Bovine Rod Outer Segment Membranes Research Articles

βγ-Transducin stimulates hydrolysis and synthesis of phosphatidylinositol 4,5-bisphosphate in bovine rod outer segment membranes

T betagamma was shown to stimulate the hydrolysis and synthesis of PtdInsP2 in dark-adapted bovine retinal rod outer segments. In contrast, T alphaGDP blocked the effect of betagamma-transducin. It was also demonstrated that T betagamma was a stimulator of 32P incorporation into PtdInsP2 in ROS. These findings explain the modulating actions of GTP and light on PtdInsP2 hydrolysis and synthesis in ROS. The possible existence of cross-talk between the cGMP and phosphoinositide cascades in retinal rods was discussed.

S-Prenylated Cysteine Analogs Inhibit Receptor-Mediated G Protein Activation in Native Human Granulocyte and Reconstituted Bovine Retinal Rod Outer Segment Membranes

We have previously shown that the S-prenylated cysteine analogue N-acetyl-S-trans,trans-farnesyl-L-cysteine (L-AFC) inhibits basal and formyl peptide receptor-stimulated binding of guanosine 5'-O-(3-thiotriphosphate) (GTP[S]) to and hydrolysis of GTP by membranes of HL-60 granulocytes and have presented evidence suggesting that this inhibition was not caused by reduced protein carboxyl methylation [Scheer, A., & Gierschik, P. (1993) FEBS Lett. 319, 110-114]. We now report a detailed analysis of the structural properties of S-prenylated cysteine analogues required for this inhibition and demonstrate that S-prenylcysteines also suppress basal and receptor-stimulated GTP[S] binding to human peripheral neutrophil and HL-60 granulocyte membranes when stimulated by formyl peptide and complement C5a, respectively. S-Prenylcysteines did not affect pertussis toxin-mediated [32P]ADP-ribosylation of Gi proteins. The inhibitory effect of L-AFC was reversible and was not mimicked by farnesylic acid. L-AFC also interfered with GTP[S] binding to retinal transducin when stimulated by light-activated rhodopsin in a reconstituted system. This inhibitory effect was fully reversed upon increasing the concentration of either the G protein beta gamma dimer or the activated receptor. On the basis of these results, we suggest that S-prenylated cysteine analogues like L-AFC inhibit receptor-mediated G protein activation by specifically and reversibly interfering with the interaction of activated receptors with G proteins, most likely with their beta gamma dimers, rather than by inhibiting alpha.beta gamma heterotrimer formation.

Regulation of bovine rod outer segment membrane guanylate cyclase by ATP, phosphodiesterase and metal ions.

In vertebrate retina, rod outer segment is the site of visual transduction. The inward cationic current in the dark-adapted outer segment is regulated by cyclic GMP. A light flash on the outer segment activates a cyclic GMP phosphodiesterase resulting in rapid hydrolysis of the cyclic nucleotide which in turn causes a decrease in the dark current. Restoration of the dark current requires inactivation of the phosphodiesterase and synthesis of cyclic GMP. The latter is accomplished by the enzyme guanylate cyclase which catalyzes the formation of cyclic GMP from GTP. Therefore, factors regulating the cyclase activity play a critical role in visual transduction. But regulation of the cyclase by some of these factors--phosphodiesterase, ATP, the soluble proteins and metal cofactors (Mg and Mn)--is controversial. The availability of different types of cyclase preparations, dark-adapted rod outer segments with fully inhibited phosphodiesterase activity, partially purified cyclase without PDE contamination, cloned rod outer segment cyclase free of other rod outer segment proteins, permitted us to address these controversial issues. The results show that ATP inhibits the basal cyclase activity but enhances the stimulation of the enzyme by soluble activator, that cyclase can be activated in the dark at low calcium concentrations under conditions where phosphodiesterase activity is fully suppressed, and that greater activity is observed with manganese as cofactor than magnesium. These results provide a better understanding of the controls on cyclase activity in rod outer segments and suggest how regulation of this cyclase by ATP differs from that of other known membrane guanylate cyclases.

