Location of two sulfhydryl groups in the rhodopsin molecule by use of the spin label technique.

Sulfhydryl groups of membrane-bound rhodopsin are studied with the spin label technique by using five maleimide derivative probes of different lengths. Two sulfhydryl groups are titrated per molecule of rhodopsin, These groups are located in protected but probably different environments, less than 12 A away from the aqueous phase. A distance of about 37 A is measured between the two groups. These results are consistent with a model in which the two groups would be located close by the external surface of the protein but embedded within the membrane layer, the strong immobilization of the label molecules resulting from phosphlipid-protein interactions.

Purification and characterization of myosin from bovine retina.

Myosin has been isolated from bovine retinae and characterised by its ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, its mobility in sodium dodecyl sulphate polyacrylamide gels and by electron microscopy. The purified myosin shows high ATPase activity in the presence of EDTA or Ca2+ and a low activity in the presence of Mg2+. The Mg2+-dependent ATPase activity is stimulated by rabbit skeletal muscle actin. The presumptive retinal myosin possesses a major component which has a mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis similar to that of the heavy chain of bovine skeletal muscle myosin. Electron microscopy showed retinal myosin to form bipolar filaments in 0.1 M KCl. It is concluded that the retina possesses a protein with enzymic and structural properties similar to those of muscle myosin.

Interplay between calcium and activated cGMP phosphodiesterase from retinal rod outer segments.

Hypotonic extraction of bovine retinal rod outer segments after bleaching in isotonic buffer yielded an extract exhibiting activated cGMP phosphodiesterase properties. Since this extract was virtually devoid of other proteins involved in the rod outer segment cGMP enzymatic cascade, it was used to study phosphodiesterase catalytic activity. The hypotonic extract required Mg2+ in the range 0.1-1.0 mM for optimal cGMP hydrolysis. At these Mg2+ concentrations hydrolysis could be effectively inhibited by Ca2+ at concentrations which might be attainable in rod outer segments. Since higher Ca2+ concentrations were required to give a chosen degree of inhibition at higher Mg2+ concentrations, this inhibition was probably due to competition by Ca2+ for Mg2+ binding site(s) on the phosphodiesterase catalytic unit. Other divalent cations were also able to inhibit cGMP hydrolysis, many of them (especially those with ionic radii close to that of magnesium) more effectively than calcium. It is suggested that Ca2+ may play a role in phototransduction by participating in the control of photoreceptor sensitivity, and that this is achieved by modulating rod outer segment cGMP hydrolysis.

Cyclic nucleotide phosphodiesterase of retinal photoreceptors. Partial purification and some properties of the enzyme.

1. A cyclic nucleotide phosphodiesterase (EC 3.1.4.16) has been partially purified from bovine rod outer segments. The enzyme preparation obtained has a very high specific activity towards cyclic GMP and is still able to hydrolyze cyclic AMP. Upon polyacrylamide gel electrophoresis, one major and three minor protein bands are seen, the enzyme activity being associated with the major band. The enzyme eluted from the gels still hydrolyzes both cyclic nucleotides. At all substrate concentrations tested, cyclic GMP was hydrolyzed at a faster rate. The enzyme eluted from the gel columns migrated as a single band upon electrophoresis in 0.1% sodium dodecyl sulfate-polyacrylamide gels corresponding to a molecular weight of 105 000. 2. A complex kinetic pattern was observed for cyclic GMP hydrolysis: the plot of velocity vs substrate concentration was hyperbolic at low and sigmoidal at higher concentrations. By contrast, simple kinetics were observed for cyclic AMP hydrolysis yielding an apparent Km of 0.1 mM. The unusual kinetics may be implicated in the regulation of cyclic GMP levels in rod outer segments. 3. Cyclic AMP stimulated the hydrolysis of cyclic GMP at low and inhibited it at higher concentrations. Addition of Mg2+ appeared to be necessary for optimum activity. The activity measured in the absence of exogenous Mg2+ was abolished by EDTA.

Molecular organization of bovine rod cGMP-phosphodiesterase 6.

