Activation of phosphodiesterase in frog rod outer segment by an intermediate of rhodopsin photolysis. II

Frog (Rana catesbeiana) rod outer segment membrane contains cyclic GMP phosphodiesterase (EC 3.1.4.1). Irradiation of dark-adapted rod outer segment membrane increased the enzyme activity by 5–20-fold in the presence of GTP. The phosphodiesterase in rod outer segment membrane is also activated by mixing a photo-product of 11-cis (regenerated), 9-cis or 7-cis rhodopsin which is stable at 0°C. However, neither opsin in the membrane nor all-trans retinal activates the enzyme. The phosphodiesterase in rod outer segment membrane is also activated by irradiation at ?4°C. Thus, we conclude that the phosphodiesterase is activated by a common photolysis intermediate of these rhodopsin isomers, perhaps before metarhodopsin II decays.     

Disaturated and dipolyunsaturated phospholipids in the bovine retinal rod outer segment disk membrane

Thin-layer chromatography was used to separate the major phospholipid headgroup classes of the rod outer segment disk membrane into subfractions which differ markedly in fatty acid composition. At least 18% of the rod outer segment phosphatidylcholine must contain two saturated fatty acids. Furthermore, two unsaturated fatty acids are found in at least 43% of the phosphatidylserine, 24% of the phosphatidylcholine, and 24% of the phosphatidylethanolamine. The unsaturated acids are predominantly polyunsaturated in all cases. A similar separation, but with less resolution, was achieved with silicic acid column chromatography.The temperature dependence of the polarization of the fluorescence of trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid) showed that the thermal behavior of aqueous dispersions of the phosphatidylcholine subfractions was consistent with their fatty acid compositions.     

Permeability to ions of bovine retinal disk membrane vesicles in the bleached state

The permeability of the bleached disk membrane of retinal rod outer segments to univalent and divalent ions is studied by light scattering. The membranes are isolated from frozen dark-adapted bovine retinae, swollen into spherical vesicles in a hypotonic medium and bleached in dilute suspension and their size is determined by elastic and quasi-elastic light scatterings. Various electrolytes are then added to the suspending medium in order to examine their osmotic activity relative to the vesicles deformation characteristics. By following the deformation behavior of the membrane vesicles by elastic light scattering in terms of the oblate ellipsoidal shell model, the osmotic activity of a given electrolyte is qualitatively deduced and thereby the permeability of the membrane to the electrolyte is ranked in reference to a chosen standard, i.e., sucrose. By this method, we show that the permeabilities to Na⁺, K⁺, Mg²⁺ and Ca²⁺ are all alike, and those to halides (F^?, Cl^?, Br^?, I^?), nitrate and phosphates (HPO₄^2?/H₂PO₄^?) are similar. Acetate, however, is about 3-times more permeative, while sulfate is less permeative than the other anions by about the same factor. The viability of our method is checked with use of an ionophore, lasolocid (X-537A), by establishing partial recovery from the osmotic deformation through the suppression of the cation osmotic effect. Ion-induced aggregation and pH-dependent size and shape changes are both found to be insignificant.     

Two forms of intermediates of frog rhodopsin in rod outer segments

Using frog rod outer segments, we measured changes of the absorption spectrum during the conversion of rhodopsin to a photosteady-state mixture composed of rhodopsin, isorhodopsin and bathorhodopsin by irradiation with blue light (440 nm) at ? 190°C and during the reversion of bathorhodopsin to a mixture of rhodopsin and isorhodopsin by irradiation with red light (718 nm) at ? 190°C. The reaction kinetics was expressed by one exponential in the former case and by two exponentials in the latter. These results suggest that rhodopsin is composed of a single molecular species, while bathorhodopsin is composed of two kinds of molecular species designated as batho₁-rhodopsin and batho₂-rhodopsin. On warming the two forms of bathorhodopsin, each bathorhodopsin converted to its own lumirhodopsin, metarhodopsin I and finally a free all-trans-retinal plus opsin. The absorption spectra of the two forms of bathorhodopsin, lumirhodopsin and metarhodopsin I were measured at ? 190°C. We infer that a rhodopsin molecule in the excited state relaxes to either batho₁-rhodopsin or batho₂-rhodopsin, and then converts to its own intermediates through one of the two parallel pathways.     

On the evaluation of photoreceptor properties by micro-fluorimetric measurements of fluorochrome diffusion

By use of the microfluorimetric technique it is possible to study the diffusion of the fluorochrome di-dansylcystine (DDC) within isolated frog rod outer segments (ros) which are immobilysed in agarose gel. For this purpose, by a short hypotonic shock a leak is applied to one end of the ros. By this open end the DDC enters the rod and migrates through the whole outer segment. Following the propagation of the fluorescence boundary with time the cytoplasmatic diffusion constant can be determined if a Chromatographic model is used to allow for the considerable binding of DDC to the inner membrane surface. With a binding constant K=5·10^–4 cm the cytoplasmatic diffusion constant was found to be D= 1.3·10^–6cm²/s whereas D _g =2·10^–6cm²/s and D _r =3.5·10^–6cm²/s were found in agarose gel or ringer solution, respectively. Using the mobility reduction factor given by D/D _r 0.4 to calculate the cytoplasmatic conductivity an inner resistance per length of 1.7 M / could be calculated for a frog rod which is in good agreement with corresponding data obtained from electrophysiological measurements.     