Altered guanine nucleoside triphosphate binding to transducin by cholera toxin-catalysed ADP-ribosylation

The influence of cholera toxin (CTX)-catalysed ADP-ribosylation on binding of guanine nucleoside triphosphates to transducin was studied by measuring the binding of the GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), to illuminated bovine rod outer segment (ROS) membranes treated with or without CTX. Besides the well-documented inhibition of the transducin GTPase activity, CTX treatment inhibited binding of GTP[gamma S] to illuminated ROS membranes. This inhibition was due to an approximately two-fold lower apparent affinity for the nucleotide, while the density of binding sites was not altered. CTX decreased the association rate of GTP[gamma S] by a factor of about two. Competition experiments with GTP, guanosine 5'-[beta, gamma]iminotriphosphate or GDP showed that the apparent affinities for both guanine nucleoside triphosphates, but not for GDP, were lowered by about two-fold upon CTX treatment. In contrast to CTX, pertussis toxin treatment of ROS membranes reduced the density of binding sites available to GTP[gamma S], while the apparent affinity of the remaining sites was unchanged. It is concluded that ADP-ribosylation of transducin by CTX not only inhibits its GTPase activity but also decreases the affinity for guanine nucleoside triphosphates, data which suggest that the arginine moiety modified by CTX is involved in both binding and hydrolysis of GTP.

Structural and Biochemical Identity of Retinal Rod Outer Segment Membrane Guanylate Cyclase

Recent molecular cloning reports show that there are at least three membrane guanylate cyclases in vertebrate retina: (1) atrial natriuretic factor receptor guanylate cyclase (ANF-RGC), (2) C-type natriuretic peptide receptor guanylate cyclase (CNP-RGC), and (3) "retinal guanylate cyclase" (RetGC). The specific cellular localization of the first two cyclases is unknown, but RetGC is apparently localized in photoreceptor cells, suggesting that it participates in visual transduction. With the overall objective of identifying the guanylate cyclase that is linked to phototransduction, we compared the structural and regulatory properties of the biochemically characterized 112 kDa bovine rod outer segment membrane guanylate cyclase (ROS-GC) with those of RetGC, ANF-RGC and CNP-RGC. The N-terminal and two internal peptide sequences of purified ROS-GC had about 90% similarity with the corresponding sequences of the RetGC; the sequence identity with natriuretic peptide receptor cyclases was about 30%. A 19 amino acid long sequence from a tryptic peptide of ROS-GC had no corresponding sequence in the other three cyclases. ROS-GC was inhibited by ATP but ANF-RGC and CNP-RGC were activated by ATP in the presence of the respective peptide hormones. These results suggest that ROS-GC represents a new subtype of the membrane guanylate cyclase family that is structurally and biochemically distinct from the other retinal cyclases.

Stimulation and inhibition of human platelet adenylylcyclase by thiophosphorylated transducin beta gamma-subunits.

The effect of beta gamma-dimers isolated from the retinal guanine nucleotide-binding protein (G protein) transducin eluted from illuminated bovine rod outer segment membranes with GTP, guanosine 5'-O-(beta, gamma-imino)triphosphate (Gpp(NH)p), or guanosine 5'-O-(gamma-thio)triphosphate (GTP gamma S) on basal and forskolin-stimulated adenylylcyclase activities in membranes of human platelets was studied. beta gamma-Subunits isolated from transducin eluted with GTP gamma S (TD beta gamma GTP gamma S) had a concentration-dependent stimulatory effect on basal adenylylcyclase activity. The stimulatory agonist prostaglandin E1 increased the potency and the maximum extent of stimulation due to TD beta gamma GTP gamma S). With a similar concentration dependence, TD beta gamma GTP gamma S exerted an inhibitory influence on forskolin-stimulated adenylylcyclase activity. At the same concentrations, beta gamma-dimers isolated with either GTP or Gpp(NH)p did not alter enzyme activities. The observed effects of TD beta gamma GTP gamma S were similar to those of directly added GTP gamma S with regard to maximum levels, time dependence, and persistence; however, TD beta gamma GTP gamma S was approximately 10-fold more potent than GTP gamma S. Treatment of TD beta gamma GTP gamma S, but not of free GTP gamma S, with hydroxylamine caused a loss of adenylylcyclase regulation by TD beta gamma GTP gamma S. The data presented indicated that TD beta gamma GTP gamma S potently and efficiently activates the stimulatory and inhibitory G proteins of adenylylcyclase in human platelet membranes. Furthermore, evidence is provided suggesting that the observed effects of TD beta gamma GTP gamma S, which can be thiophosphorylated by GTP gamma S at the beta-subunit (Wieland, T., Ulibarri, I., Gierschik, P., and Jakobs, K. H. (1991) Eur. J. Biochem. 196, 707-716), are due to formation of GTP gamma S at the G proteins.

