Effect of detergents and lipids on transducin photoactivation by rhodopsin.

Rhodopsin samples, isolated using four different extraction procedures, were used to investigate the photodependent activation of the GTPase activity of transducin. A complete inhibition of transducin light-dependent GTP hydrolytic activity was observed when rhodopsin purified in the presence of 1% digitonin, following rod outer segment (ROS) solubilization with 1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate (CHAPS), was used for its activation [0 pmol of inorganic phosphate (Pi) released/min/pmol of rhodopsin]. Rhodopsin, isolated in the presence of 1% digitonin following ROS solubilization with 1% digitonin, was capable of stimulating slightly transducin GTPase activity, with an initial rate of 1 pmol of GTP hydrolyzed/min/pmol of rhodopsin. However, rhodopsin purified in the presence of 0.2% n-dodecyl-beta-D-maltoside (DM), following ROS solubilization with either 1% CHAPS or 1% DM, stimulated the enzymatic activity of transducin in a light-dependent manner, with an initial rate of 5 pmol of Pi released/min/pmol of rhodopsin. Addition of 0.075% egg phosphatidylcholine (PC) to the four different solubilized rhodopsin samples significantly enhanced light-stimulated GTP hydrolysis by transducin, with initial rates increasing from 0 to 1, 1 to 2, and 5 to 30 pmol of Pi released/min/pmol of rhodopsin, respectively. Furthermore, DM-solubilized rhodopsin induced the hydrolysis of the maximum amount of GTP by transducin at 0.0075% PC, while digitonin-solubilized rhodopsin only stimulated the GTPase activity of transducin to a similar value, when the amount of the photoreceptor protein was increased 4-fold and 0.15% PC was added to the assay mixture. These results suggest that the effective photoactivation of transducin by rhodopsin requires phospholipids, which seem to be differentially eliminated with the detergent extraction procedure utilized during ROS membranes solubilization and photopigment isolation.     

Purification and characterization of transducin from capybara Hydrochoerus hydrochaeris

Polypeptides of approximately 39, 36 and 相似文献   

The GTP-binding protein of rod outer segments. II. An essential role for Mg2+ in signal amplification

The role of Mg2+ in the GTP hydrolytic cycle was investigated by using purified subunits (G alpha and G beta, gamma) of the GTP-binding protein isolated from Bufo marinus rod outer segments (ROS). Mg2+ markedly stimulated the rate of GTP and guanosine-5'-O-(3-thiotriphosphate) (GTP gamma-s) binding to G alpha. This effect was especially striking in the presence of very small quantities of illuminated ROS disc membranes. GTP hydrolysis could occur in the absence of Mg2+, and Mg2+ increased the rate of GTP hydrolysis only about 50%. These data indicate that Mg2+ plays a fundamental role in amplification of the photon signal by markedly stimulating the rate of formation of GTP X G alpha complexes by very small amounts of illuminated rhodopsin while producing only a modest increase in the rate of GTP hydrolysis. Following hydrolysis of GTP, GDP X G alpha could reassociate with illuminated or unilluminated ROS disc membranes in the presence or absence of Mg2+. In the absence of guanine nucleotides, release of GDP from G alpha bound to illuminated disc membranes was detected in the presence or absence of Mg2+. Moreover, Mg2+ did not affect the rate of GDP release from membrane-bound G alpha. Illumination of B. marinus crude ROS disc membrane preparations markedly reduced pertussis toxin-mediated ADP-ribosylation of a 39,000 Mr (G alpha) protein in the presence but not in the absence, of Mg2+. Moreover, extensive dialysis of illuminated (but not unilluminated) crude ROS disc membranes against a Mg2+-containing buffer caused a marked reduction in the subsequent ADP-ribosylation of G alpha, even when Mg2+ was not present during the ADP-ribosylation step. This reduction was reversed by the addition of GDP or a GDP analogue (but not GMP or hydrolysis-resistant GTP analogues) during the ADP-ribosylation step. Dialysis of crude ROS disc membrane preparations (illuminated or unilluminated) against a Mg2+ -free buffer did not reduce the subsequent ADP-ribosylation of G alpha. These data indicate that Mg2+, in the presence of photolysed rhodopsin, can stimulate the release of GDP from crude preparations of ROS disc membranes. Four lines of evidence suggest that G alpha and G beta, gamma have Mg2+-binding site(s). When stored at 4 degrees C, in the absence of glycerol, G beta, gamma was more stable in the absence than in the presence of Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

