Size changes of phosphodiesterase in bovine rod outer segments on illumination

Light activates a 3',5'-cyclic GMP phosphodiesterase (PDE) in bovine retinal rod outer segments. The light is absorbed by rhodopsin situated in the disk membranes. PDE is a three-subunit peripheral protein on the disks and appears to be activated via a guanine nucleotide binding protein (G) in the presence of activated rhodopsin and GTP. Does the activation occur by collision coupling of G and PDE? We have studied the protein-protein interactions of PDE in situ in disk membranes by radiation inactivation. Irradiation of a protein with high-energy electrons leads to loss of activity in proportion to radiation dose and the molecular weight of the protein. We see no change in the size of PDE upon activation by light and 100 microM guanosine 5'-(beta, gamma-imidotriphosphate) (Gpp[NH]p) compared with PDE in dark with 260 microM GTP. Application of statistics to our data shows that a 27 000 change in molecular weight would be significant at the 95% level but that smaller changes would go undetected. The apparent molecular weight is 176 000 +/- 27 000 (mean +/- 95% confidence limit), in agreement with the size determined by polyacrylamide gel electrophoresis. Thus there appears to be either (i) no permanent change in PDE size on activation or (ii) a small change, undetectable by the technique, or (iii) an exchange of subunits such that no net change in molecular weight is seen.     

Light- and nucleotide-dependent binding of phosphodiesterase to rod disk membranes: correlation with light-scattering changes and vesicle aggregation

Under conditions in which large guanosine cyclic 3',5'-phosphate (cGMP)- and phosphodiesterase (PDE)-dependent changes in near-infrared transmission and vesicle aggregation and disaggregation occur, we have observed a striking change in the binding of PDE to rod disk membranes. The change in PDE binding is nucleotide and light dependent as are the light-scattering changes. The cGMP- and PDE-dependent light-scattering signal can be produced by a 500-nm light flash which bleaches 1/(1 X 10(7] rhodopsin molecules. Mg ions are an essential cofactor for the nucleotide-dependent PDE binding and light-scattering changes. 3-Isobutyl-1-methylxanthine and other competitive inhibitors of PDE hydrolytic activity support increased PDE binding to the disk membrane, vesicle aggregation, and the light-scattering signal. However, treatments which block GTP-dependent activation of PDE hydrolytic activity (colchicine, GDP, or ethylenediaminetetraacetic acid) also block these phenomena. Thus, GTP-dependent activation of PDE rather than its hydrolytic activity appears to be correlated with the light-scattering signal.     

Enhancement of phototransduction g protein-effector interactions by phosphoinositides

Light responses in photoreceptor cells are mediated by the action of the G protein transducin (G(t)) on the effector enzyme cGMP phosphodiesterase (PDE6) at the surface of disk membranes. The enzymatic components needed for phosphoinositide-based signaling are known to be present in rod cells, but it has remained uncertain what role phosphoinositides play in vertebrate phototransduction. Reconstitution of PDE6 and activated G(alphat), on the surface of large unilamellar vesicles containing d-myo-phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), stimulated PDE activity nearly 4-fold above the level observed with membranes containing no phosphoinositides, whereas G protein-independent activation by trypsin was unaffected by the presence of phosphoinositides. PDE activity was similarly stimulated by d-myo-phosphatidylinositol-3,4-bisphosphate and d-myo-phosphatidylinositol-4-phosphate (PI(4)P), but much less by d-myo-phosphatidylinositol-5-phosphate (PI(5)P) or d-myo-phosphatidylinositol-3,5-bisphosphate. Incubation of rod outer segment membranes with phosphoinositide-specific phospholipase C decreased G protein-stimulated activation of endogenous PDE6, but not trypsin-stimulated PDE activity. Binding experiments using phosphoinositide-containing vesicles revealed patterns of PDE6 binding and PDE6-enhanced G(alphat)-GTPgammaS binding, consistent with the activation profile PI(4,5)P(2) > PI(4)P > PI(5)P approximately control vesicles. These results suggest that enhancement of effector-G protein interactions represents a possible mechanism for modulation of phototransduction gain by changes in phosphoinositide levels, perhaps occurring in response to longterm changes in illumination or other environmental cues.     

