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R S Molday

Publications and source records attributed to R S Molday.

At least 73 records · Page 4Linked to original sources
PubMed1988

Evidence against the role of rhodopsin in rod outer segment binding to RPE cells.

The possible role of rhodopsin in the binding and phagocytosis of rod outer segments (ROS) by cultured bovine retinal pigment epithelial (RPE) cells was studied using both quantitative phagocytosis assays and electron microscopy. In inhibition studies an immunoaffinity purified 2-39 N-terminal rhodopsin glycopeptide, a synthetic 1-16 peptide analogue of rhodopsin and purified, unsealed ROS disc membranes were found to be ineffective in inhibiting the binding of 125I-labeled ROS to RPE cells. A two-fold excess of unlabeled intact ROS, however, inhibited 125I-labeled ROS binding to RPE cells by over 40%. In another series of experiments, rhodopsin on the surface of fixed ROS was densely labeled with gold-dextran particles conjugated to an N-terminal-specific (rho 4D2) rhodopsin monoclonal antibody or its F(ab')2 fragment in an effort to block binding and phagocytosis by RPE cells. As visualized by both transmission and scanning electron microscopy using secondary and backscatter electron imaging, these antibody-gold-dextran-labeled ROS were effectively phagocytized by RPE cells. These results provide compelling evidence that rhodopsin in the ROS plasma membrane does not function as the ligand for recognition by RPE cells.

Animals↗
PubMed1987

Glycoproteins specific for the retinal rod outer segment plasma membrane.

Two ricin-specific glycoproteins have been identified on neuraminidase-treated rod outer segment plasma membranes of bovine retinal photoreceptor cells. Ricin-gold-dextran particles were observed by electron microscopy to densely label the surface of neuraminidase-treated rod outer segments. Western blotting of proteins separated by SDS-gel electrophoresis indicated that two ricin-binding glycoproteins of Mr 230,000 and 110,000 are specific for the plasma membrane and are not found in disk membranes. These glycoproteins can serve as specific probes for the purification of the rod outer segment plasma membrane.

Animals↗
PubMed1987

Expression of a synthetic bovine rhodopsin gene in monkey kidney cells.

We report here the high-level expression of a synthetic gene for bovine rhodopsin in transfected monkey kidney COS-1 cells. Rhodopsin is produced in these cells to a level of 0.3% of the cell protein, and it binds exogenously added 11-cis-retinal to generate the characteristic rhodopsin absorption spectrum. We describe a one-step immunoaffinity procedure for purification of the rhodopsin essentially to homogeneity. The COS-1 cell rhodopsin activates the GTPase activity of bovine transducin in a light-dependent manner with the same specific activity as that of purified bovine rhodopsin. Electron microscopy of immunogold-stained cells indicates that rhodopsin is located in the plasma membrane of the transfected cells and is oriented with the amino terminus on the extracellular side of the membrane. This orientation is analogous to that of rhodopsin in the disk membranes of photoreceptor cells in the bovine retina.

Animals↗
PubMed1987

Differences in the protein composition of bovine retinal rod outer segment disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method.

The plasma membrane and disk membranes of bovine retinal rod outer segments (ROS) have been purified by a novel density-gradient perturbation method for analysis of their protein compositions. Purified ROS were treated with neuraminidase to expose galactose residues on plasma membrane-specific glycoproteins and labeled with ricin-gold-dextran particles. After the ROS were lysed in hypotonic buffer, the plasma membrane was dissociated from the disks by either mild trypsin digestion or prolonged exposure to low ionic strength buffer. The dense ricin-gold-dextran-labeled plasma membrane was separated from disks by sucrose gradient centrifugation. Electron microscopy was used to follow this fractionation procedure. The dense red pellet primarily consisted of inverted plasma membrane vesicles containing gold particles; the membrane fraction of density 1.13 g/cc consisted of unlabeled intact disks and vesicles. Ricin-binding studies indicated that the plasma membrane from trypsin-treated ROS was purified between 10-15-fold. The protein composition of plasma membranes and disks was significantly different as analyzed by SDS gels and Western blots labeled with lectins and monoclonal antibodies. ROS plasma membrane exhibited three major proteins of 36 (rhodopsin), 38, and 52 kD, three ricin-binding glycoproteins of 230, 160, and 110 kD, and numerous minor proteins in the range of 14-270 kD. In disk membranes rhodopsin appeared as the only major protein. A 220-kD concanavalin A-binding glycoprotein and peripherin, a rim-specific protein, were also present along with minor proteins of 43 and 57-63 kD. Radioimmune assays indicated that the ROS plasma membrane contained about half as much rhodopsin as disk membranes.

