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

1989 ◽  
Vol 259 (1) ◽  
pp. 13-19 ◽  
Author(s):  
M M Whalen ◽  
M W Bitensky
Keyword(s):  
Catalytic Activity ◽  
Binding Sites ◽  
Outer Segment ◽  
Rod Outer Segments ◽  
Rod Outer Segment ◽  
Outer Segments ◽  
Frog Retina ◽  
Inhibitory Site ◽  
Alpha Beta ◽  
Gamma S

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].

scholarly journals The effect of the γ-subunit of the cyclic GMP phosphodiesterase of bovine and frog (Rana catesbiana) retinal rod outer segments on the kinetic parameters of the enzyme

1990 ◽  
Vol 265 (3) ◽  
pp. 655-658 ◽  
Author(s):  
M M Whalen ◽  
M W Bitensky ◽  
D J Takemoto
Keyword(s):  
Catalytic Activity ◽  
Cyclic Gmp ◽  
Active Sites ◽  
Outer Segment ◽  
Maximal Activity ◽  
Inhibitory Effect ◽  
Gamma Subunit ◽  
Rod Outer Segment ◽  
Gamma Subunits ◽  
Alpha Beta

Rod-outer-segment cyclic GMP phosphodiesterase (PDE) (subunit composition alpha beta gamma 2) contains catalytic activity in alpha beta. The gamma-subunits are inhibitors. Removal of the gamma-subunits increases Vmax. without affecting the Km. The inhibitory effect of a single gamma-subunit (alpha beta gamma) on the Vmax. of alpha beta is much greater in bovine than in frog (Rana catesbiana) PDE. Bovine PDE in the alpha beta gamma 2 state has a Vmax. that is 2.6 +/- 0.4% of the Vmax. of alpha beta. The removal of one gamma-subunit to give alpha beta gamma results in a Vmax. 5.2 +/- 1% of that for maximal activity. Frog alpha beta gamma 2 has a Vmax. 10.8 +/- 2%, and alpha beta gamma has a Vmax. 50 +/- 18%, of the Vmax. of alpha beta. These data suggest that a single gamma-subunit can inhibit the catalytic activity of active sites on both alpha- and beta-subunits in bovine, but not in frog, rod-outer-segment PDE.

scholarly journals Correlation of rhodopsin biogenesis with ultrastructural morphogenesis in the chick retina.

1975 ◽  
Vol 64 (1) ◽  
pp. 235-241 ◽  
Author(s):  
W T Mason ◽  
K J Bighouse
Keyword(s):  
Outer Segment ◽  
Receptor Cell ◽  
Rod Outer Segments ◽  
Membrane Biogenesis ◽  
Rod Outer Segment ◽  
Rods And Cones ◽  
Outer Segments ◽  
Chick Retina ◽  
Disk Membrane ◽  
Detergent Extraction

The developing chick retina from stages 39-45 has been examined by biochemical and electron microscope techniques. The levels of rhodopsin contained in the maturing chick retina were evaluated by detergent extraction and correlated with rod outer segment formation. It was found that the appearance of rhodopsin in significant levels preceded outer segment formation by at least 2 days, thus implying that rhodopsin is synthesized in the receptor cell inner segment and translocated to the outer limb when disk membrane biogenesis occurs. The level of rhodopsin continues to rise as the rod outer segment develops. Development of both rods and cones originates and proceeds most rapidly in the fundus or central region and proceeds toward the periphery. In general, rod outer segments were noted to develop far more rapidly than cone outer segments.