The GTP-binding protein of rod outer segments. I. Role of each subunit in the GTP hydrolytic cycle

The GTP-binding protein of Bufo marinus rod outer segments (ROS) is composed of 3 subunits: G alpha, 39,000; G beta, 36,000; and G gamma, approximately 6,500. A stepwise analysis of the GTP hydrolytic cycle (GTP binding, GTP hydrolysis, and GDP release) was facilitated by using purified subunits of the GTP-binding protein. When G alpha and G beta, gamma concentrations were held constant, the initial rate of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma-s) binding to G alpha was dependent upon the amount of bleached rhodopsin present (as illuminated, urea-washed ROS disc membranes). When G alpha and the quantity of these membranes was held constant, the initial rate of GTP gamma-s binding to G alpha was markedly enhanced by increasing the amount of G beta, gamma. G beta preparations (free of G gamma) also stimulated the binding of GTP gamma-s to G alpha to the same extent as G beta, gamma preparations, suggesting that G gamma is not an essential component of the G beta, gamma-dependent stimulation of the rate of GTP gamma-s binding to G alpha. Nonlinear regression analysis revealed a single class of binding sites with an apparent stoichiometry of 1 mol of site/mol of G alpha under optimal binding conditions. Following GTP binding to G alpha, the GTP X G alpha complex dissociates from G beta, gamma which remains primarily bound to the ROS disc membranes. Moreover, while GTP remains in excess, the rates of GTP hydrolysis exhibited saturation in the presence of increasing amounts of G beta, gamma. Nonlinear regression analysis of these data argues against a direct role for G beta, gamma in the hydrolysis of GTP. Thus, both topologic and kinetic data support the concept that GTP hydrolysis is carried out by G alpha alone. After hydrolysis of GTP, the GDP X G alpha complex returned to the ROS disc membrane when G beta, gamma was present on the membrane surface, in the presence and absence of light. Without guanine nucleotides GDP release occurred in the presence of illuminated ROS disc membranes and G beta, gamma. Guanine nucleotides (GTP gamma-s approximately equal to GTP approximately equal to guanosine 5'-(beta, gamma-imido)triphosphate greater than GDP) could effectively displace GDP from G alpha under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Light-regulated biochemical events in invertebrate photoreceptors. 1. Light-activated guanosinetriphosphatase, guanine nucleotide binding, and cholera toxin catalyzed labeling of squid photoreceptor membranes

The occurrence of a guanine nucleotide binding protein activated by squid rhodopsin was established by examination of GTPase activity, guanine nucleotide binding, and cholera toxin catalyzed labeling of squid photoreceptor membranes. Purified squid (Loligo opalescens) photoreceptors exhibited GTPase activity that increased 3-4-fold by illumination. Half-maximal GTPase activity was observed when 2% of the rhodopsin was photoconverted to metarhodopsin. The Km of the light-regulated activity was 1 microM GTP. Binding of the hydrolysis-resistant GTP analogue guanosine 5'-(beta, gamma-imidotriphosphate) [Gpp(NH)p] was enhanced greater than 10 times by illumination. A protein, Mr 44 000, was identified as a component of the light-activated guanine nucleotide binding protein/GTPase through its specific labeling with [32P]NAD catalyzed by cholera toxin: light increased the extent of 32P incorporation 7-fold. The addition of ATP to the membrane suspension enhanced labeling, while guanine nucleotides inhibited labeling with the relative potency GTP gamma S much greater than GDP greater than GTP greater than Gpp(NH)p. The 44 000-dalton protein was membrane bound irrespective of variations in ionic strength and divalent ion concentration over a wide range. These results suggest that a G protein, which incorporates both GTP binding and hydrolysis functions, is intimately involved in the visual process of invertebrate photoreceptors.     

