James J. Plantner

Enzymatic deglycosylation of bovine rhodopsin.

We have investigated the action of three endo N-acetylglucosaminidases on rhodopsin. The oligosaccharide chains of native and denatured opsin and rhodopsin, both solubilized and membrane-bound, were shown to be cleaved by endohexosaminidase H, endohexosaminidase F, and peptide-N-glycosidase F (PNGase F) as revealed by SDS-PAGE. These enzymes were shown to be free of protease activity. Under correct conditions, the endoglycosidases could release one or both carbohydrate chains. Rhodopsin and opsin at concentrations between 2 and 65 nmol ml-1 were cleaved, with more complete deglycosylation occurring at the higher concentrations.

Effect of tunicamycin on the glycosylation of rhodopsin.

Abstract The incorporation of [3H]glucosamine, [3H]mannose, and [35S]methionine into rhodopsin was investigated in retinas which had been incubated in the presence and absence of the antibiotic, tunicamycin. In its presence, the incorporation of glucosamine was inhibited 70% and mannose, 96% compared to controls. In the presence of tunicamycin the attachment of glucosamine to core-region sites was virtually eliminated. The formation of unglycosylated rhodopsin was also indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and concanavalin A-Sepharose chromatography. These findings are consistent with the participation of the lipid-linked pathway in the glycosylation of this well-characterized intrinsic glycoprotein of the membranes of the disk of the rod outer segment. As indicated by the incorporation of [35S]methionine, the synthesis of rhodopsin apoprotein was inhibited by a much lesser amount. This suggests that the glycosylation of rhodopsin is not required for its insertion into the disk membrane.

A microassay for proteolytic activity

A quantitative procedure for measuring proteolytic activity, utilizing azoalbumin as substrate, has been developed for use in microtiter plates. An enzyme-linked immunosorbent assay reader is used to measure absorbance. The procedure is sensitive, as well as being both rapid and economical. It is particularly convenient for measuring large numbers of samples, such as fractions from column chromatography.

Improved, rapid radioimmunoassay for rhodopsin

An improved radioimmunoassay for rhodopsin has been devised using [125I]rhodopsin, antibodyto purified bovine rhodopsin and protein A-bearing Staphylococcus aureus, serving as a solid phase immunoadsorbent in place of a second antibody. The method retains the high degree of sensitivity of the previous method (Lentrichia, Plantner and Kean, 1980), while being much more rapid and economical.

The rhodopsin content of the human eye

The content of rhodopsin in the human retina was determined using a radioimmunoassay which measures both rhodopsin and opsin. A value of 0.5 nmole of rod visual pigment per mg of extractable protein was obtained, corresponding to 3.94 +/- 0.17 (mean +/- SEM, n = 42) nmole per eye. This value is slightly higher than those previously reported which were based on spectral determinations measuring only native rhodopsin. No significant difference was seen in the population studied with respect to age or sex. Human rhodopsin was partially purified by affinity chromatography on concanavalin A, and some of its properties were studied. A comparison of the immunological reactivity of human, bovine, rat and chicken rhodopsin indicated similarity among the three mammalian species, while the chicken was some 50 fold less reactive.

Radioimmunoassay for rhodopsin

Abstract A radioimmunoassay for rhodopsin was devised using [125I]rhodopsin and antibody to purified bovine rhodopsin in a double antibody procedure that can quantify the concentration of this visual pigment when present in picomolar amounts. The method demonstrated a high degree of sensitivity, reproducibility, and specificity, and is applicable to the determination of the rhodopsin concentration in crude extracts of tissue as well as in more purified preparations.

On the existence of several isoelectric forms of bovine rhodopsin

Abstract Isoelectric focusing studies carried out on purified bovine rhodopsin revealed the presence of several forms which differed in their isoelectric points. Three major species of rhodopsin were detected having isoelectric points of 4·99, 5·33 and 5·91. Upon bleaching, only one form of opsin was generated which had an isoelectric point of 5·22.

