Sensitive light scattering probe of enzymatic processes in retinal rod photoreceptor membranes

Light excitation of as little as 0.05% of the rhodopsin in a retinal rod membrane suspension reduces the near-IR optical transmission by 25%. This transmission decrease requires the presence of guanosine triphosphate, is opposite in sign and 25 times larger in amplitude than a GTP-dependent light-scattering signal previously reported in rod outer segment suspensions [Kuhn, H., Bennett, N., Michel-Vallez, M. & Chabre, M. (1981) Proc. Natl. Acad. Sci. USA, 78, 6873-6877], and is kinetically complex. The initial phase of the optical transmission decrease begins after about a 50-ms lag (at 0.05% bleach) and has a first-order time constant of 300-500 ms. The scattering signal returns to the preactinic baseline in a time dependent on the amount of GTP added. A nonhydrolyzable GTP analogue, guanylyl imidodiphosphate, produces a scattering signal that does not return to the preactinic baseline. Adenosine triphosphate strongly inhibits the return of the GTP-dependent transmission decrease to the preactinic baseline. This effect of ATP on the GTP signal apparently requires ATP hydrolysis because it is inhibited by the simultaneous presence of adenylyl imidodiphosphate, a nonhydrolyzable analogue of ATP. The light-scattering signal and the velocity of the activation of a rod outer segment phosphodiesterase saturate when >0.05% of the rhodopsin is bleached and both show nearly identical dependence on light stimulus. It is suggested that these nucleotide-dependent light-scattering signals arise from changes in the state of membrane aggregation that are controlled by enzymatic processes. This hypothesis is supported by the large amplitude of the signals, sedimentation experiments, and a strong membrane concentration dependence. The ATP effects can be rationalized within the above hypothesis as being due to ATP-dependent rhodopsin phosphorylation that adds negative charges to the membrane surface and tends to keep the membranes disaggregated. An additional signal, which increases light transmission, is produced by a second, much more intense flash. The latter signal is interpreted as the result of proton binding by bleached rhodopsin molecules that decreases the negative charge repulsion between the membranes and allows increased aggregation.