X-ray diffraction analysis of wet isolated bovine rod outer segment disks. A dehydration study

Sequences of X-ray diffraction patterns were obtained from dehydrating, artificially oriented multilayers of isolated, bovine rod outer segment disks. A direct-phase analysis was applied to highly hydrated specimens to determine sequences of low resolution (approx. 30 Å) electron density profiles of the disks as dehydration proceeded. The profiles were found to evolve smoothly as the multilayer lattice simultaneously shrank and became increasingly ordered. The bilayer profiles were largely invariant under dehydration and the evolution of the diffraction consistent with simple decreases in fluid spacings. The specimens were observed to phase separate into characteristic primary and a secondary lattices when the multi-layer became too dehydrated. The small unit cell size of the secondary lattice was suggestive of a lipid phase. Large changes in the diffraction patterns from phase separated specimens were observed upon bleaching of the specimen. The changes were consistent with a reversible disordering of the primary lattice.     

Activation of phosphodiesterase by rhodopsin and its analogues

Activation of guanosine 3,5-cyclic monophosphate (cGMP) phosphodiesterase (EC 3.1.4.35.) in frog rod outer segment membrane by rhodopsin and its analogues was investigated. The Schiff-base linkage between opsin and retinal in rhodopsin was not always necessary for the phosphodiesterase activation. The binding of -ionone ring of retinal to a hydrophobic region of opsin was not enough to induce the enzyme activation. A striking photo-activation of the enzyme was induced by photo-isomerization of rhodopsin analogues from cis to trans form. It seems probable that an expanded conformation of opsin around the retinylidene chromophore induced by the cis to trans isomerization may be the trigger for the activation of phosphodiesterase. On the other hand, the phosphodiesterase in frog rod outer segment was activated by warming of bathorhodopsin to –12 C and then incubating it at the same temperature. Thus, metarhodopsin II or an earlier intermediate than metarhodopsin II should be a direct intermediate for the enzyme activation.Based on material presented at the Fifth International Congress of Eye Research, Eindhoven, October 1982     

Lipid composition of photoreceptor membranes from goldfish retinas

Isolation and biochemical characterization of goldfish retinal photoreceptor outer segment membranes are described. The lipid fraction is composed primarily of phospholipids (68 mol%) with substantial amounts of neutral lipids (32 mol%). Sterols account for only about 2 wt% of the membranes (about 9 mol% of the total lipids). The phospholipid class composition and fatty acid composition are similar to those of other vertebrate photoreceptor membranes. Two novel findings were the high levels of free fatty acids (21 mol% of the total lipids, primarily palmitic and docosahexaenoic acids) and the presence of relatively significant amounts of a C-32 diacylglycerol molecular species.     

Identification and Immunolocalization of Phospholipase C in Bovine Rod Outer Segments

Bovine rod outer segments (ROS) contain a phospholipase C (PLC) that hydrolyzes phosphatidylinositol 4,5-bisphosphate. Approximately 60-70% of PLC activity is recovered in soluble extracts of ROS. Moreover, the specific activity of this soluble PLC is approximately 10-fold higher than that of resealed ROS enzyme activity. Peptide-specific antiserum (Ab 1109) directed against a highly conserved sequence of the Y-region found in several PLC isozymes was used to detect any PLC belonging to this family. This antibody specifically recognized a protein of apparent molecular mass of approximately 140 kDa present in immunoblots of soluble extracts of both ROS and whole retina. The elution profile of this 140-kDa antigen from a Sephadex G-150 column coincided with the peak of PLC activity, suggesting PLC activity is associated with the 140-kDa protein. Immunocytochemical studies of bovine retina using Ab 1109 showed pronounced immunoreactive labeling in the photoreceptor layer. In resealed ROS and washed ROS membranes, Ab 1109 recognized an additional protein of apparent molecular mass of 70 kDa not usually detectable in soluble extracts of ROS, suggesting the presence of at least two isozymes of PLC in ROS.     

Distinct states of lipid mobility in bovine rod outer segment membranes Resolution of spin label results

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0°C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24°C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0°C and a considerable temperature dependence. This component which is not resolved at high temperatures (24–35°C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62–64 G at 0°C, with very little temperature dependence (61–62 G at 35°C), and is attributed to protein aggregation.     

An extended Ca2+-hypothesis of visual transduction with a role for cyclic GMP

A model is described having the following features: Light induces Ca2+ release from vertebrate rod outer segments discs via pores composed of multimeric rhodopsin. Cytoplasmic Ca2+ reversibly blocks Na+ channels of the surface membrane, with the time course of development and amplitude of the response to light being influenced by restrictions on intradiscal Ca2+ diffusion. The falling phase of response reflects a decline in cytoplasmic [Ca2+] due to a Ca2+-binding protein controlled by cyclic GMP so that its binding capacity is increased by the reduction in cytoplasmic [cyclic GMP] which follows rhodopsin bleaching.     