Identification of an isoprenylated cysteine methyl ester hydrolase activity in bovine rod outer segment membranes.

Proteins from eucaryotic cells which have a carboxyl-terminal CAAX motif are posttranslationally modified by isoprenylation. The pathway involves the linkage of an all-trans-farnesyl (C15) or an all-trans-geranylgeranyl (C20) moiety to the cysteine residue followed by proteolysis which generates the modified cysteine as the carboxyl-terminal residue. Carboxylmethylation of the modified cysteine residue completes the pathway. This latter methylation reaction is the only potentially reversible reaction in the pathway and thus of possible regulatory significance. A specific esterase is required to reverse the methylation. It is demonstrated here that simple isoprenylated cysteine derivatives, such as N-acetyl-S-farnesyl-L-cysteine methyl ester (L-AFCM) and N-acetyl-S-geranylgeranyl-L-cysteine methyl ester (L-AGGCM), are substrates for a rod outer segment (ROS) membrane esterase activity. The KM and Vmax values for L-AFCM and L-AGGCM are 186 microM and 2.2 nmol mg-1 min-1 and 435 microM and 4.8 nmol mg-1 min-1, respectively. The enzyme(s) is stereoselective rather than stereospecific because D-AFCM is enzymatically hydrolyzed with KM and Vmax values of 157 microM and 0.46 nmol mg-1 min-1, respectively. The enzyme(s) does not process N-acetyl-L-cysteine methyl ester, demonstrating that the isoprenyl moiety is required for substrate activity. Ebelactone B is a potent mechanism-based inactivator of the enzyme with a KI = 42 microM and a kinh = 3.7 x 10(-3) s-1. Importantly, L-AFCM, L-AGGCM, and ebelactone B all inhibit the demethylation of the endogenous ROS substrates, showing that the same enzymatic activity is involved in the processing of the synthetic and physiological substrates.

Subunit interactions of GTP-binding proteins.

Fluorescence energy transfer [cf. Förster, T. (1948) Ann. Phys. 6, 55-75] was tested for its suitability to study quantitative interactions of subunits of G0 with each other and these subunits or trimeric G0 with the beta 1-adrenoceptor in detergent micelles or after reconstitution into lipid vesicles [according to Feder, D., Im, M.-J., Klein, H. W., Hekman, M., Holzhöfer, A, Dees, C., Levitzki, A., Helmreich, E. J. M. & Pfeuffer, T. (1986) EMBO J. 5, 1509-1514]. For this purpose, alpha 0- and beta gamma-subunits and trimeric G0 purified from bovine brain, the beta gamma-subunits from bovine rod outer segment membranes and the beta 1-adrenoceptor from the turkey erythrocyte were all labelled with either tetramethylrhodamine maleimide or fluorescein isothiocyanate under conditions which leave the labelled proteins functionally intact. In the case of alpha 0- and beta gamma-interactions, specific high-affinity binding interactions (Kd approximately 10 nM) and nonspecific low-affinity binding interactions (Kd approximately 1 microM) could be readily distinguished by comparing fluorescence energy transfer before and after dissociation with 10 microM guanosine 5'-O-[gamma-thio]triphosphate and 10 mM MgCl2 where only low-affinity binding interactions remained. Interactions between alpha 0- and beta gamma-subunits from bovine brain or from bovine retinal transducin did not differ much. The beta gamma-subunits from bovine brain were found to bind with high transfer efficiency and comparable affinities to the hormone-activated and the nonactivated beta 1-receptor reconstituted in lipid vesicles: Kd = 100 +/- 20 and 120 +/- 20 nM, respectively; however, beta gamma-subunits from transducin appeared to bind more weakly to the beta 1-adrenoceptor than beta gamma-subunits from bovine brain. Separated purified homologous alpha 0- and beta gamma-subunits from bovine brain interfered mutually with each other in binding to the beta 1-adrenoceptor presumably because they had a greater affinity for each other than for the receptor. These findings attest to the suitability of fluorescence energy transfer for studying protein-protein interactions of G-proteins and G-protein-linked receptors. Moreover, they supported the previous finding [Kurstjens, N. P., Fröhlich, M., Dees, C., Cantrill, R. C., Hekman, M. & Helmreich, E. J. M. (1991) Eur. J. Biochem. 197, 167-176] that beta gamma-subunits can bind to the nonactivated beta 1-adrenoceptor.