Reconstitution of rhodopsin and the cGMP cascade in polymerized bilayer membranes

The successful reconstitution of rhodopsin, the rod outer segment (ROS) G protein, and the ROS phosphodiesterase (PDE) into partially polymerized bilayer membranes is described. Purified bovine rhodopsin (Rh) was inserted into performed partially polymerized lipid vesicles. Sonicated vesicles composed of approximately equal moles of dioleoylphosphatidylcholine (DOPC) (or 1-palmitoyl-2-oleoyl-phosphatidylcholine) and 1,2-bis(octadeca-2,4-dienoyl)phosphatidylcholine (DENPC) were photolyzed with 254-nm light to polymerize the DENPC and form domains of DOPC and polyDENPC in the vesicle wall. Rh-octyl glucoside (OG) micelles were slowly added to the vesicle suspension to give 15 mM OG (below the OG critical micelle concentration). The suspension was incubated and then dialyzed and purified on a sucrose gradient. Ultracentrifugation revealed a major Rh-lipid band which was harvested and found to contain a 100 +/- 10 phosphatidylcholine to rhodopsin ratio (Rh-polyDENPC/DOPC). The orientation of Rh in the membrane was determined by limited proteolytic digestion of Rh and by competitive inhibition of monoclonal antibody binding to solubilized disk membranes. Results were compared with control membranes of Rh-DOPC (1:43) prepared by insertion and Rh-phospholipid membranes prepared by detergent dialysis. Visual inspection of thermolysin proteolytic patterns of Rh indicates one major population cleaved at the carboxy terminus, as is found in disk membranes with an asymmetric arrangement of Rh. In contrast, proteolysis of a Rh-egg PC/PE (1:50/50) membrane (detergent dialysis) produced two Rh populations, which indicates a symmetric arrangement of Rh. The Rh-polyDENPC/DOPC (1:100) membranes were allowed to compete with solubilized, immobilized disk membranes for the monoclonal antibody R2-15 (specific for the amino-terminal region of Rh). They were intermediate between the asymmetric ROS disk membranes and the symmetric dialysis membranes in their ability to bind the R2-15 monoclonal antibody. The data indicate approximately 80% of the Rh's in Rh-polyDENPC/DOPC are in the normal orientation found in disks. These Rh-containing polymerized bilayer membranes demonstrated functionality as determined by chemical regeneration, kinetic spectrophotometry, and cGMP cascade reconstitution experiments. In the latter experiments the peripheral proteins, ROS G protein and PDE, bound with comparable efficiency to both the polymerized PC bilayers and egg PC bilayers. Thus the biocompatibility of the phosphatidylcholine membrane surface was maintained after polymerization of DENPC.     

In vitro isoprenylation and membrane association of mouse rod photoreceptor cGMP phosphodiesterase alpha and beta subunits expressed in bacteria.

We investigated the specificity of CAAX box-related isoprenylation of rod photoreceptor cGMP phosphodiesterase (PDE) subunits expressed in bacteria and the consequences of this modification on rod disk membrane association. Full-length cDNA sequences of the alpha and beta subunits of mouse PDE, inserted into bacterial pET expression vectors, were overexpressed as fusion proteins containing 28 (bMP-alpha) and 26 (bMP-beta) additional amino acid residues at their N termini. Both fusion proteins were overexpressed and stored in inclusion bodies. Purified bMP-alpha and bMP-beta were recognized by bovine PDE-specific polyclonal antibodies, but did not associate with depleted rod disk membranes and were catalytically inactive. Using bovine brain or retina extracts as sources of protein prenyltransferases and tritiated farnesyl- or geranylgeranylpyrophosphate as donors, bMP-alpha (CAAX sequence CCIQ) was exclusively farnesylated, and bMP-beta (CAAX sequence CCIL) was exclusively geranylgeranylated. After isoprenylation, bMP-alpha and bMP-beta each associated with rod photoreceptor outer segment disk membranes under isotonic, but not under hypotonic, conditions. The results indicate that isoprenylated bMP-alpha and bMP-beta each interact independently with membranes and that isoprenylation is the key modification that facilitates membrane association.     

