Paul A. Liebman

Visual pigments of frog and tadpole (Rana pipiens)

Abstract Photoreceptor cell outer segments of five different morphologies from frog and tadpole retinas have been examined by microspectrophotometry. Photosensitive pigments were found with the following λ max : Frog: red rod 502, green rod 432, principal cone 575, accessory cone 502, single cone 575; Tadpole: red rod 527, green rod 438, principal cone 620, accessory cone 527, and single cone 620. Axial densities of pigments are computed from their measured transverse densities. A 2 derived pigments have wider bandwidths on a wavenumber plot than A 1 based pigments. Red rod wavenumber plots match published nomograms but other pigments do not. All pigments are dichroic and have comparable photosensitivities. Molar concentration of visual pigments is nearly the same in all cells. However, the small size and number of cones in frog retinas probably makes the total relative contribution of cone pigment molecules too small for successful spectroscopic studies in extracts. Animals in the visual pigment phase of metamorphosis have identical pigment mixtures in all red rods.

Lateral Diffusion of Visual Pigment in Photoreceptor Disk Membranes

Visual pigment molecules are found to move transversely, but not longitudinally, in both rod and cone outer segments of mud puppy and frog. This is consistent with the idea that they are immersed in a two-dimensional fluid disk membrane. The diffusion coefficient for the motion is about 5 x 10-9 square centimeters per second at 20�C, corresponding to a root-mean-square molecular displacement of 0.3 micrometer in 1 second.

The control of phosphodiesterase in rod disk membranes: Kinetics, possible mechanisms and significance for vision

The past seven years have witnessed a most exciting transition in thinking and research into the mechanism of visual transduction and adaptation. We have moved from a very long era where our attention was glued to the dominating quantity of visual pigment molecules in the receptors and their spectral changes upon bleaching to an era of minority-molecule wet biochemistry. The beginning of the latter saw introduction of the Ca” hypothesis (Hagins, 1972) and at the same time the important discoveries that light is able to indirectly modulate the behavior of nucleoside phosphate enzymes that control rhodopsin phosphorylation (Kuhn, Cook and Dreyer. 1973: Bownds, Dawes. Miller and Stahlman, 1972) and the phosphorylation of other proteins (Bitensky, Gorman and Miller, 1971: Bitensky, IMiki, Keirns, Keirns, Baraban, Freeman. Wheeler, Lacy and Marcus, 1975). Important also has been the focusing of attention upon the quantitative aspects of transduction and adaptation, i.e. specification of amplification (Yoshikami and Hagins. 1973) and speed (Penn and Hagins, 1972; Baylor, Hodgkin and Lamb, 1974) required of any model seeking to explain these properties of visual receptors. Several years ago, like other laboratories, we began to seek experimental evidence that Ca” might be released in sufficient quantity and with sufficient speed to verify the CaZc hypothesis. We used both steady-state and kinetic approaches. the latter by using the Ca’* sensitive metalochromic indicator dye. Arsenazo III. Though we found rods to contain Ca* i in total amount consistent with the quantitative needs of the Ca’hypothsis (Liebman, 1974), experiments in which we were very careful to exclude visible light before measurement consistently failed to demonstrate significant light-induced release of Ca” (Liebman. 1978). In the hope of reloading Ca” into rod disk vesicles that might be leaky to CaZC, we added a variety of energy providing compounds including ATP and cyclic nucleotides in the presence of Ca”. These provided neither evidence favoring presence of a Ca” pump nor subsequent light-mediated release (Liebman, 1978). However, addition of cyclic GMP (cGMP) in the presence of light and ATP produced a dramatic response in the Arsenazo III measurement that proved to be caused by protons associated with rapid hydrolysis of the cGMP, i.e. Hz0 + cGMP-’ = 5’GMP-’ + H’ (Liebman 1978). Though failing to make progress with intended work on Ca2’ regulation, we thus discovered a method of monitoring hydrolysis of cGMP using H’ recording methods that yield intrinsically great kinetic resolution and provide a continuous view of regulation of this reaction. Our subsequent work proved unequivocally that we were monitoring the activation of rod disk membrane cyclic GMP phosphodiesterase (Liebman and Evanczuk. 1979). On frog material, examination of intitial rates of hydrolysis as a function of concentration of cGMP showed a K, of 50-75 fiXI, pH optimum at 8.0, inhibition by theophylline and IBMX. requirement for MgzC or MnzC and nucleoside triphosphate cofactors, lack of effect of proton ionophores. much reduced rate and K, of 2.5mM in CAMP instead of cGMP, stoichiometric recovery of protons or S’GMP product equal to amount of cGMP added, light control of V,,, and not K, and very high light sensitivity (Liebman and Evanczuk, 1979; Yee and Liebman. 1979). Thus we confirmed nearly all the properties of the light-activated phosphodiesterase reported by other laboratories where slow but highly specific methods had been used to identify cGMP hydrolysis (Miki, Baraban, Keirns. Boyce and Bitensky, 1975). Further work has defined additional parameters such as specificity for GTP as activation cofactor, a light-triggered GTP’ase and high light-dark control ratio (Yee and Liebman, 1979). In the present paper we report similar measurements on bovine disk membrane preparations, and describe the discovery of an ATP-mediated phosphodiesterase (PDE) shut-off mechanism. We propose a detailed biochemical model of visual transduction incorporating facts discovered in our own and in other laboratories. We anticipate a number of objections to the model by showing that its early stages have the light sensitivity, speed, gain and specificity requisite of any mechanism that mediates visual transduction.

