Functional equivalence of metarhodopsin II and the Gt-activating form of photolyzed bovine rhodopsin.
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)
Kibelbek
J
J
Department of Biochemistry, University of Virginia Health Sciences Center, Charlottesville 22908.
Mitchell
D C
DC
Beach
J M
JM
Litman
B J
BJ
D000818:Animals; D002417:Cattle; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006868:Hydrolysis; D010777:Photochemistry; D011487:Protein Conformation; D012243:Rhodopsin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D013057:Spectrum Analysis
C013110:metarhodopsins; D012243:Rhodopsin; D019204:GTP-Binding Proteins