Enhancement of phototransduction protein interactions by lipid surfaces

The G protein cascade of vision depends on two peripheral membrane proteins: the G protein, transducin (G(t)), and cGMP phosphodiesterase (PDE). Each has covalently attached lipids, and interacts with transduction components on the membrane surface. We have found that their surface interactions are critically dependent on the nature of the lipid. Membranes enhance their protein-protein interactions, especially if electrostatic attraction is introduced with positively charged lipids. These interactions are less enhanced on highly curved surfaces, but are most enhanced by unsaturated or bulky acyl chains. On positively charged membranes, G(t) assembles at a high enough density to form two-dimensional arrays with short-range crystalline order. Cationic membranes also support extremely efficient activation of PDE by the GTPgammaS (guanosine 5'-O-(thiotriphosphate)) form of Galpha(t) (Galpha(t)-GTPgammaS), minimizing functional heterogeneity of transducin and allowing activation with nanomolar Galpha(t)-GTPgammaS. Quantification of PDE activation and of the amount of Galpha(t)-GTPgammaS bound to PDE indicated that G(t) activates PDE maximally when bound in a 1:1 molar ratio. No cooperativity was observed, even at nanomolar concentrations. Thus, under these conditions, the one binding site for Galpha(t)-GTPgammaS on PDE that stimulates catalysis must be of higher affinity than one or more additional sites which are silent with respect to activation of PDE.     

Purification and characteristics of photoreceptor light-activated guanosinetriphosphatase

We describe a reconstitution of light-activated vertebrate photoreceptor GTPase and a purification of the GTP-binding protein (G protein), which is a component of the GTPase and modulates the light-activated phosphodiesterase (PDE) enzyme system. Rod outer segments (ROS) of bull frogs were treated with ethylenediaminetetraacetic acid (EDTA), and the GTPase and PDE fractions were solubilized (EDTA supernatant). When the EDTA supernatant and EDTA-treated membrane fraction (EDTA-washed membranes) were recombined, light-dependent GTPase activity appeared. In the reconstituted system, the Km for GTP as substrate was 0.5 microM; the optimum pH was 7.5-8.0. The isoelectric point of GTPase in EDTA supernatant was 4.8. G protein was purified 400-fold from ROS, and the molecular weight of G protein was determined to be 40 000 by polyacrylamide gel electrophoresis. The amount of G protein in ROS was calculated as at least 1 molecule per 400 rhodopsin molecules. By recombining (in the presence or absence of GTP) purified G protein, PDE, H fraction (an additional component of GTPase), and illuminated or unilluminated EDTA-washed membranes (as a source of rhodopsin), we showed that illuminated rhodopsin, G protein, PDE, and GTP are the minimum requirements for light-dependent PDE activity. We discuss the significance of these findings in the regulation of the light-activated GTPase and PDE activities, especially with regard to the mechanism of activation.     

The GDI-like solubilizing factor PDEδ sustains the spatial organization and signalling of Ras family proteins

We identify a role for the GDI-like solubilizing factor (GSF) PDEδ in modulating signalling through Ras family G proteins by sustaining their dynamic distribution in cellular membranes. We show that the GDI-like pocket of PDEδ binds and solubilizes farnesylated Ras proteins, thereby enhancing their diffusion in the cytoplasm. This mechanism allows more effective trapping of depalmitoylated Ras proteins at the Golgi and polycationic Ras proteins at the plasma membrane to counter the entropic tendency to distribute these proteins over all intracellular membranes. Thus, PDEδ activity augments K/Hras signalling by enriching Ras at the plasma membrane; conversely, PDEδ down-modulation randomizes Ras distributions to all membranes in the cell and suppresses regulated signalling through wild-type Ras and also constitutive oncogenic Ras signalling in cancer cells. Our findings link the activity of PDEδ in determining Ras protein topography to Ras-dependent signalling.     