Animals↗
PubMed1987

Peripherin. A rim-specific membrane protein of rod outer segment discs.

Monoclonal antibodies were used with radioimmune assays and immunocytochemical techniques to identify and localize membrane proteins in bovine rod outer segment (ROS) disc membranes. When ROS membrane proteins were separated by SDS-polyacrylamide gel electrophoresis in the presence of the sulfhydryl reducing agent, 2-mercaptoethanol, two monoclonal antibodies designated as 3B6 and 2B6 were found to bind to a polypeptide having an apparent molecular weight (Mr) of 33,000 daltons. In the absence of 2-mercaptoethanol, these monoclonal antibodies bound to a doublet having Mr of 67,000 and 69,000. Immunogold-dextran labeling of ROS sections embedded in Lowicryl resin indicated that this protein is localized around the periphery of the ROS organelle where the discs come in close contact to the ROS plasma membrane. Immunogold labeling of morphologically intact isolated discs prepared by mild trypsinization of ROS fragments confirmed that this disc membrane protein is localized along the rim region of discs. On the basis of these localization studies, the authors have named this protein peripherin. Immunogold-dextran markers were also used with previously characterized antirhodopsin monoclonal antibodies to visualize the distribution of rhodopsin on isolated discs. Dense labeling was observed along the lamellar region of the discs, but little if any labeling was observed on the extreme edges of the discs. These results are consistent with the view that the lamellar region of discs containing rhodopsin is a distinct membrane domain from the rim region which contains peripherin, a high Mr rim protein and possibly other proteins involved in disc-disc and disc-plasma membrane interactions.

Animals↗
PubMed1986

A spectrin-like protein in retinal rod outer segments.

Biochemical and immunochemical studies indicate that rod outer segments (ROS) of bovine photoreceptor cells contain a Mr 240,000 polypeptide related to the alpha-subunit of red blood cell (RBC) spectrin. With the use of sodium dodecyl sulfate gel electrophoresis in conjunction with the immunoblotting technique, monoclonal antibody 4B2 was found to bind to a Mr 240,000 polypeptide in ROS that is distinct from the prominent Mr 220,000 concanavalin A binding glycoprotein. The Mr 240,000 polypeptide is highly susceptible to degradation by endogenous proteases. It does not appear to be an integral membrane protein but is tightly membrane associated since it can be partially extracted from ROS membranes with urea in the absence of detergent. The 4B2 antibody cross-reacted with RBC ghosts and bovine brain microsomal membranes. Radioimmune assays and immunoblotting analysis of purified bovine RBC spectrin further revealed that the 4B2 antibody predominantly labeled the alpha-chain of RBC spectrin having an apparent molecular weight of 240,000. Polyclonal anti-spectrin antibody that bound to both the alpha- and beta-chain of RBC spectrin predominantly labeled a Mr 240,000 polypeptide of ROS membranes. Two faintly labeled bands in the molecular weight range of 210,000-220,000 were also observed. These components may represent variants of the beta-chain of spectrin that are weakly cross-reacting or present in smaller quantities than the alpha-chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Differential immunogold-dextran labeling of bovine and frog rod and cone cells using monoclonal antibodies against bovine rhodopsin.