Proton Uptake by Light Induced Interaction between Rhodopsin and G-Protein

1985 ◽  
Vol 40 (5-6) ◽  
pp. 400-405 ◽  
Author(s):  
Andreas Schleicher ◽  
Klaus Peter Hofmann
Keyword(s):  
G Protein ◽  
Outer Segment ◽  
Bromocresol Purple ◽  
Rod Outer Segments ◽  
Rod Outer Segment ◽  
Outer Segments ◽  
Proton Uptake ◽  
Indicator Dye ◽  
Ph Electrode

Abstract The light-induced proton uptake of rod outer segment disc membranes has been investigated in the absence and presence of G-protein. Proton uptake was measured as the alkalisation of the suspending medium using a pH electrode and/or the indicator dye bromocresol purple. It was found that besides the known proton uptake of photolysed rhodopsin additional uptake of one proton accompanies formation of the complex between rhodopsin and G-protein. No measurable proton uptake was found under conditions of rapid redissociation of the complex indicating an only transient protonation during its lifetime. Proton uptake was the same in washed membra­nes recombined with G-protein and in ordinarily stacked rod outer segments. The additional proton uptake reported here is not due to enhanced metarhodopsin II.

scholarly journals Rhodopsin in the rod outer segment plasma membrane.

1976 ◽  
Vol 69 (1) ◽  
pp. 29-42 ◽  
Author(s):  
S Basinger ◽  
D Bok ◽  
M Hall
Keyword(s):  
Plasma Membrane ◽  
Gel Electrophoresis ◽  
Visual Pigment ◽  
Outer Segment ◽  
Gel Filtration ◽  
Acid Mixture ◽  
Rod Outer Segments ◽  
Gel Filtration Chromatography ◽  
Rod Outer Segment ◽  
Outer Segments

Isolated frog retinas were incubated in vitro with a 4-h pulse of [3H]leucine, then chased for 32 h with a nonradioactive amino acid mixture. At the end of the incubation, light and electron microscope autoradiograms were prepared from some of the retinas. The autoradiograms revealed: (a) intense radioactivity in the basal disks of the rod outer segments, (b) diffuse label evenly distributed throughout the rod outer segments, and (c) a high concentration of label in the entire rod outer segment plasma membrane. Incubation under identical conditions, but with puromycin added, significantly inhibited the labeling of all of these components. To identify the labeled proteins, purified outer segments from the remaining retinas were analyzed biochemically by SDS disc gel electrophoresis and gel filtration chromatography. SDS gel electrophoresis showed that about 90% of the total rod outer segment radioactivity chromatographed coincident with visual pigment, suggesting that the radiolabeled protein in the plasma membrane is visual pigment. Gel filtration chromatography demonstrated that the radiolabeled protein co-chromatographed with rhodopsin rather than opsin, and that the newly synthesized visual pigment is both the basal disks and the plasma membrane is present in the native configuration.

scholarly journals Differential effects of protease digestion on photoreceptor lectin binding sites.

1985 ◽  
Vol 33 (7) ◽  
pp. 642-646 ◽  
Author(s):  
J G Wood ◽  
L Napier-Marshall
Keyword(s):  
Binding Sites ◽  
Outer Segment ◽  
Lectin Binding ◽  
Rod Outer Segments ◽  
Proteolytic Digestion ◽  
Experimental Conditions ◽  
Intense Staining ◽  
Outer Segments ◽  
Intracellular Compartments ◽  
Lectin Binding Sites

The use of lectin cytochemistry together with proteolytic digestion techniques to partially characterize lectin binding sites of several intracellular compartments in frog photoreceptors was studied. Uniform access of reagents to all intracellular compartments was obtained by performing the experiments directly on semithin sections of retinal tissue embedded in a hydrophilic plastic resin. Protease pretreatment of sections of Xenopus laevis eyecup leads to a loss of wheat germ agglutinin (WGA) binding sites from most of the rod outer segment. Under experimental conditions used here, cone outer segment WGA binding sites are resistant to proteolytic digestion. Another major difference between rod and cone under segments is that rod outer segments are heavily labeled with succinylated WGA, whereas cone outer segments are barely labeled except for a region of intense staining thought to be at the connecting cilium. WGA binding sites in the shed outer segment tip (phagosome) are also relatively resistant to proteolytic digestion, as is the tip region of a few rod outer segments. This difference in lectin binding properties between the bulk of the outer segment membrane and the shed outer segment membrane is the only distinction we have observed between the two compartments in terms of their glycoconjugates. These results may be useful in terms of designing experiments to isolate cone and rod outer segments separately. They indicate that a change in outer segment glycoconjugates may accompany the shedding and phagocytosis events, as previously suggested, but this change does not necessarily involve the addition of saccharides to outer segment glycoproteins.

scholarly journals Preparation and characterization of monoclonal antibodies to several frog rod outer segment proteins.