Rhodopsin kinase prepared from bovine rod disk membranes quenches light activation of cGMP phosphodiesterase in a reconstituted system

Rhodopsin kinase was extracted into a buffer containing 200 mM KCl and no MgCl2. The activity of the enzyme was stabilized with the use of a mixture of protease inhibitors, aprotinin, benzamidine, leupeptin, and pepstatin. The extract consisted of three major proteins of molecular weight (Mr) 65,000, 56,000, and 37,000, of which the Mr 65,000 protein was identified with the kinase activity since preparations containing the other proteins had no kinase activity and the Mr 65,000 protein was phosphorylated when the extract was incubated with ATP. A reconstituted cGMP phosphodiesterase (PDE) system consisting of peripheral protein-depleted rod disk membranes (RDM), GTP binding protein (G-protein), and PDE was used to test the effectiveness of the rhodopsin kinase preparation in mediating the ATP-dependent quench of light activation of PDE. In the absence of kinase, light-activated PDE activity lasted several minutes. In its presence, ATP and to a lesser extent GTP quenched the activation about as rapidly as in rod disk membranes. The influence of kinase was unaffected by increasing G-protein or PDE content of the reconstituted system but was slowed down by brighter flashes, showing that quench was caused by the inactivation of bleached rhodopsin and not of PDE or G-protein.     

Membrane stimulation of cGMP phosphodiesterase activation by transducin: comparison of phospholipid bilayers to rod outer segment membranes.

To clarify the role of phospholipids in G protein-effector interactions of vertebrate phototransduction, transducin activation of cGMP phosphodiesterase (PDE) has been reconstituted on the surface of well-defined phosphatidylcholine (PC) vesicles, using purified proteins from bovine rod outer segments (ROS). PC vesicles enhanced PDE stimulation by the GTP-gamma S-bound transducin alpha subunit (T alpha-GTP gamma S) as much as 17-fold over activation in the absence of membranes. In the presence of 3.5 microM accessible PC in the form of large (100 nm) unilamellar vesicles, 500 nM T alpha-GTP gamma S stimulated PDE activity to more than 70% of the maximum activity induced by trypsin. Activation required PC, PDE, and T alpha-GTP gamma S, but did not require prior incubation of any of the components, and occurred within 4 s of mixing. The PC vesicles were somewhat more efficient than urea-washed ROS membranes in enhancing PDE activation. Half-maximal activation occurred at accessible phospholipid concentrations of 3.8 microM for PC vesicles, and 13 microM for ROS membranes. Titrations of PDE with T alpha-GTP gamma S in the presence of membranes indicated a high-affinity (Kact less than 250 pM) activation of PDE by a small fraction (0.5-5%) of active T alpha-GTP gamma S, as did titrations of ROS with GTP gamma S. When activation by PC vesicles was compared to PDE binding to membranes, the results were consistent with activation enhancement resulting from formation of a T alpha-GTP gamma S-dependent PDE-membrane complex with half-maximal binding at phospholipid concentrations in the micromolar range. The value of the apparent dissociation constant, KPL, associated with the activation enhancement was estimated to be in the range of 2.5 nM (assuming an upper limit value of 1600 phospholipids/site) to 80 nM (for a lower limit value of 50 phospholipids/site). Another component of membrane binding was more than 100-fold weaker and was not correlated with activation by T alpha-GTP gamma S. Low ionic strength disrupted the ability of ROS membranes, but not PC vesicles, to bind and activate PDE. Removal of PDE's membrane-binding domain by limited trypsin digestion eliminated both the binding of PDE to vesicles and the ability of PDE to be activated by T alpha-GTP gamma S and membranes. These results suggest that ROS membrane stimulation of PDE activation by T alpha-GTP gamma S is due almost exclusively to the phospholipids in the disk membrane.     

