Rod outer segment (ROS) disks, either stacked or freely floating, respond to flash illumination to yield a specific, ATP-dependent, light-scattering signal AL. In broken ROS AL signals occur only when AD signals have preceded them. The degree to which the preceding AD signal has been completed determines the amplitude of the following AL signal. However, in freshly detached ROS from dark-adapte...

Cone visual pigments are responsible for color vision. Using low-temperature spectroscopy and linear/logarithmic time scale flash photolysis, we studied the photochemistry of the cone visual pigment P521 from the lizard Gekko gekko. We found both meta I and meta II intermediates in this cone pigment; meta I absorbs at 480 nm, and meta II absorbs at ca. 380 nm. The formation of meta I is a fast ...

We reported a rapid, light-stimulated release of calcium from isolated rod outer segments that is apparent only when both the disk membrane and the plasma membrane are made permeable to calcium by adding the ionophore A23187 [Kaupp, U. B., Schnetkamp, P. P. M., & Junge, W. (1979) Biochim. Biophys. Acta 552, 390-403]. In this paper, we have investigated the light-sensitive diskal binding sites a...

Phosphorylation reduces the lifetime and activity of activated G protein-coupled receptors, yet paradoxically shifts the metarhodopsin I-II (MI-MII) equilibrium (K(eq)) of light-activated rhodopsin toward MII, the conformation that activates G protein. In this report, we show that phosphorylation increases the apparent pK for MII formation in proportion to phosphorylation stoichiometry. Decreas...

The effect of cholesterol on rod outer segment disk membrane structure and rhodopsin activation was investigated. Disk membranes with varying cholesterol concentrations were prepared using methyl-beta-cyclodextrin as a cholesterol donor or acceptor. Cholesterol exchange followed a simple equilibrium partitioning model with a partition coefficient of 5.2 +/- 0.8 in favor of the disk membrane. Re...

The effect of cholesterol on rod outer segment disk membrane structure and rhodopsin activation was investigated. Disk membranes with varying cholesterol concentrations were prepared using methyl-cyclodextrin as a cholesterol donor or acceptor. Cholesterol exchange followed a simple equilibrium partitioning model with a partition coefficient of 5.2 0.8 in favor of the disk membrane. Reduced cho...

Rhodopsin mutants responsible for autosomal dominant retinitis pigmentosa (ADRP) were prepared by site-directed mutagenesis and characterized. The aim was to evaluate ADRP mutations that occur at three locations on the cytoplasmic surface of rhodopsin: Thr-58 near the cytoplasmic border of helix A, the tetrapeptide Leu-68 to Pro-71 in the first cytoplasmic loop, and Arg-135 at the cytoplasmic b...

The biological function of Glu-181 in the photoactivation process of rhodopsin is explored through spectroscopic studies of site-specific mutants. Preresonance Raman vibrational spectra of the unphotolyzed E181Q mutant are nearly identical to spectra of the native pigment, supporting the view that Glu-181 is uncharged (protonated) in the dark state. The pH dependence of the absorption of the me...

Phosphatidyl inositol-phospholipase C (PI-PLC) in squid retina was studied by immunoblotting and its activities were determined using [3H]phosphatidyl inositol bisphosphate ([3H]PIP2) as substrate. PI-PLC activity was found mostly in soluble fraction when the retina homogenate was treated with 400 mM KCl, but was associated with rhabdomal membranes under low salt conditions (20 mM Hepes). A pro...