Rhodopsin in dimyristoylphosphatidylcholine-reconstituted bilayers forms metarhodopsin II and activates Gt.
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)
Mitchell
D C
DC
Department of Biochemistry, University of Virginia Health Sciences Center, Charlottesville 22908.
Kibelbek
J
J
Litman
B J
BJ
D000818:Animals; D002417:Cattle; D004134:Dimyristoylphosphatidylcholine; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D007700:Kinetics; D008051:Lipid Bilayers; D010777:Photochemistry; D010782:Photolysis; D012243:Rhodopsin / Q000031:analogs & derivatives* / Q000378:metabolism*; D013053:Spectrophotometry; D013816:Thermodynamics
D008051:Lipid Bilayers; D006165:Guanylyl Imidodiphosphate; C013110:metarhodopsins; D012243:Rhodopsin; D019204:GTP-Binding Proteins; D004134:Dimyristoylphosphatidylcholine