The role of cholesterol in rod outer segment membranes

The photoreceptor rod outer segment (ROS) provides a unique system in which to investigate the role of cholesterol, an essential membrane constituent of most animal cells. The ROS is responsible for the initial events of vision at low light levels. It consists of a stack of disk membranes surrounded by the plasma membrane. Light capture occurs in the outer segment disk membranes that contain the photopigment, rhodopsin. These membranes originate from evaginations of the plasma membrane at the base of the outer segment. The new disks separate from the plasma membrane and progressively move up the length of the ROS over the course of several days. Thus the role of cholesterol can be evaluated in two distinct membranes. Furthermore, because the disk membranes vary in age it can also be investigated in a membrane as a function of the membrane age. The plasma membrane is enriched in cholesterol and in saturated fatty acids species relative to the disk membrane. The newly formed disk membranes have 6-fold more cholesterol than disks at the apical tip of the ROS. The partitioning of cholesterol out of disk membranes as they age and are apically displaced is consistent with the high PE content of disk membranes relative to the plasma membrane. The cholesterol composition of membranes has profound consequences on the major protein, rhodopsin. Biophysical studies in both model membranes and in native membranes have demonstrated that cholesterol can modulate the activity of rhodopsin by altering the membrane hydrocarbon environment. These studies suggest that mature disk membranes initiate the visual signal cascade more effectively than the newly synthesized, high cholesterol basal disks. Although rhodopsin is also the major protein of the plasma membrane, the high membrane cholesterol content inhibits rhodopsin participation in the visual transduction cascade. In addition to its effect on the hydrocarbon region, cholesterol may interact directly with rhodopsin. While high cholesterol inhibits rhodopsin activation, it also stabilizes the protein to denaturation. Therefore the disk membrane must perform a balancing act providing sufficient cholesterol to confer stability but without making the membrane too restrictive to receptor activation. Within a given disk membrane, it is likely that cholesterol exhibits an asymmetric distribution between the inner and outer bilayer leaflets. Furthermore, there is some evidence of cholesterol microdomains in the disk membranes. The availability of the disk protein, rom-1 may be sensitive to membrane cholesterol. The effects exerted by cholesterol on rhodopsin function have far-reaching implications for the study of G-protein coupled receptors as a whole. These studies show that the function of a membrane receptor can be modulated by modification of the lipid bilayer, particularly cholesterol. This provides a powerful means of fine-tuning the activity of a membrane protein without resorting to turnover of the protein or protein modification.     

Cholesterol heterogeneity in bovine rod outer segment disk membranes

Rod outer segment disk membranes have been used to study visual transduction events. Numerous studies have also focused on protein-lipid interactions in these membranes. The possible heterogeneity of the disk membrane composition has not been addressed in such studies. Freeze fracture studies (Andrews, L. D., and Cohn, A. I. (1979) J. Cell Biol. 81, 215-220; Caldwell, R., and McLaughlin, B. (1985) J. Comp. Neurol. 236, 523-537) suggest a difference in cholesterol content between newly formed and old disks. This potential heterogeneity in disk membrane composition was investigated using digitonin. Osmotically intact bovine rod outer segment disk membranes prepared by Ficoll flotation were separated based on the cholesterol content of the disks. The addition of digitonin to disk membrane suspensions in a one-to-one molar ratio with respect to cholesterol produced an increase in the density of the membranes in proportion to the amount of cholesterol present. The digitonin-treated disks were separated into subpopulations using a sucrose density gradient. Disks were shown to vary in cholesterol to phospholipid ratio from 0.30 to 0.05. The ratio of phospholipid to protein remained constant in all disk subpopulations at approximately 65 phospholipids per protein. No significant change in the fatty acid composition of the disks was observed as a function of change in cholesterol content. This work demonstrates compositional heterogeneity in disk membranes which may ultimately affect function.     

cGMP phosphodiesterase dependent light-induced scattering changes in suspensions of retinal disc membranes.

Light-induced GTP-dependent scattering changes are studied in suspensions of retinal disc membranes to which one or both of the purified proteins involved in the phototransduction mechanism (G-protein and cGMP phosphodiesterase) are reassociated; a scattering change which depends on the presence of both G-protein (G) and inhibited cGMP phosphodiesterase (PDE) and on an ATPase-dependent process, previously described in Bennett [(1986) Eur. J. Biochem. 157, 487-495] is compared to the signal observed in the absence of PDE or of ATP and to PDE activity. The same signal can also be induced either in the dark or in the light by addition of preactivated G in the presence of inhibited PDE. This PDE-dependent scattering change is composed of two components (fast and slow); the variation of the amplitude and kinetics of both components with PDE or G concentration is similar to the variation of the active PDE state with two activator GGTP molecules (G with GTP bound), calculated with dissociation constants previously reported for the interaction between GGTP and PDE [Bennett, N., & Clerc, A. (1989) Biochemistry 28, 7418-7424]. The two components are therefore proposed to be associated with processes which depend on the formation of the active PDE state with two activators.     