Purification of visual arrestin from squid photoreceptors and characterization of arrestin interaction with rhodopsin and rhodopsin kinase

Invertebrate visual signal transduction involves photoisomerization of rhodopsin, activating a guanine nucleotide binding protein (G protein) of the G(q) class, iG(q), which stimulates a phospholipase C, increasing intracellular Ca2+. Arrestin binding to photoactivated rhodopsin is a key mechanism of desensitization. We have previously reported the cloning of a retina-specific arrestin cDNA from Loligo pealei displaying 56-64% sequence similarity to other reported arrestin sequences. Here, we report the purification of the 55-kDa squid visual arrestin. Purified squid visual arrestin is able to inhibit light-activated GTPase activity dose-dependently in arrestin-depleted rhabdomeric membranes and associate with the membrane in a light-dependent manner. Membrane association can be partially inhibited by inositol 1,2,3,4,5,6-hexakisphosphate (IP6), a soluble analog of the membrane lipid phosphatidylinositol 3,4,5-triphosphate. In reconstitution assays, we demonstrate arrestin phosphorylation by squid rhodopsin kinase, a novel function among the G protein-coupled receptor kinase family. Phosphorylation of purified arrestin requires squid rhodopsin kinase, membranes, light-activation, and the presence of Ca2+. This is the first large-scale purification of an invertebrate arrestin and biochemical demonstration of arrestin function in the invertebrate visual system.     

cGMP suppresses GTPase activity of a portion of transducin equimolar to phosphodiesterase in frog rod outer segments. Light-induced cGMP decreases as a putative feedback mechanism of the photoresponse.

In rod photoreceptor cells, the light response is triggered by an enzymatic cascade that causes cGMP levels to fall: excited rhodopsin (Rho*)----rod G-protein (transducin, Gt)----cGMP-phosphodiesterase (PDE). This results in the closure of plasma membrane channels that are gated by cGMP. PDE activation by Gt occurs when GDP bound to the alpha-subunit of Gt (Gt alpha) is exchanged with free GTP. The interaction of Gt alpha-GTP with the gamma-subunits of PDE releases their inhibitory action and causes cGMP hydrolysis. Inactivation is thought to be caused by subsequent hydrolysis of Gt alpha-GTP by an intrinsic Gt-GTPase activity. Here we report that there are two portions of Gt in frog rod outer segments (ROS) expressing different rates of GTP hydrolysis: 19.5 +/- 3 mmol of Gt/mol of Rho, equivalent to that amount which participates in PDE activation, hydrolyzing GTP at a rate of approximately 0.6 turnover/s ("fast") and the remaining Gt (80.5 +/- 3 mmol/mol Rho) hydrolyzing GTP at a rate of 0.058 +/- 0.009 turnover/s. Fast GTPase activity is abolished in the presence of cGMP. This effect occurs over the physiological range of cGMP concentration changes in ROS, half-saturating at approximately 2 microM and saturating at 5 microM cGMP. cGMP-dependent suppression of GTPase is specific for cGMP; cAMP in millimolar concentration does not affect GTPase, while the poorly hydrolyzable cGMP analogue, 8-bromo-cGMP, mimics the effect. GTPase regulation by cGMP is not affected by Ca2+ over the concentration range 5-500 nM, which spans the physiological changes in cytoplasmic Ca2+ in rod cells. We suggest that the fast cGMP-sensitive GTPase activity is a property of the Gt that activates PDE. In this model, cGMP serves not only as a messenger of excitation but also modulates GTPase activity, thereby mediating negative feedback regulation of the pathway via PDE turnoff: a light-dependent decrease in cGMP accelerates the hydrolysis of GTP bound to Gt, resulting in the rapid inactivation of PDE.     

Functional homology between signal-coupling proteins. Cholera toxin inactivates the GTPase activity of transducin