The cGMP-gated channel of the rod photoreceptor cell characterization and orientation of the amino terminus.

The molecular properties and orientation of the cGMP-gated cation channel of bovine rod outer segment membranes were studied using biochemical and immunochemical methods. Western blots labeled with anti-channel monoclonal antibodies indicate that the channel has a subunit Mr of 63,000 in bovine rod outer segment membranes prepared in the presence and absence of protease inhibitors and in rod outer segments from other mammalian retinas. The channel has an apparent Mr of 78,000 in both COS-1 cells and Xenopus oocytes expressing the cloned cDNA. NH2-terminal sequence analysis indicates that the lower Mr of the channel in rod outer segments is caused by the absence of the first 92 amino acids predicted by cDNA sequence analysis. Immunofluorescent and immunogold labeling has confirmed that the 63,000 form of the channel is present in rod outer segments. These results indicate that photoreceptor cell-specific co-translational or post-translational cleavage of the NH2-terminal segment of the channel occurs prior or during the incorporation of the channel into the rod outer segment plasma membrane. Immunogold labeling studies using site-directed antibodies also indicate that the NH2-terminal segment of the rod outer segment channel is exposed on the cytoplasmic side of the plasma membrane.

Activation of signal‐transducing guanine‐nucleotide‐binding regulatory proteins by guanosine 5′‐[γ‐thio]triphosphate

Signal-transducing guanine-nucleotide-binding regulatory proteins (G proteins) are heterotrimers, composed of the nucleotide-binding alpha subunit and a beta gamma dimer. The influence of beta gamma dimer preparations of the retinal G protein transducin (TD) was studied on formylpeptide-receptor--G-protein interactions in membranes of differentiated HL 60 cells. For this, TD was prepared from bovine rod outer segment (ROS) membranes with either GTP or its analogs, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imino]triphosphate (Gpp[NH]p). After removal of free nucleotides, TD beta gamma was separated from TD alpha and its function analyzed. Addition of TD beta gamma isolated from TD prepared with GTP[S] (TD beta gamma GTP[S]) to HL 60 membranes abolished high-affinity binding of fMet-Leu-[3H]Phe (fMet, N-formylmethionine) to its receptor. In contrast, TD beta gamma isolated from TD prepared with GTP (TD beta gamma GTP), boiled TD beta gamma GTP[S] and TD alpha prepared with GTP[S] had no or only slight effects. The inhibitory effect of TD beta gamma GTP[S] on fMet-Leu-[3H]Phe receptor binding was potentiated by GDP at low concentrations but not by GTP[S]. Furthermore, TD beta gamma GTP[S], but not TD beta gamma GTP or TD beta gamma isolated from TD prepared with Gpp[NH]p (TD beta gamma Gpp[NH]p), prevented fMet-Leu-Phe-stimulated binding of [35S]GTP[S] to G proteins in HL 60 membranes, measured in the presence of GDP. When TD beta gamma GTP was incubated with GTP [S] and TD-depleted illuminated ROS membranes, and subsequently separated from the membranes and free GTP[S], this TD beta gamma GTP, similar to TD beta gamma GTP[S], abolished high-affinity binding of fMet-Leu-[3H]Phe to its receptor, fMet-Leu-Phe-stimulated binding of [35S]GTP[S], and fMet-Leu-Phe-stimulated GTP hydrolysis in HL 60 membranes. Inhibition of [35S]GTP[S] binding by TD beta gamma was not seen in the presence of the metabolically stable GDP analog, guanosine 5'-[beta-thio]diphosphate. In order to obtain an insight into the modification of TD beta gamma apparently caused by GTP[S], and into its mechanism of action in HL 60 membranes, TD, TD alpha and TD beta gamma, all prepared in the presence of GTP, were incubated with [35S]GTP[S] and TD-depleted illuminated ROS membranes. Fluorographic analysis of the supernatant proteins revealed 35S labelling of the beta band of the G protein. When apparently thiophosphorylated TD beta gamma was incubated with [3H]GDP in the presence of HL 60 membranes, [3H]GTP[S] was rapidly formed.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of recombinant rho A GTP-binding proteins with photoexcited rhodopsin