1984 ◽  
Vol 84 (2) ◽  
pp. 251-263 ◽  
Author(s):  
P L Witt ◽  
H E Hamm ◽  
M D Bownds
Keyword(s):  
Monoclonal Antibodies ◽  
G Protein ◽  
Protein Fraction ◽  
Outer Segment ◽  
Solid Phase ◽  
Detection System ◽  
Rod Outer Segments ◽  
Western Blots ◽  
Rod Outer Segment ◽  
Outer Segments

Monoclonal antibodies to proteins important in phototransduction in the frog rod outer segment have been obtained. These include 6 different antibodies to rhodopsin, 50 to a guanine nucleotide binding protein (G-protein; 40,000 daltons), and 2 to cytoplasmic proteins. The antigens used were Percoll-purified rod outer segments, a rod outer segment soluble protein fraction, or a soluble plus peripheral membrane protein fraction. Antibodies were assayed by solid phase assay using a fluorogenic detection system. Proteins to which antibodies bound were assayed on Western blots, and the sensitivities of three different detection systems were compared. Most antibodies bound to only one rod outer segment protein band on Western blots. Immunofluorescence microscopy demonstrated binding of both anti-rhodopsin and anti-G-protein to isolated frog rod outer segments. Antibodies were purified from either culture supernatants or ascites fluid on protein A affinity columns. Two purified anti-G-protein antibodies have binding affinities to 125I-labeled G-protein of less than 10(-6) M-1. Of 11 antibodies to frog or bovine G-protein tested in solid phase and Western blot assays, all bind to the alpha rather than the beta or gamma subunits. Procedures developed here are being used in preparing other antibodies that affect reactions in the phototransduction pathway.

scholarly journals ULTRASTRUCTURAL LOCALIZATION OF RHODOPSIN IN THE VERTEBRATE RETINA

1974 ◽  
Vol 62 (2) ◽  
pp. 257-273 ◽  
Author(s):  
Lily Yeh Jan ◽  
Jean-Paul Revel
Keyword(s):  
Plasma Membrane ◽  
Outer Segment ◽  
Sodium Dodecyl ◽  
Ultrastructural Localization ◽  
Rod Outer Segments ◽  
Membrane Particles ◽  
Outer Segments ◽  
Bovine Rhodopsin ◽  
Frog Retina ◽  
Extreme Care

Early work by Dewey and collaborators has shown the distribution of rhodopsin in the frog retina. We have repeated these experiments on cow and mouse eyes using antibodies specific to rhodopsin alone. Bovine rhodopsin in emulphogene was purified on an hydroxyapatite column. The purity of this reagent was established by spectrophotometric criteria, by sodium dodecyl sulfate (SDS) gel electrophoresis, and by isoelectric focusing. This rhodopsin was used as an immunoadsorbent to isolate specific antibodies from the antisera of rabbits immunized with bovine rod outer segments solubilized in 2% digitonin. The antibody so prepared was shown by immunoelectrophoresis to be in the IgG class and did not cross-react with lipid extracts of bovine rod outer segments. Papain-digested univalent antibodies (Fab) coupled with peroxidase were used to label rhodopsin in formaldehyde-fixed bovine and murine retinas. In addition to the disk membranes, the plasma membrane of the outer segment, the connecting cilium, and part of the rod inner segment membrane were labeled. We observed staining on both sides of the rod outer segment plasma membrane and the disk membrane. Discrepancies were observed between results of immunolabeling experiments and observations of membrane particles seen in freeze-cleaved specimens. Our experiments indicate that the distribution of membrane particles in freeze cleaving experiments reflects the distribution of membrane proteins. Immunolabeling, on the other hand, can introduce several different types of artifact, unless controlled with extreme care.

scholarly journals Protein kinase C in rod outer segments: effects of phosphorylation of the phosphodiesterase inhibitory subunit