Both the light-stimulated cGMP phosphodiesterase of retinal rod outer segments (ROS) and hormone-stimulated adenylate cyclase are regulated by guanine nucleotide-binding regulatory proteins (N). Transducin serves as the signal-carrying regulatory protein in ROS, and the N protein (also called G or G/F) performs this role in the adenylate cyclase system. The GTP form of these regulatory proteins activates the corresponding enzyme, whereas the GDP form does not. Both transducin and the N protein possess a GTPase activity that restores the regulatory protein to the unstimulated state. Cholera enterotoxin catalyzes the transfer of ADP-ribose from NAD+ to the N protein, which inhibits its GTPase activity and activates adenylate cyclase. We report here that the toxin also catalyzes ADP-ribosylation of the alpha-subunit of transducin in ROS membranes. This modification of the guanine nucleotide-binding subunit of transducin is markedly enhanced by the bleaching of rhodopsin and by the addition of guanosine-5'-(beta, gamma-imino)triphosphate. In contrast, GDP, GTP, and guanosine-5'-(3-O)thiotriphosphate inhibit the reaction, while GMP and ATP have no effect. Under optimal conditions, toxin catalyzes labeling of 0.7 mol of the alpha-subunit of transducin/mol of bound [3H]guanosine-5'-(beta, gamma-imido)triphosphate and causes 70% inhibition of the light-dependent GTPase activity of transducin in ROS. These results indicate close functional homology between transducin of ROS and the N protein of adenylate cyclase.     

Activation of cGMP phosphodiesterase by purified green rod pigment from frog retina

Activation of cGMP phosphodiesterase(PDE) of frog rod outer segments (ROS) by purified green rod pigment (GRP) was analyzed. GRP activated PDE in a similar manner to purified rhodopsin. This activation required illumination of the pigment and presence of GTP.     

Rhodopsin-G-protein interactions monitored by resonance energy transfer

Resonance energy transfer measurements were implemented to monitor the specific interactions between G-protein and rhodopsin in phospholipid vesicles reconstituted with the purified proteins. Fluorescently labeled G-protein was extracted from bleached rod outer segments (ROS) reacted with several sulfhydryl reagents: N-(1-pyrenyl)maleimide (P), monobromobimane (B), 7-(diethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin (C), and N-(4-anilino-1-naphthyl)maleimide (A). Limited labeling of ROS, resulting in the modification of less than a single -SH residue per G-protein molecule and less than 0.2 residue per rhodopsin, did not impair the specific in situ interactions between rhodopsin and G-protein. This was demonstrated by preservation of their light-activated tight association and Gpp(NH)p binding and their fast dissociation with excess GTP. The distribution of fluorescent label among the three subunits of G-protein revealed a highly reactive -SH group in the gamma subunit accessible to labeling when G-protein was bound specifically to bleached rhodopsin. Recombination of purified fluorescent derivatives of G-protein with purified rhodopsin reconstituted in lipid vesicles restored the light-activated Gpp(NH)p binding to a level comparable to that measured with unlabeled G-protein. Similar observations were obtained with ROS depleted of peripheral proteins. Likewise, modification of up to two -SH groups per rhodopsin molecule with the fluorescent reagents did not affect the functional recombination of G-protein with rhodopsin in reconstituted lipid vesicles or in depleted ROS. Interactions between rhodopsin and G-protein were monitored by resonance energy transfer measurements, with the following fluorescent conjugates as donor/acceptor couples: P-rhodopsin/C-G-protein, P-rhodopsin/B-G-protein, and P-G-protein/C-rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterase. II. Its role in the turnoff of phosphodiesterase in vivo

Retinal cGMP phosphodiesterase (PDE) is regulated by Pgamma, the regulatory subunit of PDE, and GTP/Talpha, the GTP-bound alpha subunit of transducin. In the accompanying paper (Matsuura, I., Bondarenko, V. A., Maeda, T., Kachi, S., Yamazaki, M., Usukura, J., Hayashi, F., and Yamazaki, A. (2000) J. Biol. Chem. 275, 32950-32957), we have shown that all known Pgammas contain a specific phosphorylation motif for cyclin-dependent protein kinase 5 (Cdk5) and that the unknown kinase is Cdk5 complexed with its activator. Here, using frog rod photoreceptor outer segments (ROS) isolated by a new method, we show that Cdk5 is involved in light-dependent Pgamma phosphorylation in vivo. Under dark conditions only negligible amounts of Pgamma were phosphorylated. However, under illumination that bleached less than 0.3% of the rhodopsin, approximately 4% of the total Pgamma was phosphorylated in less than 10 s. Pgamma dephosphorylation occurred in less than 1 s after the light was turned off. Analysis of the phosphorylated amino acid, inhibition of Pgamma phosphorylation by Cdk inhibitors in vivo and in vitro, and two-dimensional peptide map analysis of Pgamma phosphorylated in vivo and in vitro indicate that Cdk5 phosphorylates a Pgamma threonine in the same manner in vivo and in vitro. These observations, together with immunological data showing the presence of Cdk5 in ROS, suggest that Cdk5 is involved in light-dependent Pgamma phosphorylation in ROS and that the phosphorylation is significant and reversible. In an homogenate of frog ROS, PDE activated by light/guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) was inhibited by Pgamma alone, but not by Pgamma complexed with GDP/Talpha or GTPgammaS/Talpha. Under these conditions, Pgamma phosphorylated by Cdk5 inhibited the light/GTPgammaS-activated PDE even in the presence of GTPgammaS/Talpha. These observations suggest that phosphorylated Pgamma interacts with and inhibits light/GTPgammaS-activated PDE, but does not interact with GTPgammaS/Talpha in the homogenate. Together, our results strongly suggest that after activation of PDE by light/GTP, Pgamma is phosphorylated by Cdk5 and the phosphorylated Pgamma inhibits GTP/Talpha-activated PDE, even in the presence of GTP/Talpha in ROS.     