The small molecular mass GTP-binding proteins rho A, B and C are targets for ADP-ribosyltransferase activity of the botulinum exoenzyme C3. The possible interaction of recombinant rho A proteins expressed in E. coli with photoexcited rhodopsin was studied by reconstitution with bovine rod outer segment (ROS) membranes depleted of endogenous GTP-binding proteins by treatment with urea. As reported for C3 substrates present in untreated ROS membranes, ADP-ribosylation of recombinant rho A proteins, both normal and Val-14 mutant, by C3 was inhibited when reconstituted with illuminated compared to dark-adapted ROS membranes pretreated with urea. GDP reduced the light-induced inhibition, while GTP[S] and light inhibited ADP-ribosylation of rho A proteins in a synergistic manner.

Calcium ion-regulated phospholipase C activity in bovine rod outer segments

Bovine retinal rod outer segment membranes are enriched in a phosphoinositide-specific phosphodiesterase (phospholipase C) activity strictly modulated by free calcium ion concentration. The enzyme(s) was highly active on phosphatidylinositol 4,5-bisphosphate: maximal hydrolysis rate was attained at 10(-5)M Ca2+ and accounted for 91 +/- 4 nmoles hydrolyzed/min/mg of protein. The results support the notion that in vivo the enzyme(s) is regulated so as to conform to the phototransduction events.

Cation selectivity of and cation binding to the cGMP-dependent channel in bovine rod outer segment membranes.

The properties of the cGMP-dependent channel present in membrane vesicles prepared from intact isolated bovine rod outer segments (ROS) were investigated with the optical probe neutral red. The binding of neutral red is sensitive to transport of cations across vesicular membranes by the effect of the translocated cations on the surface potential at the intravesicular membrane/water interface (Schnetkamp, P. P. M. J. Membr. Biol. 88: 249-262). Only 20-25% of ROS membrane vesicles exhibited cGMP-dependent cation fluxes. The cGMP-dependent channel in bovine ROS carried currents of alkali and earth alkali cations, but not of organic cations such as choline and tetramethylammonium; little discrimination among alkali cations (K greater than Na = Li greater than Cs) or among earth alkali cations (Ca greater than Mn greater than Sr greater than Ba = Mg) was observed. The cation dependence of cGMP-induced cation fluxes could be reasonably well described by a Michaelis-Menten equation with a dissociation constant for alkali cations of about 100 mM, and a dissociation constant for Ca2+ of 2 mM. cGMP-induced Na+ fluxes were blocked by Mg2+, but not by Ca2+, when the cations were applied to the cytoplasmic side of the channel. cGMP-dependent cation fluxes showed a sigmoidal dependence on the cGMP concentration with a Hill coefficient of 2.1 and a dissociation constant for cGMP of 92 microM. cGMP-induced cation fluxes showed two pharmacologically distinct components; one component was blocked by both tetracaine and L-cis diltiazem, whereas the other component was only blocked by tetracaine.

Identification of a C-terminal protein carboxyl methyltransferase in rat liver membranes utilizing a synthetic farnesyl cysteine-containing peptide substrate.

