The rhodopsin photointermediate metarhodopsin-II (M_II) catalyzes an exchange of GDP to GTP on its cognate G-protein transducin (G_t) that results in splitting of the G_t heterotrimer into G_t,α- and G_t,βγ-subunits. The rate of this M_II-catalyzed reaction was studied on isolated rod outer segment disks and on rhodopsin reconstituted into model membranes at rhodopsin/lipid molar ratios from 1/5,000 up to 1/70. Membrane-incorporated, dark-adapted rhodopsin was photoactivated with a flash. In the presence of radioactive [³⁵S]-GTPγS, the photointermediate M_II splits accessible G_t molecules into subunits. The concentration of the [³⁵S]GTPγS-G_t,α complex was measured with a scintillation counter. It was observed that pre-association of heterotrimeric GDP-G_t with the lipid matrix containing rhodopsin is a critical step for high reaction rates. Every receptor in the M_II state may split over a hundred membrane-bound G_t molecules in rapid succession. By small angle neutron scattering (SANS) it was established that reconstituted, dark adapted rhodopsin molecules in membranes remained monomeric up to rhodopsin/lipid molar ratios of 1/300. While the amount of M_II was high and steady in the range from 1/5,000 down to 1/300, there was a continuous decline of the fraction of M_II formed from 1/300 – 1/70. The splitting of G_t into subunits was followed on life samples by SANS. Rates of M_II-catalyzed splitting of G_t into G_t,α- and G_t,βγ-subunits were highest when rhodopsin was monomeric. Bleached rhodopsin in a lipid matrix, in the absence of G_t, tends to oligomerize even at low concentration.

Jonathan Nickels

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