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)     

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.     

Limited trypsin proteolysis of photoreceptor GTP-binding protein. Light- and GTP-induced conformational changes

The amphibian photoreceptor rod outer segment contains a guanine nucleotide-binding complex which consists of a 39,000-dalton polypeptide that binds guanine nucleotides (G protein), a 36,000-dalton polypeptide (H protein), and an approximately 6,500-dalton polypeptide. Sensitivity to trypsin proteolysis was utilized as a probe of structure-function relationships for these polypeptides. Digestion of the H protein generated fragments of 26,000 and 15,000 daltons whose proteolytic susceptibility was not altered by guanosine triphosphates, light, or membranes. The approximately 6,500-dalton polypeptide was not trypsin sensitive. When the G protein was eluted from illuminated membranes by GTP, trypsin proteolysis cleaved a terminal 1,000-dalton fragment (G1) to yield a 38,000-dalton fragment (G38). With increased digestion time, a 6,000-dalton fragment (G6) was removed from G38 to yield a 32,000-dalton fragment (G32). G32 was subsequently digested to fragments of 23,000 and 12,000 daltons. However, when the G protein was eluted from illuminated membranes by hydrolysis-resistant analogues of GTP, G32 was protected from further digestion. This is consistent with a GTP-induced conformational change in the G protein which is altered by GTP hydrolysis. Proteolysis of the G protein after covalent labeling with a photoaffinity analogue of GTP demonstrated that the analogue is bound to first G38 and then G32, indicating the GTP-binding site is associated with G32. Fragment G6 was cleaved when the G protein was soluble or bound to unilluminated membranes. However, when bound to illuminated membranes, fragments were generated reflecting the loss of 7,500, 9,000, or 11,000 daltons from the G protein. This light-induced alteration in proteolytic susceptibility indicates there is a light-induced conformational change in the G protein. Fragment G1 was not removed from the G protein when it was membrane bound, suggesting G1 is involved in binding to a membrane structure. These data suggest that the light-induced binding of the G protein to illuminated membranes and the reversal of this binding by GTP are mediated through conformational changes in the G protein and that three conformations exist: 1) a basal, inactive conformation; 2) a primed conformation induced by binding to photolyzed rhodopsin, with a high affinity for GTP; and 3) an active conformation, induced by binding of GTP, which activates the catalytic complex of light-activated phosphodiesterase.     

A monoclonal antibody to guanine nucleotide binding protein inhibits the light-activated cyclic GMP pathway in frog rod outer segments

A monoclonal antibody that blocks the light-activated cyclic GMP (cGMP) pathway in frog photoreceptor outer segments (ROS) has been obtained. The antibody (4A) inhibits guanine nucleotide binding to G-protein, the intermediate that links rhodopsin excitation to cGMP phosphodiesterase (PDE), inhibiting light-induced PDE activity as a consequence. Antibody inhibition of the light-activated cGMP pathway is complete at a stoichiometry of approximately one antibody per G-protein in the mixture, which indicates high specificity of the inhibition. Inhibition is more pronounced than that caused by PDE inhibitors such as isobutylmethylxanthine (IBMX) or Ro 20-1724. Antibody 4A has the further effect of inhibiting the phosphorylation of two low molecular weight proteins, components I and II, whose phosphorylation normally can be stimulated by raising cGMP levels. The inhibition is not overridden by adding cGMP, which suggests that the G-protein influences these phosphorylations by a pathway distinct from its action on cGMP concentration. Antibody 4A may prove useful as a probe of the relevance of the cGMP pathway to visual transduction in living photoreceptors. Six other monoclonal antibodies to G-protein, as well as six monoclonal antibodies to rhodopsin and one to PDE, do not block light-activated guanine nucleotide binding, PDE activity, or ROS protein phosphorylations.     