Microspectrophotometric measurements of visual pigments in two species of turtle, Pseudemys scripta and Chelonia mydas

Abstract Determination of visual pigment content of turtle outer segments allows arguments concerning colored oil drop function in color vision to be more satisfactorily evaluated. Our microspectrophotometric measurements detect in Pseudemys scripta elegans : P518 2 in rods and P450 2 , P518 2 or P620 2 in cones. In Chelonia mydas mydas we find: P502 1 in rods, P440 1 , P502 1 and P562 1 in cones. These findings rule out the simple theory that turtles see color through the use of a single detector visual pigment coupled with a number of differently colored discriminator filters. In each animal, a pigment that would naively be expected to occur in rods only, was found in 3 types of receptor-rods, oil-drop-bearing cones and oil-drop-free cones. The finding of Vitamin A 1 derived pigments in a sea turtle and Vitamin A 2 derived pigments in a fresh water turtle is reminiscent of the distribution of visual pigments among fresh and sea water fish.

[72] Real time assay of rod disk membrane cGMP phosphodiesterase and its controller enzymes

Publisher Summary This chapter discusses the real time assay of rod disk membrane cyclic guanosine monophosphate (cGMP) phosphodiesterase and its controller enzymes. The assay originates from an accidental discovery that rod disk membrane suspensions generate H + in amounts stoichiometrically equal to the quantity of added cyclic GMP. Standard sucrose flotation methods yield rod disk membranes (RDM) appropriate to this assay. As the enzymes involved are membrane peripheral proteins that are easily lost at low ionic strength and low Mg 2+ , precautions appropriate to their preservation should be observed. In the instance of a membrane-bound enzyme that is under the control of other enzymes, allosteric regulators, and substrate or product regulation, this is particularly important, for there may be rapid changes in enzymatic activity, even when attempts are made to keep these conditions constant. A steady-state measurement under such conditions gives an “integral” turnover rate having very little analytical value.

Identification of a G-protein in depolarizing rod bipolar cells

Synaptic transmission from photoreceptors to depolarizing bipolar cells is mediated by the APB glutamate receptor. This receptor apparently is coupled to a G-protein which activates cGMP-phosphodiesterase to modulate cGMP levels and thus a cGMP-gated cation channel. We attempted to localize this system immunocytochemically using antibodies to various components of the rod phototransduction cascade, including Gt (transducin), phosphodiesterase, the cGMP-gated channel, and arrestin. All of these antibodies reacted strongly with rods, but none reacted with bipolar cells. Antibodies to a different G-protein, G(o), reacted strongly with rod bipolar cells of three mammalian species (which are depolarizing and APB-sensitive). Also stained were subpopulations of cone bipolar cells but not the major depolarizing type in cat (b1). G(o) antibody also stained certain salamander bipolar cells. Thus, across a wide range of species, G(o) is present in retinal bipolar cells, and at least some of these are depolarizing and APB-sensitive.