Polypeptides synthesized in eucaryotic cells with a C-terminal -Cys-Xaa-Xaa-Xaa (-CXXX) sequence are candidates for post-translational modifications that include the removal of the last 3 amino acids and the lipidation and methyl esterification of the cysteinyl residue. To characterize the methylation reaction in vitro, the peptide Leu-Ala-Arg-Tyr-Lys-Cys (LARYKC) and its S-isoprenylated and S-alkylated derivatives were synthesized and assayed as methyl-accepting substrates with subcellular fractions of rat tissues including liver microsomal membranes. While little or no peptide-specific methyltransferase activity was detected in the latter preparation using the unmodified hexapeptide, the C10, C15, and C20 isoprenylated derivatives were substrates with Km values of 389 microM for S-geranyl-LARYKC, 2.2 microM for S-farnesyl-LARYKC, and approximately 10.9 microM for S-geranylgeranyl-LARYKC. The methyl-acceptor activities of a variety of n-alkyl S-derivatives of LARYKC (C8, C10, C13, C15) were also tested; all of these compounds were poorer substrates than the S-geranyl derivative. This enzyme activity uses S-adenosyl-L-methionine as the methyl donor (Km = 2.1 microM) and can be inhibited by S-adenosylhomocysteine (Ki = 9.2 microM), a product of the methylation reaction. The S-farnesyl-LARYKC peptide can inhibit the carboxyl methylation of bovine retinal rod outer segment membrane proteins that was previously shown to occur at the alpha-carboxyl group of C-terminal cysteine residues, demonstrating that the same enzyme can methylate both peptides and proteins. These results suggest that the methyl esterification of proteins containing a C-terminal -CXXX sequence requires not only the removal of the 3 terminal amino acids, but the isoprenylation of the sulfhydryl group as well.

Immunological characterization and localization of the Na+/Ca2(+)- exchanger in bovine retina

The sodium/calcium exchanger was purified from bovine retinal rod outer segment membranes and used for the immunization of New Zealand White rabbits. A polyclonal antibody was produced which was found to bind specifically to the 230 kDa Na+/Ca2(+)-exchanger protein as assessed by Western blotting. The antibody did not bind to the high-molecular-weight "rim protein," thereby demonstrating that this protein is distinct from the rod outer segment of Na+/Ca2(+)-exchanger. We used the polyclonal antibody for immunohistochemically localizing the exchange protein in bovine retina. Fluorescent light microscopy revealed intensive immunolabeling of the photoreceptor outer segments, whereas other retinal cell layers exhibited minimal binding. Using the electron microscopic immunogold method, we found specific antibody binding to the extracellular side of rod outer segment plasma membrane. Rod disk membranes, rod inner segments, and cone photoreceptors displayed no significant labeling. We therefore conclude that the Na+/Ca2(+)-exchanger is localized primarily in the rod outer segment plasma membrane, the most appropriate localization considering its proposed role in the process of vertebrate phototransduction.

Interaction of small G proteins with photoexcited rhodopsin

Bovine rod outer segment (ROS) membranes contain in addition to the heterotrimeric G protein transducin, several small GTP-binding proteins (23–27 kDa). Furthermore, these membranes contain two substrate proteins (about 22 and 24 kDa) for botulinum C3 ADP-ribosyltransferase known to ADP-ribosylate small G proteins in any mammalian cell type studied so far. Most interestingly, [ 32P]ADP-ribosylation of ROS membrane small G proteins by C3 is regulated by light and guanine nucleotides in a manner similar to pertussis toxin-catalyzed [ 32P]ADP-ribosylation of the α-subunit of transducin. These findings suggest that not only the heterotrimeric G protein transducin but also the C3 substrate small G proteins present in ROS membranes interact with photoexcited rhodopsin and thus contribute to its signalling action.

Enzymatic Methylation of 23-29-kDa Bovine Retinal Rod Outer Segment Membrane Proteins: Evidence for Methyl Ester formation at Carboxyl-Terminal Cysteinyl Residues

A group of 23-29-kDa polypeptides in the membranes of bovine rod outer segments are substrates for S-adenosylmethionine-dependent methylation reactions. The bulk of the methyl group incorporation is in base-labile ester-like linkages, and does not appear to be due to the widespread D-aspartyl/L-isoaspartyl methyltransferase (EC 2.1.1.77). To determine the site(s) of methylation, 3H-methylated proteins separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate were eluted and digested with papain, leucine aminopeptidase-M, and prolidase. After performic acid oxidation of the digest, a base-labile radioactive material was recovered that coeluted with a synthetic standard of cysteic acid methyl ester upon cation exchange and G-15 gel filtration chromatography, as well as in two thin-layer electrophoresis and two thin-layer chromatography systems. These results provide direct evidence for the methylation of the alpha-carboxyl group of a carboxyl-terminal cysteinyl residue, a modification that has been proposed for the 21-kDa Ha-ras product and other cellular proteins (Clarke, S., Vogel, J. P., Deschenes, R. J., and Stock, J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4643-4647).