Early changes in retinal 5'-nucleotidase activity in hereditary retinal degeneration

Activity of soluble cGMP phosphodiesterase (PDE) and of two membrane enzymes, 5'-nucleotidase and Na,K-ATPase, was studied in the developing retina of rats with inherited retinal degeneration. It was found that by day 10 of life, the content of 5'-nucleotidase in the afflicted rats was significantly reduced as compared with controls. This difference was unchanged throughout the subsequent animals' life. Na,K-ATPase activity in the afflicted and normal animals was the same. Within the first 45 days of life, PDE calculated with respect to the rhodopsin content was not different as regards both the afflicted and normal rats. When calculated with respect to protein, the changes in PDE corresponded with the reported data. The data obtained allowed a suggestion to be made that changes in 5'-nucleotidase in inherited retinal degeneration are disease-specific. They are accounted for by changes in the enzymes of nonphotoreceptor retinal membranes. The changes in PDE may be regarded as secondary, correlating with variation in the number of the photoreceptor membranes.     

Differential scanning calorimetry of bovine rhodopsin in rod-outer-segment disk membranes.

Rhodopsin-containing retinal rod disk membranes from cattle have been examined by differential scanning calorimetry. Under conditions of 67 mM phosphate pH 7.0, unbleached rod outer segment disk membranes gave a single major endotherm with a temperature of denaturation (Tm) of 71.9 +/- 0.4 degrees C and a thermal unfolding calorimetric enthalpy change (delta Hcal) of 700 +/- 17 kJ/mol rhodopsin. Bleached rod outer segment disk membranes (membranes that had lost their absorbance at 498 nm after exposure to orange light) gave a single major endotherm with a Tm of 55.9 +/- 0.3 degrees C and a delta Hcal of 520 +/- 17 kJ/mol opsin. Neither bleached nor unbleached rod outer segment disk membranes gave endotherms upon thermal rescans. When thermal stability is examined over the pH range of 4-9, the major endotherms of both bleached and unbleached rod outer segment disk membranes were found to show maximum stability at pH 6.1. The observed delta Hcal values for bleached and unbleached rod outer segment disk membranes exhibit membrane concentration dependences which plateau at protein concentrations beyond 1.5 mg/mL. For partially bleached samples of rod outer segment disk membranes, the calorimetric enthalpy change for opsin appears to be somewhat dependent on the degree of bleaching, indicating intramembrane nearest neighbor interactions which affect the unfolding of opsin. Delta Hcal and Tm are particularly useful for assessing stability and testing for completeness of regeneration of rhodopsin from opsin. Other factors such as sample preparation and the presence of low concentrations of ethanol also affect the delta Hcal values while the Tm values remain fairly constant. This shows that the delta Hcal is a sensitive parameter for monitoring environmental changes of rhodopsin and opsin.     

Activated cGMP phosphodiesterase of retinal rods. A complex with transducin alpha subunit.

Purified G-protein (transducin) activated with the nonhydrolyzable analog guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and cGMP phosphodiesterase (PDE) from retinal rods are added to protein-stripped disc membranes. Specific binding of the mainly soluble alpha subunit of G-protein with GTP gamma S bound (G alpha GTP gamma S, activator of the PDE) to the disc membrane in the presence of PDE is measured from gel scans or experiments with labeled G-protein alpha subunit (G alpha). Its variation as a function of G concentration matches the theoretical variation of G alpha involved in the activation of PDE calculated with previously estimated dissociation constants (Bennett, N., and Clerc, A. (1989) Biochemistry 28, 7418-7424), and the G alpha bound/PDE ratio at saturation is close to 2. No increase of G alpha binding to the membrane is observed when purified inhibitory subunit of PDE (PDE gamma) is added together with or instead of total PDE, and excess PDE gamma remains soluble. These results suggest that activated PDE is a complex with the activator G alpha GTP rather than PDE from which the inhibitory subunits have been removed. A method for purifying PDE gamma with a high yield of recovery and activity is described.     