Studies of mucin-type glycoproteins: Olefinic amino acids, products of the β-elimination reaction

Abstract The carbohydrate moieties of mucin-type glycoproteins are attached to the polypeptide core by O -glycosyl linkages of N -acetylgalactosamine to the hydroxyl groups of both serine and threonine. Alkali catalyzes the release of the carbohydrate groups by a β-elimination reaction. Studies reported here on the relative rates of the β-elimination reaction have demonstrated that serine-linked glycosides are released more rapidly than threonine-linked glycosides. The ratio of glycosylated serine to glycosylated threonine can readily be determined after β-elimination. The olefinic amino acids, which are products of the β-elimination reaction, are converted into pyruvate and α-ketobutyrate by acid hydrolysis and then are assayed spectrophotometrically with lactate dehydrogenase. In addition, the olefinic amino acids formed upon alkali treatment of pig submaxillary mucin have been further identified by preparation of the dinitrophenylhydrazone derivatives of the α-keto acids and their conversion to α-amino acids by catalytic hydrogenolysis.

The influence of carbohydrates on the binding of rod outer-segment (ROS) disc membranes and intact ROS by the cells of the retinal pigment epithelium of the embryonic chick*

The role of carbohydrates in mediating the interaction of rhodopsin-containing membranes with retinal pigment epithelium (RPE) cells was investigated by studying the influence of various monosaccharides on their binding by RPE cells of the embryonic chick maintained in cell culture. Rod outer-segment (ROS) disc membranes were selected as a model rhodopsin-containing membrane system for these studies in view of their high concentration of rhodopsin and the relative purity with which they can be isolated. Disc membranes, frozen and thawed in order to expose the carbohydrate groups of rhodopsin which are oriented intraluminally in situ, were incubated with monolayers of RPE cells under various conditions, and the binding of the membranes by the cells was quantitated by radioimmunoassay for rhodopsin. Cell-membrane association was also verified by indirect immunofluorescence microscopy. The surface accessibility of the sugars in frozen-thawed discs was verified by succinyl concanavalin A-binding studies. From 15- to 20-fold increase in carbohydrate-reactive sites was obtained after freezing and thawing the discs. The RPE cell-membrane binding process was saturable, and time- and temperature-dependent. By means of competition studies carried out in the presence of high concentrations of various monosaccharides, and also by comparing the binding of disc membranes whose carbohydrate groups were either exposed (frozen-thawed) on the surface or inaccessible (native), it was concluded that the carbohydrates of rhodopsin, mannose and N-acetylglucosamine, were not involved in the interaction with the RPE. The possibility was also examined that enzymatically galactosylated rhodopsin might serve as a site for recognition by the RPE cell. The binding of ROS disc membranes modified in this manner was not enhanced, indicating that the presence of galactose groups on rhodopsin did not serve as a site for recognition by the RPE. The influence of monosaccharides on the binding of intact ROS by the RPE cells was also investigated. Similar to the results with the disc membranes, the process was not blocked by the presence in the incubation medium of high concentrations (up to 30,000-fold higher than that of rhodopsin) of mannose or GlcNAc, as with the disc membranes, or by glucose or galactose. Thus, from these studies it is concluded that a lectin-like carbohydrate-recognition process may not be involved in the interaction between rhodopsin-containing membranes and the RPE cells.

Biosynthesis of mannose-containing heteropolymers by cell-free preparations of bovine retina

Abstract Cell-free preparations of bovine retinas were shown to catalyze the transfer of mannose from the sugar nucleotide, GDP-[ 14 C]mannose to endogenous acceptors. When fractionated by adsorption and affinity chromatography, and gel filtration, relatively little radioactivity was found in visual pigment. A major product synthesized by the retina under these conditions had properties of a large molecular weight oligosaccharide-lipoidal component which may be an intermediate involved in the biosynthesis of visual pigment.