Multiple steps of phosphorylation of activated rhodopsin can account for the reproducibility of vertebrate rod Single-photon responses

Single-photon responses (SPRs) in vertebrate rods are considerably less variable than expected if isomerized rhodopsin (R*) inactivated in a single, memoryless step, and no other variability-reducing mechanisms were available. We present a new stochastic model, the core of which is the successive ratcheting down of R* activity, and a concomitant increase in the probability of quenching of R* by arrestin (Arr), with each phosphorylation of R* (Gibson, S.K., J.H. Parkes, and P.A. Liebman. 2000. Biochemistry. 39:5738–5749.). We evaluated the model by means of Monte-Carlo simulations of dim-flash responses, and compared the response statistics derived from them with those obtained from empirical dim-flash data (Whitlock, G.G., and T.D. Lamb. 1999. Neuron. 23:337–351.). The model accounts for four quantitative measures of SPR reproducibility. It also reproduces qualitative features of rod responses obtained with altered nucleotide levels, and thus contradicts the conclusion that such responses imply that phosphorylation cannot dominate R* inactivation (Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836–1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733–747.). Moreover, the model is able to reproduce the salient qualitative features of SPRs obtained from mouse rods that had been genetically modified with specific pathways of R* inactivation or Ca2+ feedback disabled. We present a theoretical analysis showing that the variability of the area under the SPR estimates the variability of integrated R* activity, and can provide a valid gauge of the number of R* inactivation steps. We show that there is a heretofore unappreciated tradeoff between variability of SPR amplitude and SPR duration that depends critically on the kinetics of inactivation of R* relative to the net kinetics of the downstream reactions in the cascade. Because of this dependence, neither the variability of SPR amplitude nor duration provides a reliable estimate of the underlying variability of integrated R* activity, and cannot be used to estimate the minimum number of R* inactivation steps. We conclude that multiple phosphorylation-dependent decrements in R* activity (with Arr-quench) can confer the observed reproducibility of rod SPRs; there is no compelling need to invoke a long series of non-phosphorylation dependent state changes in R* (as in Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836–1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733–747.). Our analyses, plus data and modeling of others (Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836–1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733–747.), also argue strongly against either feedback (including Ca2+-feedback) or depletion of any molecular species downstream to R* as the dominant cause of SPR reproducibility.

Visual receptor pigments in the african cichlid fish,Haplochromis burtoni

Abstract The retinal receptors of the African cichlid fish, Haplochromis burtoni , are arranged in a mosaic of square arrays which have four double cones around a central cone. Microspectrophotometric measurement of spectral absorbance reveals that the short single cones contain the blue photopigment, P455, while the larger double cones contain “red-green” pigment pairs of P562 and P523. Rods are interspersed throughout the cone array and contain rhodopsin, P500. Spectral shape, pigment absorbance and the λ max of photo-products are characteristic of A 1 based pigments. The retinal receptors of H. burtoni appear to be well matched to important visual recognition tasks.

Procion yellow: A marker dye for outer segment disc patency and for rod renewal

Abstract When Procion Yellow is injected into the vitreous cavity of eyes of living animals, it does two things: (1) it stains the length of the outer segment of cones and, (2) it stains only the new-forming basal lamellae of rod outer segments. Thus it is possible to discriminate rods from cones by color difference and to monitor the renewal of rod outer segment by observing the stained leaflets move toward the sclera as new, unstained lamellae form.

Variation in the λmax of rhodopsin from individual frogs

Abstract The λmax of rhodopsin was determined for individual frogs using intact, isolated retinae and single rod outer segments. It was found that the λmax varied from animal to animal over an 8-nm range with a mean spectral position of 504.5 nm. This variation is not the result of other visual pigments or photoproducts and probably represents differences in the degree of opsin-chromophore interaction.