Role of membrane integrity on G protein-coupled receptors: Rhodopsin stability and function

Rhodopsin is a prototypical G protein-coupled receptor (GPCR) - a member of the superfamily that shares a similar structural architecture consisting of seven-transmembrane helices and propagates various signals across biological membranes. Rhodopsin is embedded in the lipid bilayer of specialized disk membranes in the outer segments of retinal rod photoreceptor cells where it transmits a light-stimulated signal. Photoactivated rhodopsin then activates a visual signaling cascade through its cognate G protein, transducin or Gt, that results in a neuronal response in the brain. Interestingly, the lipid composition of ROS membranes not only differs from that of the photoreceptor plasma membrane but is critical for visual transduction. Specifically, lipids can modulate structural changes in rhodopsin that occur after photoactivation and influence binding of transducin. Thus, altering the lipid organization of ROS membranes can result in visual dysfunction and blindness.     

Kinetic studies suggest that light-activated cyclic GMP phosphodiesterase is a complex with G-protein subunits

Cyclic GMP phosphodiesterase (PDE) in rod disk membranes has three subunits of molecular weight 88 000 (alpha), 84 000 (beta), and 13 000 (gamma). Physiological activation of the enzyme by light is mediated by a GTP binding protein (G protein). The enzyme can also be activated by controlled digestion with trypsin, which destroys the gamma subunit, leaving the activated enzyme as PDE alpha beta [Hurley, J. B., & Stryer, L. (1982) J. Biol. Chem. 257, 11094-11099]. Addition of purified gamma subunit to PDE alpha beta inhibited the enzyme fully. This suggested the possibility that G protein could also activate PDE by removing the gamma subunit and leaving the active enzyme in the form of PDE alpha beta. Should this be true, the properties of light- and trypsin-activated enzymes should be comparable. We found this not to be the case. The Km of light-activated enzyme for cyclic GMP was about 0.9-1.4 mM while that of trypsin-activated enzyme was about 140 microM. The cyclic AMP Km was also different for the two enzymes: 6.7 mM for light-activated enzyme and 2.0 mM for trypsin-activated enzyme. The inhibition of both enzymes by the addition of purified gamma subunit also differed significantly. Trypsin-activated enzyme was fully inhibited by the addition of about 200 nM gamma, but light-activated enzyme could not be fully inhibited even with 2600 nM inhibitor subunit. The Ki of the trypsin-activated enzyme for gamma was 15 nM and of the light-activated enzyme 440 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial characterization of six chlorophyll a-protein complexes isolated from a blue-green alga by a nondetergent method

Incorporation of cholesterol hemisuccinate into thylakoid membranes decreased the membrane fluidity as measured by polarized fluorescence from 1,6-diphenyl-1,3,5-hexatriene. Increasing membrane viscosity in this manner did not inhibit the thylakoid membrane protein kinase. In contrast the effects of the protein phosphorylation on State I-State II transitions, which were observed in untreated membranes, were abolished. This observation is interpreted as indicating that protein phosphorylation-induced energy transfer changes are sensitive to membrane viscosity because they might require a lateral migration of the light-harvesting complex serving Photosystem II from grana to stromal lamellae. Cation effects on room- and low-temperature fluorescence emission properties and membrane adhesion were not abolished in these cholesterol hemisuccinate-treated membranes.     

Manipulation of cholesterol levels in rod disk membranes by methyl-beta-cyclodextrin: effects on receptor activation

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. Reduced cholesterol in disk membranes resulted in a higher proportion of photolyzed rhodopsin being converted to the G protein-activating metarhodopsin II (MII) conformation, whereas enrichment of cholesterol reduced the extent of MII formation. Time-resolved fluorescence anisotropy measurements using 1,6-diphenyl-1,3,5-hexatriene showed that increasing cholesterol reduced membrane acyl chain packing free volume as characterized by the parameter f(v). The level of MII formed showed a positive linear correlation with f(v) over the range of 4 to 38 mol % cholesterol. In addition, the thermal stability of rhodopsin increased with mol % of cholesterol in disk membranes. No evidence was observed for the direct interaction of cholesterol with rhodopsin in either its agonist- or antagonist-bound form. These results indicate that cholesterol mediates the function of the G protein-coupled receptor, rhodopsin, by influencing membrane lipid properties, i.e. reducing acyl chain packing free volume, rather than interacting specifically with rhodopsin.