Specificity of the functional interactions of the beta-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles

We have assessed the functional interactions of two pure receptor proteins with three different pure guanine nucleotide regulatory proteins in phosphatidylcholine vesicles. The receptor proteins are the guinea pig lung beta-adrenergic receptor (beta AR) and the retinal photon receptor rhodopsin. The guanine nucleotide regulatory proteins were the stimulatory (Ns) and inhibitory (Ni) proteins of the adenylate cyclase system and transducin (T), the regulatory protein from the light-activated cyclic GMP phosphodiesterase system in retinal rod outer segments. The insertion of Ns with beta AR in lipid vesicles increases the extent of binding of [35S] GTP gamma S to Ns and in parallel, the total GTPase activity. However, there is little change in the actual rate of catalytic turnover of GTPase activity (defined as mol of Pi released/min/mol of Ns-guanine nucleotide complexes). Enhancement of this turnover rate requires the beta-agonist isoproterenol and is accounted for by an isoproterenol-promoted increase in the rate and extent of [35S]GTP gamma S binding to Ns. The co-insertion of the beta AR with Ni or transducin results in markedly lower stimulation by isoproterenol of both the GTPase activity and [35S]GTP gamma S binding to these nucleotide regulatory proteins indicating that their preferred order of interaction with beta AR is Ns much greater than Ni greater than T. This contrasts with the preferred order of interaction of these different nucleotide regulatory proteins with light-activated rhodopsin which we find to be T approximately equal to Ni much greater than Ns. Nonetheless the fold stimulation of GTPase activity and [35S]GTP gamma S binding in T, induced by light-activated rhodopsin, is significantly greater than the "fold" stimulation of these activities in Ni. This reflects the greater intrinsic ability of Ni to hydrolyze GTP and bind guanine nucleotides (at 10 mM MgCl2, 100-200 nM GTP or [35S] GTP gamma S) compared to T. The maximum turnover numbers for the rhodopsin-stimulated GTPase in both Ni and T are similar to those obtained for isoproterenol-stimulated activity in Ns. This suggests that the different nucleotide regulatory proteins are capable of a common upper limit of catalytic efficiency which can best be attained when coupled to the appropriate receptor.     

Predictive value of the analogy between hormone-sensitive adenylate cyclase and light-sensitive photoreceptor cyclic GMP Phosphodiesterase: A specific role for a light-sensitive GTPase as a component in the activation sequence

We report experiments which involve a light sensitive GTPase in the light dependent activation of retinal rod 3′5′-cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE). The data suggest that the light activated GTPase is intermediate between rhodopsin and PDE in the light-dependent activation sequence. We list the many striking similarities between hormone sensitive adenylate cyclase and light activated PDE in order to emphasize that the findings presented herein may have predictive value for ongoing studies of the hormone sensitive adenylate cyclase specifically regarding the role of the hormone activated GTPase in the activation sequence.     

Transient response of retinal rod outer segment phosphodiesterase to actinic light pulses. I. Simple quantitative kinetic model