Phosphodiesterase 6 (PDE6), a multisubunit (alphabetagamma(2)delta) enzyme, plays a major role in visual function by hydrolysing cGMP in response to a light stimulus. Solubilized bovine rod PDE6 molecules depleted of their gamma subunits were purified to homogeneity from bovine retinal rods and their molecular organization was investigated by electron microscopy. Image analysis of single particles revealed the three-dimensional dimeric arrangement of the purified alphabetadelta complex, and the internal organization of each catalytic subunit into three distinct domains at a resolution of 2.8 nm. The relative volume of each domain is consistent with sequence analysis and functional data, which suggest that these domains correspond to the catalytic and two GAF domains. This hypothesis was confirmed by immunolabelling experiments, which located the N-terminal part of the catalytic subunit where the major interaction between the two alphabeta subunits was found to occur. The 3D molecular organization of human platelet PDE5 appears highly homologous to that of bovine rod PDE6, as predicted by similarities in their primary sequences. These observations describe the quaternary organization of the catalytic PDE6 alphabeta complex, and place the catalytic and regulatory domains on a structural model.

Localization of light-induced conformational changes in bovine rhodopsin.

Conformational changes in the extradiscal regions of rhodopsin induced by illumination were investigated by modifying the visual pigment by mild treatment with cyanogen bromide prior to and after light exposure. Light induced an increased yield of cleavage of the Met bond 253-254 and a new cleavage at the Met bond 155-156 of the rhodopsin polypeptide chain. These residues, located at the beginnings of the membrane-buried helices 6 and 4, respectively, were concluded to become extradiscally exposed upon illumination.

Light-induced conformational change in rhodopsin detected by modification of G-protein binding, GTP gamma S binding and cGMP phosphodiesterase activation.

CNBr treatment of rod outer segments was performed in dark and in light conditions. With the subsequent modified rhodopsin and opsin the cGMP phosphodiesterase activation system was reconstituted. The recombination systems exhibited greatly reduced G-protein binding, GTP gamma S binding and cGMP phosphodiesterase activation. The reduction in activity of these three steps of the PDE activation cascade is most significant with modified opsin and is shown to be due to its inability to bind the G alpha subunit. The correlation between the localization of CNBr cleavage in dark and light conditions and these results is strongly indicative that a light-induced conformational change occurs in two extradiscal regions of rhodopsin.

Mono ADP-ribosylation of transducin catalyzed by rod outer segment extract.

Transducin is the retinal rod outer segment (ROS)-specific G protein coupling the photoexcited rhodopsin to cyclic GMP-phosphodiesterase. The alpha subunit of transducin is known to be ADP-ribosylated by bacterial toxins. We investigated the possibility that transducin is modified in vitro by an endogenous ADP-ribosyltransferase activity. By using either ROS, cytosolic extract of ROS or purified transducin in the presence of [alpha-32P]nicotinamide adenine dinucleotide (NAD+), the alpha and beta subunits of transducin were found to be radiolabeled. The labeling was decreased by snake venom phosphodiesterase I (PDE I). The modification was shown to be mono ADP-ribosylation by analyses on thin layer chromatography of the PDE I-hydrolyzed products which revealed only 5'AMP residues. In addition we report that sodium nitroprusside activates the ADP-ribosylation of transducin.

Amino-acid sequence determination of a hydrophobic region of bovine rhodopsin.

The 50 amino acid residues sequence of a hydrophobic region of bovine rhodopsin, a membrane protein of retinal rod photoreceptors of molecular weight 39,000 was determined. This primary structure determination was performed on the S5 fragment (about 12,000 molecular weight) obtained from 2-(2 nitrophenylsulfenyl)-3-methyl-3'bromo-indolenine cleavage of the protein. Automatic Edman degradation used in liquid phase was performed in presence of N-cetyl-N,N,N-trimethyl-ammoniumbromide, a cationic detergent incorporated in the proteic film. S5 is a C-terminal rhodopsin fragment and contains the phosphorylation sites. The covalent structure determined overlaps with the sequence of an already known fragment [1]; thus 25 per cent of the rhodopsin primary structure is now elucidated. Our results are in agreement with and chiefly refine the topological model for rhodopsin which correlates its membrane location and its functional sites.

Chemical cleavage of bovine rhodopsin at tryptophanyl bonds; characterization of the polypeptide fragments and the phosphorylated site.

Bovine rhodopsin from retinal rod photoreceptors, a protein of 39,000 molecular weight, was cleaved by BNPS-Skatole at the level of tryptophanyl bonds. This hydrolysis yields five fragments S1, S2, S3, S4 and S5 (molecular weights: 35,000, 28,000, 19,500, and 15,500 and 12,000, respectively) and four peptides. Large fragments were purified by polyacrylamide gel electrophoresis in SDS. S2, S3 and S4 contain the glycanes of native rhodopsin and their N-termini are blocked. S5 has the same C-terminal extremity as rhodopsin and contains the phosphorylated site. Phosphate groups are incorporated in this fragment on serines and threonines.