Eleven monoclonal antibodies against different segments of bovine rhodopsin were used with immunogold-dextran markers to label Lowicryl thin sections of bovine and frog retinal photoreceptor cells for visualization by transmission electron microscopy. Antibodies against the C-terminus, F1-F2 loop and N-terminus of rhodopsin were all observed to label bovine rod outer segments (ROS) densely, but to label rod inner segments (RIS) only sparsely. Most antibodies bound 200-600 gold particles per micron2 in the ROS, 10-60 gold particles per micron2 in the RIS and 5-20 particles per micron2 on the Lowicryl resin. One antibody against the N-terminus and one antibody against the C-terminus resulted in the binding of over 1000 particles per micron2 in bovine ROS. Cone outer segments (COS) were labeled with only one antibody, rho 3D6, having a specificity for the 1'-4' C-terminus of bovine rhodopsin. Ninety per cent of the COS were observed to be labeled with this antibody. Immunogold-dextran labeling was also used to study the cross-reactivity of these antibodies to rhodopsin in red and green frog ROS and COS. Monoclonal antibodies directed against sites along the F1-F2 loop, and the N-terminus labeled red frog ROS densely, but did not label either green ROS or COS. Three C-terminal specific antibodies against binding sites along the 1'-8' segment labeled both green and red ROS, but a higher extent of labeling was observed on the green ROS. Antibody rho 3D6, which bound to bovine COS, also labeled frog COS. These results indicate that the F1-F2 loop and segments along to the N-terminus and the C-terminus of bovine rhodopsin show a high degree of homology with corresponding regions of frog rhodopsin from red ROS; the C-terminal 1'-8' segment of bovine rhodopsin is closely related to the corresponding segment of frog rhodopsin from green ROS; and the 1'-4' C-terminus of bovine rhodopsin is highly homologous to segments of opsin from most bovine and frog COS. The labeling of frog red ROS in relation to multiple forms of rhodopsin observed by SDS-gel electrophoresis is discussed.

Animals↗
PubMed1985

Inhibition of monoclonal antibody binding and proteolysis by light-induced phosphorylation of rhodopsin.

Light-induced phosphorylation of rhodopsin in bovine rod outer segment disk membranes inhibits the binding of three carboxyl-terminal-specific anti-rhodopsin antibodies and the cleavage of the carboxyl-terminal region of rhodopsin by trypsin and Staphylococcus aureus V-8 protease. Two monoclonal antibodies, rho 3A6 and rho 1C5, which previously have been shown to preferentially bind to the 8'-12' and the 9'-18' carboxyl-terminal segments of rhodopsin, respectively, are both highly sensitive to phosphorylation. When an average of one phosphate is incorporated per rhodopsin, the binding reactivity of rhodopsin for these antibodies decreases to 30% that of nonphosphorylated rhodopsin as measured in radioimmune competition assays. Reactivity of the rho 1D4 antibody whose primary binding site is localized in the 1'-8' C-terminal segment of rhodopsin is unaffected at this level of phosphorylation but decreases to 30% when three phosphates on average are incorporated per rhodopsin. Direct binding studies using 125I-labeled antibodies indicate that phosphorylation of rhodopsin decreases the maximum extent of rho 3A6 and rho 1C5 binding to rhodopsin. For rho 1D4, the maximum extent of binding is unaffected by phosphorylation, but the dissociation constant is increased by 10-fold. Phosphorylation of rhodopsin also inhibits cleavage of the 1'-9' and 1'-7' carboxyl-terminal peptides by trypsin and S. aureus V-8 protease, respectively. When an average of one phosphate per rhodopsin is incorporated, cleavage decreases to 40% that of nonphosphorylated rhodopsin as measured by high-performance liquid chromatography. Phosphorylation of rhodopsin had no effect on S. aureus cleavage of rhodopsin into the F1 (Mr 25 000) and F2 (Mr 12 000) fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Localization of lectin receptors on bovine photoreceptor cells using dextran-gold markers.