1996 ◽  
Vol 317 (1) ◽  
pp. 291-295 ◽  
Author(s):  
Igor P. UDOVICHENKO ◽  
Jess CUNNICK ◽  
Karen GONZALEZ ◽  
Alexander YAKHNIN ◽  
Dolores J. TAKEMOTO
Keyword(s):  
Protein Kinase C ◽  
Catalytic Activity ◽  
Protein Kinase ◽  
Direct Interaction ◽  
Inhibitory Effect ◽  
Rod Outer Segments ◽  
Visual Transduction ◽  
Gtp Hydrolysis ◽  
Outer Segments ◽  
Kinase C

The inhibitory subunit (PDEγ) of the cGMP phosphodiesterase (PDEαβγ2) in rod outer segments (ROS) realizes its regulatory role in phototransduction by inhibition of PDEαβ catalytic activity. The photoreceptor G-protein, transducin, serves as a transducer from the receptor (rhodopsin) to the effector (PDE) and eliminates the inhibitory effect of PDEγ by direct interaction with PDEγ. Our previous study [Udovichenko, Cunnick, Gonzalez and Takemoto (1994) J. Biol. Chem. 269, 9850–9856] has shown that PDEγ is a substrate for protein kinase C (PKC) from ROS and that phosphorylation by PKC increases the ability of PDEγ to inhibit PDEαβ catalytic activity. Here we report that transducin is less effective in activation of PDEαβ(γp)2 (a complex of PDEαβ with phosphorylated PDEγ, PDEγp) than PDEαβγ2. PDEγp also increases the rate constant of GTP hydrolysis of transducin (from 0.16 s-1 for non-phosphorylated PDEγ to 0.21 s-1 for PDEγp). These data suggest that phosphorylation of the inhibitory subunit of PDE by PKC may regulate the visual transduction cascade by decreasing the photoresponse.

scholarly journals MEMBRANE CHARACTERISTICS AND OSMOTIC BEHAVIOR OF ISOLATED ROD OUTER SEGMENTS

1973 ◽  
Vol 56 (2) ◽  
pp. 389-398 ◽  
Author(s):  
Juan I. Korenbrot ◽  
Dennis T. Brown ◽  
Richard A. Cone
Keyword(s):  
Plasma Membrane ◽  
Outer Segment ◽  
Receptor Cell ◽  
Water Flux ◽  
Rod Outer Segments ◽  
Repeat Distance ◽  
Permeability Constant ◽  
Outer Segments ◽  
Length Reduction ◽  
Osmotic Behavior

Freshly isolated frog rod outer segments are sensitive osmometers which retain their photosensitivity; their osmotic behavior reveals essentially the same light-sensitive Na+ influx observed electrophysiologically in the intact receptor cell. Using appropriate osmotic conditions we have examined freeze-etch replicas of freshly isolated outer segments to identify the membrane which regulates the flow of water and ions. Under isosmotic conditions we find that the disc to disc repeat distance is almost exactly twice the thickness of a disc. This ratio appears to be the same in a variety of vertebrate rod outer segments and can be reliably measured in freeze-etch images. Under all our osmotic conditions the discs appear nearly collapsed. However, when the length of the outer segment is reduced by hyperosmotic shocks the discs move closer together. This markedly reduces the ratio of repeat distance to disc thickness since disc thickness remains essentially constant. Thus, the length reduction of isolated outer segments after hyperosmotic shocks primarily results from reduction of the extradisc volume. Since the discs are free floating and since they undergo negligibly small changes in volume, the plasma membrane alone must be primarily responsible for regulating the water flux and the light-sensitive Na+ influx in freshly isolated outer segments. On this basis we calculate, from the osmotic behavior, that the plasma membrane of frog rod outer segment has a Na+ permeability constant of about 2.8 x 10-6 cm/s and an osmotic permeability coefficient of greater than 2 x 10-3 cm/s.

Simple sugars inhibit rod outer segment disc shedding by the frog retina

1983 ◽  
Vol 23 (12) ◽  
pp. 1371-1377 ◽  
Author(s):  
Ashley R. Heath ◽  
Scott F. Basinger
Keyword(s):  
Outer Segment ◽  
Rod Outer Segment ◽  
Frog Retina
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