Absorption of a photon by the visual pigment rhodopsin leads to the formation of an activated conformational state, denoted rho*, which is capable of activating the visual G-protein, Gt. The bleaching of rhodopsin can be resolved into a series of spectrally distinct photointermediates. Previous studies suggest that the photointermediate metarhodopsin II (meta II, lambda max of 380 nm) corresponds to the physiologically active form rho*. In the studies reported herein, spectral and enzymological data were analyzed and compared so as to evaluate the temporal correspondence between meta II and rho*. This information was obtained by direct observation of the meta II and rho* decay times in parallel experiments utilizing identical preparations of urea-stripped, bovine retinal rod outer segment disk membranes at pH 8.0, 20 degrees C. Postflash spectra were deconvolved to resolve the meta II absorbance at 380 nm, and a decay time for the loss of meta II of 8.2 min (SD = 0.5 min) was obtained from fitting these data to a single-exponential decay process. The diminishing ability of bleached rhodopsin to activate Gt was measured by monitoring the level of catalyzed exchange of Gt-bound GDP for a nonhydrolyzable GTP analogue. Analysis of the decrease in the initial velocity of nucleotide exchange, measured at various postflash incubation times, yielded a rho* decay time of 7.7 min (SD = 0.5 min) when analyzed as a single-exponential process. The similarity of these decay times provides direct evidence that meta II and rho* are present over the same time regime, and further supports the equivalence of these two forms of photoactivated rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)
The photochemical intermediate metarhodopsin II (meta II; lambda max = 380 nm) is generally identified with rho*, the conformation of photolyzed rhodopsin which binds and activates the visual G-protein, Gt [Emeis, D., & Hoffman, K.P. (1981) FEBS Lett. 136, 201-207]. Purified bovine rhodopsin was incorporated into vesicles consisting of dimyristoylphosphatidylcholine (DMPC), and the rapid formation of a photochemical intermediate absorbing maximally at 380 nm was quantified via both flash photolysis and equilibrium spectral measurements. Kinetic and equilibrium spectral measurements performed above the Tm of DMPC showed that Gt, in the absence of GTP, enhances the production of the 380-nm-absorbing species while reducing the concentration of the 478-nm-absorbing species, metarhodopsin I (meta I), in a manner similar to that observed in the native rod outer segment disk membrane. This Gt-induced shift in the equilibrium concentration of photointermediates indicated that the species with an absorbance maximum at 380 nm was meta II. The presence of rho* in the DMPC bilayer was established via measurements of photolysis-induced exchange of tritiated GMPPNP, a nonhydrolyzable analogue of GTP, on Gt. Above Tm, the metarhodopsin equilibrium is strongly shifted toward meta I relative to the native rod outer segment disk membrane; however, at 37 degrees C, 40% of the photointermediates are in the form of meta II. The formation of meta II above Tm is slowed by a factor of ca. 2 relative to the disk membrane. Below Tm, the equilibrium is shifted still further toward meta I, and meta II forms ca. 7 times slower than in the disk membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
The superfamily of membrane-bound receptors, which function in signal transduction by activating a guanine nucleotide binding protein or G-protein in response to agonist binding, shares a number of structural and mechanistic properties. Among these similarities is downregulation of functional activity via receptor phosphorylation. In this study, the effects of intermediate levels of phosphorylation (greater than or equal to 4 added phosphates per receptor molecule) on receptor conformational equilibria are examined by comparing the photochemical properties of phosphorylated and unphosphorylated rhodopsins which were incorporated separately into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine vesicles. Postflash spectra reflecting the contributions of metarhodopsins I, II, and III (meta I, meta II, and meta III) were obtained from these samples. Deconvolution of appropriate difference spectra allowed a determination of the concentration of the photointermediates of interest. Meta II is the form of photolyzed rhodopsin which binds and activates the visual G-protein (Gt); thus, its relative abundance at equilibrium and temporal stability are important parameters in determining the efficiency of visual signal transduction. The effects of pH and temperature on the meta I in equilibrium with meta II equilibrium constant (Keq) and the rate of decay of meta II to meta III were examined for the reconstituted phosphorylated and unphosphorylated rhodopsin samples. Keq was essentially unaffected by phosphorylation when measured at pH 7.0 and 8.0 and 20 and 37 degrees C. The decay time (lifetime) of meta II----meta III had a value of approximately 4.7 min in both phosphorylated and unphosphorylated samples.(ABSTRACT TRUNCATED AT 250 WORDS)
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