Using a newly developed dextran-gold particle preparation combined with both pre- and post-embedding labeling techniques, three lectins were investigated for their surface and intracellular binding patterns in bovine retinal photoreceptor cells. Concanavalin A staining revealed heavy labeling of both the inner and outer segment plasma membranes (preembedded labeling), and intense labeling of the outer segment with lower levels in the inner segment (postembedded labeling). Rods and cones had similar patterns. Ricinus communis agglutinin-120 revealed only sparse to moderate labeling of the rod inner and outer segments (preembedded labeling). Much heavier labeling was apparent on the cone outer and inner segments. Direct labeling of thin sections revealed generally sparse labeling of inner and outer segments, but there was a noticeably greater intensity of binding in the basal region of rod outer segments. Wheat germ agglutinin revealed heavy labeling of rod and cone outer segments, with much reduced binding in the corresponding inner segments (preembedded labeling). A similar pattern was visible in directly labeled thin sectioned tissue, with the additional appearance of heavy binding to the proximal region of the connecting cilium. This latter binding is suggestive of a specialized connecting cilium region, possibly involved in regulation of rhodopsin transport to or prevention of backflow from the outer segment.

Animals↗
PubMed1984

Localization of binding sites for carboxyl terminal specific anti-rhodopsin monoclonal antibodies using synthetic peptides.

The binding sites for four monoclonal antibodies, rho 1D4, rho 3C2, rho 3A6, and rho 1C5, have been localized within the C-terminal region of bovine rhodopsin: Asp18'-Glu-Ala16'-Ser-Thr-Thr-Val12'-Ser-Lys-Thr-Gl u8'-Thr-Ser-Gln-Val4'-Ala-Pr o -Ala1'. Antibody binding sites were localized by using synthetic C-terminal peptides in conjunction with solid-phase competitive inhibition assays and limited proteolytic digestion of rhodopsin in conjunction with electrophoretic immunoblotting techniques. Binding of the rho 1D4 and rho 3C2 antibodies to immobilized rhodopsin was inhibited with peptides of length 1'-8' and longer. Antibody rho 1D4 binding was not inhibited by peptides 2'-13' or 3'-18', indicating that the C-terminal alanine residue of rhodopsin was required. Similar competitive inhibition studies indicated that the antibody rho 3A6 required peptides of length 1'-12' and longer whereas rho 1C5 required peptide 1'-18'. Peptide 3'-18' was as effective as 1'-18' in inhibiting rho 3A6 binding to rhodopsin, but replacement of glutamic acid in position 8' with glutamine abolished competition. This substitution had little effect on the binding of antibody rho 1C5. Thus, Glu8' was essential for rho 3A6 binding but not for the binding of the rho 1C5 antibody. Cleavage of the seven amino acid C-terminus from rhodopsin and further cleavage to F1 (Mr 25 000) and F2 (Mr 12 000) fragments with Staphylococcus aureus V8 protease abolished binding of rho 1D4 antibody to the membrane-bound rhodopsin fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1984

Separation of cells labeled with immunospecific iron dextran microspheres using high gradient magnetic chromatography.

Immunospecific magnetic microspheres, consisting of ferromagnetic iron dextran conjugated to Protein A, were used to specifically label red blood cells (RBC) for cell separation studies using high gradient magnetic chromatography ( HGMC ). When 10(7)-10(8) RBC labeled with Protein A-iron dextran microspheres were applied to a column containing 30 mg stainless steel wire placed in a 7.5 kilogauss magnetic field, 96 +/- 2% of the cells were retained in the column. These cells could be eluted by removing the magnetic field and mechanically agitating the column. The retention of labeled cells by HGMC was shown to be dependent on the applied magnetic field and the amount of wire packed into the column. HGMC in conjunction with cell labeling with immunospecific iron dextran microspheres have useful applications for the separation of specific cell types.

Cell Separation↗
PubMed1983

Interaction of Escherichia coli F1-ATPase with dicyclohexylcarbodiimide-binding polypeptide.

Antibody raised against the N,N'-dicyclohexylcarbodiimide (DCCD)-binding polypeptide of Escherichia coli bound to the cytoplasmic surface of the cell membrane. A weak reaction was seen with everted vesicles of the thermophile PS3. Rat-liver mitochondrial membranes did not react with the antibody. Reaction of the isolated DCCD-binding polypeptide with the antibody was prevented by oxidation of methionine residues or cleavage of the polypeptide with cyanogen bromide. Modification of the arginine residues of the DCCD-binding polypeptide did not affect interaction with the antibody. Purified F1-ATPase of E. coli bound to the isolated DCCD-binding polypeptide as shown by solid-phase radioimmune assay. Binding involved the alpha and/or beta subunits of F1 and the arginine residues of the polar central region of the DCCD-binding polypeptide. Our results are consistent with a looped arrangement of the DCCD-binding polypeptide in the membrane in which the carboxyl- and amino-terminal regions of the molecule are at the periplasmic surface and the polar central region, interacting with F1, is at the cytoplasmic surface of the cell membrane.

