Protein complement of rod outer segments of the frog retina

Biochemistry ◽  
1986 ◽  
Vol 25 (16) ◽  
pp. 4512-4523 ◽  
Author(s):  
Heidi E. Hamm ◽  
M. Deric Bownds
Keyword(s):  
Rod Outer Segments ◽  
Outer Segments ◽  
Frog Retina ◽  
Protein Complement

scholarly journals Ionic blockage of the light-regulated sodium channels in isolated rod outer segments.

1978 ◽  
Vol 71 (6) ◽  
pp. 657-681 ◽  
Author(s):  
C M Wormington ◽  
R A Cone
Keyword(s):  
Continuous Flow ◽  
High Sensitivity ◽  
Rod Outer Segments ◽  
Na Channels ◽  
Anionic Sites ◽  
Outer Segments ◽  
N2 Atmosphere ◽  
Frog Retina ◽  
Membrane Oxidation ◽  
Ca Ions

We have investigated, with osmotic techniques, the light-regulated Na+ channels in rod outer segments (ROS) and ROS fragments freshly isolated from the frog retina. Values of Na+ permeability (PNa) similar to those observed electrophysiologically in the retina were observed using the osmotic technique (continuous flow) described by Korenbrot and Cone. In the other osmotic techniques that we explored, PNa was greatly diminished, if not completely suppressed; however, we found with these techniques that antioxidant conditions (N2 atmosphere or EDTA) significantly increased PNa, suggesting that the Na+ channels are highly sensitivive to membrane oxidation. Using the continuous flow technique, we investigated the H+ and Ca++ dependence of the Na+ channels and found that both of these ions, at micromolar activities, can block the channels. Raising the external H+ activity decreases PNa (reversibly) in a single "sigmoidal" response with an apparent pKa of 5.8. Similarly, in the presence of the ionophores X537A or A23187 which allow equilibration of Ca++ across membranes, the Na+ channels are blocked when the external Ca++ activity is increased from 10(-7) to 10(-5) M. This high sensitivity to both H+ and Ca++ ions suggests that high field strength anionic sites may exist in or near the Na+ channels and that the channels are blocked when these sites bind H+ or Ca++ ions.

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 Localization of antibody binding sites in ultrathin sections of unembedded frog retinal tissue.

1983 ◽  
Vol 31 (1) ◽  
pp. 29-34 ◽  
Author(s):  
D C Pease ◽  
I Nir ◽  
V Clark ◽  
M Hall
Keyword(s):  
Rabbit Antiserum ◽  
External Support ◽  
Rod Outer Segments ◽  
General Technique ◽  
Rods And Cones ◽  
Retinal Tissue ◽  
Outer Segments ◽  
Frog Retina ◽  
Rabbit Igg ◽  
Ultrathin Sections

Much ultrastructural detail is retained in tissue fixed only with aldehydes and subsequently air-dried after suspension in a polyvinyl acetate emulsion. The latter provides an external support only, but permits ultrathin sectioning; thus, an exposure of intracellular contents for potential immunocytochemical reactions is achieved. Sections of unembedded frog retina so prepared have been studied with success. The tissue was incubated first with a rabbit antiserum prepared against gradient purified bovine rod outer segments. Following incubation, reacted sites were labeled with ferritin-conjugated goat anti-rabbit IgG and stained with phosphotungstic acid. Intense labeling of the rod outer segments was clearly achieved, whereas the cone outer segments were without label. Other parts of the retina, including the ellipsoid region of both rods and cones, were also without significant label. These regions provided an intrinsic control for the specificity of the antiserum and established the validity of the general technique.

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.

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