Conformational changes of cytosolic loops of bovine rhodopsin during the transition to metarhodopsin-II: an investigation by Fourier transform infrared difference spectroscopy.

In order to assign the structural changes of the protein, observed in the Fourier transform infrared (FT-IR) difference spectra of the rhodopsin-metarhodopsin-II transition, to specific regions of the protein, rhodopsin was treated by proteases. Nonilluminated and bleached rhodopsin was treated with protease K and papain. Rhodopsin digested in the bleached state was subsequently regenerated with 11-cis-retinal. From these modified samples the rhodopsin-metarhodopsin-II FT-IR difference spectra were measured. Comparing the difference spectra with that of unmodified rhodopsin, clear deviations in the amide-I and amide-II spectral range are observed. This indicates that in the unmodified pigment conformational changes of those parts of the cytosolic surface take place which are susceptible to the proteases. From the larger spectral changes obtained with samples digested in the bleached state it is concluded that the extent of modification is larger. The difference spectra of rhodopsin modified with 10 mM dithiothreitol support the existence of the 4th loop which also undergoes conformational changes. The spectral changes are interpreted in terms of a transition of an ordered structure of the loops in rhodopsin to a more random structure in metarhodopsin-II. The results demonstrate that by combining FT-IR spectroscopy with protein modification by specific proteases, conformational changes of the protein can be localized to specific regions.

On a soluble system for studying light activation of rod outer segment cyclic GMP phosphodiesterase.

Bovine rod outer segment (ROS) cyclic GMP phosphodiesterase (PDE) could be activated about 6-fold by light, an effect that could be simulated by isolated bleached rhodopsin. About 90% of PDE activity in ROS could be extracted with 10 mM Tris-HCl, pH 7.5, but light is ineffective in activating the soluble enzyme. However, bleached rhodopsin could activate it in the presence of a very low concentration of ATP, strongly suggesting the mediation of rhodopsin in the light activation of the enzyme in ROS. Direct evidence is presented to suggest that the phosphorylation of opsin (bleached rhodopsin) is unrelated to the activation of PDE by bleached rhodopsin and ATP. The reconstitution of the light activation of PDE in a soluble system presented here opens up a new direction to future investigations on the mechanism of light regulation of cyclic GMP levels in retina and its implication in the photoreceptor function.

Light-stimulated phosphorylation of rhodopsin in the retina: the presence of a protein kinase that is specific for photobleached rhodopsin.

A protein kinase has been extracted from bovine rod outer segments by a mild procedure. The enzyme acts specifically on photobleached, not unbleached, rhodopsin and will not catalyze the phosphorylation of histones, phosvitin, or casein. We propose the name "opsin kinase" for the enzyme, which is not affected by cyclic nucleotides but which is inhibited by theophylline. Preparations of purified rod outer segments, however, appear to contain only low concentration of opsin phosphatase activity.

[Purification and partial sequence of a hydrophobic polypeptide from BNPS-skatole cleaved bovine rhodopsin]. / Purification et séquence partielle d'un polypeptide hydrophobe de la rhodopsine bovine fragmentée par le BNPS-scatole.

Rhodopsin isolated from outer segments of cattle retinas was cleaved at tryptophan residues by the BNPS-skatole. One of the polypeptides obtained S 5 (molecular weight about 12,000) of hydrophobic nature was isolated and the sequence of its 50 first residues revealed 60% of hydrophobic AA.

Light-induced conformational changes in the extradiscal regions of bovine rhodopsin.

Light-induced conformational changes occurring at the cytosolic surface of rhodopsin were investigated by performing limited digestions of native and illuminated visual pigment with thermolysin, Arg-C endoproteinase, papain and proteinase K. A higher susceptibility of the extradiscal regions of the bleached pigment to the proteases were observed together with altered capacities of the digested bleached rhodopsins to activate the cGMP phosphodiesterase. The overall results strongly suggest that light induces conformational changes not only in the C-terminal end but also in the second and the third extradiscal loop of rhodopsin.

Some observations on bipolar filaments formed by non-muscle myosins.

Adrenal medullary and retinal myosins formed bipolar filaments in vitro. These filaments showed features suggesting flexibility in the rod region of the myosin molecules composing such filaments; in certain cases the myosin heads spread away from the filament backbone, in others the backbone itself was twisted. In addition the bare central backbone showed transverse striations.