We present a quantitative kinetic model for the transient velocity (microM of cGMP hydrolyzed/s) response of retinal rod outer segment (ROS) cGMP phosphodiesterase (v(t) versus t) to a stimulating light pulse in the linear response range. The model gives an excellent fit to experimental v(t) versus t data for ROS suspensions at different concentrations of GTP and GDP and clarifies experimental results which are difficult to understand in the absence of such a model. It contains the minimum number of steps required to fit our experimental data and consists of one rate-limiting step with specific rate kL for the production of active phosphodiesterase (PDE), PDE*, by photoactivated rhodopsin, R*, and deactivation processes for R* and PDE* with lifetimes tau R and tau P, respectively. The experimental graphs of v(t) versus t at each concentration of GTP and GDP are characterized by a fast rise to a peak value, vpeak, followed by a slow decay to zero level. The minimal kinetic model allows us to characterized completely the effects of GTP and GDP, and any other pertinent species, in terms of their effects on the parameters kL, tau R, and tau P. Our kinetic model indicates that for "washed" ROS preparations (a) the risetime of v(t) is determined by tau P which has a value of about 2 s and is insensitive to [GTP]. (b) The decay of v(t) is determined by tau R which decreases with [GTP] and has a value greater than 300 s at low [GTP] and a limiting value of 50 s at high [GTP]. We attribute the greater than 300 s lifetime to the complex R*G (where G is ROS G protein) and the 50-s lifetime to free R*. (c) The rate kL increases hyperbolically with [GTP] with a half-maximal value of 56 microM and kL.max = 22-45 s-1. (d) Peak velocity is given by the expression vpeak alpha kL tau P which is consistent with the dependence of kL on [GTP] and the experimental finding that vpeak varies hyperbolically with [GTP]. The minimal model has also allowed us to (a) develop clear definitions of amplification for the light-triggered enzymatic cascade and (b) clarify experimental methods for measuring gain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Contribution of the guanosinetriphosphatase activity of G-protein to termination of light-activated guanosine cyclic 3',5'-phosphate hydrolysis in retinal rod outer segments

Light activation of GTP binding to G-protein and its eventual hydrolysis are hypothesized to lead to activation and inactivation of cGMP phosphodiesterase (PDE) in vertebrate rod disk membranes (RDM). However, the reported GTPase rate of 3 per minute is too slow to account for the observed rapid inactivation of PDE. Our investigations on GTPase activity showed that RDM isolated in the dark have considerable dark GTPase activity, which is enhanced by light. In dark and light, the enzyme exhibits biphasic substrate dependence with two Km's for GTP of 2-3 and 40-80 microM at 22 degrees C and less than 1 and 10-25 microM at 37 degrees C. The Km's were not influenced by light. On the basis of G-protein content of the RDM, the Vmax's for the two activities at 37 degrees C in light are 4-5 and 20-30 GTPs hydrolyzed per minute per G-protein. RDM washed free of soluble and peripheral proteins do not have measurable GTPase activity in the dark or light. Purified G-protein alone also did not turn over GTP, apparently because bleached rhodopsin is required for it to bind GTP. Reconstitution of washed membranes with purified G-protein restores both the low- and high-Km GTPase activities. Inactivation of G-protein as measured by PDE turnoff and dissociation signal recovery is found to be faster at higher than lower [GTP], consistent with the observation that the higher GTPase activity associated with the higher Km alos resides in the G-protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction between the main components of the vertebrate retinal rod phototransduction system in solutions of detergent n-nonyl-β-D-glucoside

cGMP-Specific phosphodiesterase (PDE6) is the key enzyme of the phototransduction system of vertebrate retinal rod outer segments (ROS). The properties of PDE in extracts prepared by solubilization of bovine ROS in a high concentration (0.5% w/v) of detergent n-nonyl-β-D-glucoside (NG) and following centrifugation (ROS-NG) have been studied. Basal PDE activity of the preparations was low, but it greatly (>50-fold) increased (up to ∼20 μmol cGMP hydrolyzed/min per mg rhodopsin (R)) in the presence of trypsin. In bleached GTPγS-containing preparations the specific PDE activity was dependent on ROS-NG concentration and was half-maximal at about 0.8 μM of ROS G protein transducin (Gt). In dark-adapted GTPγS-containing ROS-NG preparations bleaching of 0.2% of the rhodopsin resulted in half-maximal PDE activation. The same result was obtained when PDE in dark-adapted ROS-NG preparations was activated by addition of a highly purified bleached rhodopsin solubilized by 0.5% solution of NG. The results demonstrate that the presence of NG has no significant influence either on the properties of the main ROS phototrans-duction system elements (R, Gt and PDE) or on the interaction between photoactivated R and Gt and suggest that the detergent NG can be used for crystallization of the rhodopsin-transducin complex.