhodopsin (9) at room temperature. Bathorhodopsin is the only intermediate during the bleaching of bovine rhodopsin known to absorb strongly at 561 nm. The energy of the 530-nm pump pulse was about 10 4J the energy of the 561-nm probe pulse was about 10 7J. The beam sizes were about 1 mm2 for the pump and 0.5 mm2 for the probe. The samples (about 1.5 mL) were held in 0.5-cm cuvettes. The concentrations were about 4 A cm I at the absorption peaks near 500 nm the ratios A Ajjj were about 0.3 and ratios ASsa. As<)o were about 0.7 for rhodopsin and 0.5 for isorhodopsin. Each data point shown is the average of six (rhodopsin) and nine (isorhodopsin) laser shots. Typical mean standard deviations are 0.03. The zero time is located using a 0.5 cm CS2 Kerr optical shutter at the sample site. The half width at half maximum for the CS2 shutter prompt response curve is about 6 ps.

Fig. 5. (A) Bleaching intermediates in the photolysis of bovine rhodopsin extracts. Adapted from Yoshizawa and Horiuchi [61]. (B) Bleaching intermediates in the photolysis of squid rhodopsin extracts. Adapted from Shichida et al. [105]. Photoreactions are symbolized by wavy lines, thermal reactions by straight lines.

In spite of the differences in the behavior of the pigments, in situ, as compared to extracts, studies on extracts provide useful information, not only on the structure of the bleaching intermediates, but also on the possible roles they may be playing in the photoreceptors. The bleaching intermediates from several species have been investigated extensively by using flash photolysis techniques and both low temperature and ultrafast kinetic spectroscopy. As an example, Fig. 5 shows the sequence of the intermediates in the photolysis of bovine and squid rhodopsin extracts. 

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