Light-induced axial and radial shrinkage effects and changes of the refractive index in isolated bovine rod outer segments and disc vesicles

Flash-induced transients in the near-infrared scattering of bovine rod outer segments and isolated discs are investigated. Their common characteristic is the saturation at a rhodopsin bleaching of ca. 10%, which was previously described for the so-called signalP. The theory is based on the Rayleigh-Gans-approximation and on a cylindrical particle shape. This treatment is shown to be applicable in the measured angular range (in general30), in spite of the polydisperse shape of the real particles. Using the angular dependence of the relative intensity change (difference scattering curve), changes of the polarizability (refractive index) and of the particle shape can be distinguished. Model difference scattering curves are calculated for the dimensions of the rod outer segments. Static scattering measurements are used for an estimation of the average particle shape: the isolated disc samples appear to contain flat discs as well as an admixture of rod-like structures (ca. 1% of the total scattering mass); in rod outer segment preparations, a contribution of non-rodlike scattering is found which is strongly dependent on the treatment of the sample. The flash induced transients were measured using randomly oriented particles (discs and rod outer segments) and axially oriented rod outer segments. The angular dependence of the amplitude yields its difference scattering curve. On suspensions of isolated discs, which were re-loaded with the proteins extracted at low ionic strength, one single signal is observed (termedP _D, first order,=0.6–1.2 s). Using randomly oriented rod outer segments, a signal with complex millisecond kinetics (termed signalP) and a slow signal (termedP _S, first order,=5–25 s) can be distinguished kinetically. In the axially oriented rod outer segments, theP-signal splits into a fast axial (10 ms) and a slower radial component (50–100 ms). The slow signalP _S observed in ROS and the signalP _D in discs have one common physical interpretation as local changes of the polarizability, directly observed in light-scattering as a change of the refractive index. The fast signalP in ROS, however, has no detectable local component but represents a pure shrinkage effect. On the axially oriented system, this shrinkage turns out to be axial and radial with different kinetics. Only rough estimations for the relative shrinkage effects and refractive index changes can be given. One obtains for 1% rhodopsin bleaching:n/n10^–4,L/L10^–2,R/R5×10^–4. Assuming a fluid plane for the disc membrane, the planar shrinkage induced by one bleached rhodopsin is estimated from the radial shrinkage as ca. 300 å². This high value is discussed in relation to the binding of rhodopsin to the GTP-binding protein which is involved in comparable effects described by Kühn et al. (1981). According to our data, a chemical binding process in milliseconds is only indicated in the isolated disc; in the closed disc stack of the rod outer segment, only weak (fast) local interactions are consistent with the difference scattering data. A turn or lift of the GTPase would better satisfy this condition and explain the above high value for the individual shrinkage effect.Abbreviations ROS rod outer segments - RGA Rayleigh-Gans-approximation     

Catabolism of myo-Inositol to Precursors Utilized for De Novo Glycerolipid Biosynthesis

Systemic injection of [2-3H]myo-inositol into frogs resulted in the incorporation of more than half of the label into glycerolipid classes other than phosphoinositides in retinal rod outer segment membranes. Following methanolysis and differential extraction of isolated lipid classes, radioactivity was recovered primarily in the aqueous phase. After phospholipase C hydrolysis of the total membrane lipids, 97% of the radioactivity was extractable with organic solvents, and 70% of the label in lipids was in 1,2-diglycerides. These results indicate that the label was incorporated primarily into the glyceryl moiety of the membrane glycerolipids. Intraocular injection of frog eyes or in vitro incubation of frog retinas with [2-3H]myo-inositol resulted in the incorporation of radioactivity almost exclusively into phosphoinositides in rod outer segment membranes. Incubation of retinas with [U-14C]glucuronic acid did not result in the formation of labeled retinal lipids. These results suggest that myo-inositol can be catabolized systemically to precursors utilized for glycerolipid biosynthesis in the retina.     

Can metarhodopsin I activate rod outer segment phosphodiesterase?

The rod photoreceptors of vertebrate retinas contain a cGMP phosphodiesterase (PDE) that is activated by light. The light is absorbed by rhodopsin that activates an intermediate GTP-binding protein; this species then activates the PDE. Photo-excited rhodopsin passes through a series of transient states, and the purpose of this study is to identify the earliest state that interacts with the GTP-binding protein and thus activate the PDE. The majority of evidence points to this state being metarhodopsin II (MII), but PDE activation is seen at low temperatures where the rhodopsin reaction sequence is not expected to pass beyond the metarhodopsin I (MI) stage. Light thresholds for PDE activation have been determined under conditions where little MII is generated, and these are compared with the concentration of MII. The conclusion is that for a criterion threshold of PDE activity, the MII concentration is constant, irrespective of the amount of MI present, which suggests that MI cannot activate the PDE system.