Amino Acid Sequence↗
PubMed1982

Immunospecific ferromagnetic iron-dextran reagents for the labeling and magnetic separation of cells.

Ferromagnetic iron dextran particles were prepared by reacting a mixture of ferrous chloride and ferric chloride with dextran polymers under alkaline conditions. Particles purified by gel filtration chromatography were in the size range of 30-40 nm, had an electron dense core of about 15 nm, were stable against aggregation in physiological buffer, showed little non-specific binding to cells and had a magnetic moment. Protein A from Staphylococcus aureus was covalently coupled to periodate-oxidized ferromagnetic iron-dextran particles. These conjugates were used to indirectly label antigen sites on human red blood cells and thymocytes for visualization by scanning and transmission electron microscopy. Cells labeled with these immunospecific ferromagnetic particles are were quantitatively retained by a simple permanent magnet and could be separated from unlabeled cells. Applications of these novel reagents in the separation of cells, cell membranes and receptors in drug targeting studies are discussed.

Animals↗
PubMed1982

Organization of rhodopsin and a high molecular weight glycoprotein in rod photoreceptor disc membranes using monoclonal antibodies.

Four monoclonal antibodies obtained from the fusion of mouse myeloma cells with lymphocytes of mice immunized with bovine rod outer segment disc membranes were shown to bind to the surface of sealed discs. Radioimmune labeling of rod outer segment membrane proteins separated by sodium dodecyl sulfate gel electrophoresis indicated that two monoclonal antibodies (3D6 and 4B4) were against rhodopsin. Limited proteolysis of rod outer segment membranes with trypsin and Streptomyces griseus protease indicated that the 3D6 antibody bound to the trypsin-sensitive region close to the carboxyl-terminal end of rhodopsin. The 4B4 antibody bound at a trypsin insensitive, but S. griseus protease-sensitive internal region of rhodopsin accessible on the cytoplasmic surface of discs. Two other monoclonal antibodies (3D12 and 4B2) were found to bind to different regions of the Mr = 220,000 concanavalin A binding glycoprotein of rod outer segment disc membranes. Proteolysis studies indicated that these antibodies also bound to a Mr = 140,000 fragment which does not contain the concanavalin A binding site. Immunoferritin-labeling studies for transmission electron microscopy confirm the location of the 3D6 and 4B2 antigens on the cytoplasmic or interdisc surface of disc membranes.

Animals↗
PubMed1981

Analysis of lectin-specific cell surface glycoprotein on neuroblastoma cells.

The binding sites for the lectins wheat germ agglutinin, Ricinus communis agglutinin and concanavalin A on mouse neuroblastoma cell membranes were identified using SDS-gel electrophoresis in combination with fluorescent lectins. Ricinus communis agglutinin and wheat germ agglutinin were found to bind almost exclusively to a single polypeptide with an apparent molecular weight of 30 000. Concanavalin A labeled over 20 different polypeptides, most with molecular weights greater than 50 000. However, when the neuroblastoma cells were treated with concanavalin A so as to internalize all the concanavalin A binding sites visible at the level of the fluorescent microscope and the purified plasma membranes analyzed for their concanavalin A binding polypeptides, only four of the 20 glycopolypeptides were missing or significantly reduced in amount. Thus, these four high molecular weight concanavalin A-binding polypeptides appear to be the major cell surface receptors for concanavalin A. Binding studies with iodinated concanavalin A indicated that these polypeptides represented the high affinity concanavalin A binding sites (Kd = 2 . 10(-7) M). Low affinity concanavalin A binding sites were present on the cell surface after internalization of high affinity concanavalin A binding sites.

Animals↗