Fourier transform infrared spectroscopic investigation of rhodopsin structure and its comparison with bacteriorhodopsin

FT-IR spectroscopy has been used to investigate the conformation of rhodopsin in bovine rod outer segment membranes, dispersed in aqueous suspension in both 2H2O and H2O. Detailed analysis of the amide I band was made, using second-derivative and deconvolution procedures. The frequency of the major amide I component is consistent with the presence of predominantly alpha-helices within the rhodopsin structure. A spectroscopic change occurs at acidic pH with the membranes in both 2H2O and H2O. The results for the membranes dispersed in H2O at pH 7 were used to estimate a value of 0.67 for w (amide II/amide I intensity ratio in H2O). This value of w gives an estimate of the unexchanged amide protons, in rhodopsin, of 51%. The extent of amide proton exchange at acidic p2H (p2H 5 and 2), in 2H2O was also determined. The conformation of rhodopsin in its unbleached and bleached states was investigated but no significant difference in the secondary structure was observed. A comparison, after second-derivative and deconvolution analysis, of the spectra of rhodopsin with that of bacteriorhodopsin shows that both proteins exhibit a similar number of amide I components. However, with bacteriorhodopsin the amide I band occurs at a higher frequency. Bacteriorhodopsin under similar conditions, in 2H2O, has 20% more unexchanged amide protons than does rhodopsin.

Comparison of the phosphodiesterase inhibitory subunit interactions of frog and bovine rod outer segments

The rod outer segments of the bovine and frog retina possess a cyclic GMP phosphodiesterase (PDE) that is composed of two larger subunits, alpha and beta (P alpha beta), which contain the catalytic activity and a smaller gamma (P gamma) subunit which inhibits the catalytic activity. We studied the binding of P gamma to P alpha beta in both the bovine and frog rod outer segment membranes. Analysis of these data indicates that there are two classes of P gamma binding sites per P alpha beta in both species. The activation of PDE by the guanosine 5'-[gamma-thio]triphosphate form of the alpha subunit of transducin, T alpha.GTP gamma S, was also studied. These data indicate that the two classes of P gamma binding sites contribute to the formation of two classes of binding sites for T alpha.GTP gamma S. We demonstrate solubilization of a portion of the P gamma by T alpha.GTP gamma S in both species. There is also present, in both species, a second class of P gamma which is not solubilized even when it is dissociated from its inhibitory site on P alpha beta by T alpha.GTP gamma S. The amount of full PDE activity which results from release of the solubilizable P gamma is about 50% in the frog PDE but only approx. 17% in the bovine PDE. We also show that activation of frog rod outer segment PDE by trypsin treatment releases the PDE from the membranes. This type of release by trypsin has already been demonstrated in bovine rod outer segments [Wensel & Stryer (1986) Proteins: Struct. Funct. Genet. 1, 90-99].

TRANSDUCIN ACTIVATION BY MOLECULAR SPECIES OF RHODOPSIN OTHER THAN METARHODOPSIN II*

Decay of metarhodopsin II was accelerated by hydroxylamine treatment or dark incubation of metarhodopsin II at 30 degrees C. The products thus obtained after decay of metarhodopsin II induced GTPase activity on transducin as well as metarhodopsin II suggesting that rhodopsin could activate transducin after the decay of metarhodopsin II intermediate. After urea-treated bovine rod outer segment membrane was completely bleached, rhodopsin in the membrane was regenerated by the addition of 11-cis retinal at various temperatures between 0 and 37 degrees C. The capacity to induce GTPase activity on transducin and phosphate incorporating capacity catalyzed by rhodopsin kinase were measured on such rhodopsins. The results showed that: (1) Regeneration of alpha band of rhodopsin was complete regardless of regeneration temperature; (2) When regenerated at temperatures below 10 degrees C, rhodopsins induced a GTPase activity on transducin in the dark even after treatment with hydroxylamine, whereas rhodopsins after regeneration at temperatures above 13 degrees C did not; (3) When regenerated at 0 degrees C, rhodopsin was phosphorylated if incubated with rhodopsin kinase and ATP in the dark, whereas the spectrally regenerated rhodopsin at 30 degrees C was not. The complete quenching of functions of photoactivated rhodopsin was achieved by recombination with 11-cis retinal at temperatures above 13 degrees C but not below 10 degrees C suggesting the existence of a low temperature intermediate upon regeneration.