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J J Plantner

Publications and source records attributed to J J Plantner.

31 records · Page 2Linked to original sources
PubMed1983

Multiple isoelectric forms of detergent-solubilized bovine rhodopsin. I. Identity, composition and properties.

Isoelectric focusing was performed on crude bovine rhodopsin (a detergent extract of rod outer segments) and on rhodopsin after purification by adsorption chromatography on calcium phosphate-Celite and affinity chromatography on Con A-Sepharose. The purified unbleached (native) material resolved into three forms of nearly equal amounts having isoelectric points (pI) of 5.19, 5.58 and 6.14. Each form was stable to refocusing. The composition of the three forms as determined by amino acid analysis, phosphate analysis and polyacrylamide gel electrophoresis was the same as that found for unfocused rhodopsin. Each isoelectric form cross-reacted with rabbit antibody to bovine rhodopsin. After photobleaching, one form of opsin was observed, having an isoelectric point of 5.58. While crude rhodopsin showed a single species in the native state (pI of 6.20), multiple forms were revealed after treatment with phospholipase. Purified rhodopsin, after reduction, showed a single major isoelectric point of 6.09 rather than the multiple forms. Exposure of rod outer segments or rhodopsin to antioxidants or rcrude rhodopsin showed a single species in the native state (pI of 6.20), multiple forms were revealed after treatment with phospholipase. Purified rhodopsin, after reduction, showed a single major isoelectric point of 6.09 rather than the multiple forms. Exposure of rod outer segments or rhodopsin to antioxidants or removal of phospholipids led to an alteration in the relative abundance of the multiple isoelectric forms, but not to a change in their isoelectric point.

Amino Acids↗
PubMed1983

Multiple isoelectric forms of detergent-solubilized bovine rhodopsin. II. Spectral analysis.

The absorption and circular dichroic spectra of three stable isoelectric forms of purified rhodopsin (in Emulphogene BC 720) with isoelectric points of 5.19, 5.58 and 6.14 were analysed over the accessible wavelength region of 190-800 nm. It was found from the spectral analysis of the 5.58 and 6.14 forms that the tertiary structure of these isoelectric forms was different, without any change being observed in the secondary structure of these proteins. The difference in structure was removed by denaturation with SDS. Specifically, the aromatic amino acid residues of the 6.14 isoelectric form were suggested to be in a more polar environment as compared to those for the 5.58 isoelectric form. The changes of structure in the microenvironment of retinal in these proteins were minor in comparison to the tertiary changes observed in the proteins. This indicated that the site of charge perturbation is not localized in the retinylidene microenvironment. Furthermore, the spectral features of these isoelectric forms were features common to the spectra of both purified unfocused rhodopsin and crude rhodopsin, suggesting that the isoelectric forms are not a consequence of purification. Spectral analysis of the 5.19 isoelectric form indicated that this form of rhodopsin had an increased tendency to aggregate after focusing. Based on the denaturation properties of these isoelectric forms, it was shown that the 5.19 and 5.58 isoelectric forms had different conformations. It was concluded from this study that preparations of 'purified' rhodopsin are a heterogeneous mixture of stable proteins with different tertiary structures and different isoelectric points.

Animals↗
PubMed1983

The enzymatic cleavage of rhodopsin by the retinal pigment epithelium. I. Enzyme preparation, properties and kinetics: characterization of the glycopeptide product.

An enzyme system has been detected in the bovine retinal pigmented epithelium (RPE) which carries out the degradation of rhodopsin. The substrate for these studies was bovine rhodopsin labeled by in vitro techniques with [3H]-glucosamine or [3H]-mannose, the former being the derivative most extensively examined. Labeled rhodopsin was purified by adsorption and affinity chromatography followed by preparative isoelectric focusing. Cell-free preparations from bovine RPE cleaved rhodopsin to a single glycopeptide. The pH optimum for the reaction was about 3.5. The apparent Km for rhodopsin was about 5 microns. Rhodopsin was cleaved about 55% under saturating conditions, the limited extent of reaction due primarily to its partial inactivation for use as a substrate during the course of the reaction. Inhibition by cleavage products was not observed. Among several tissues which were examined, the RPE was the most active in rhodopsin-cleaving activity. Although stable as a crude extract, the activity of the rhodopsin-cleaving enzyme, as well as the bovine serum albumin (BSA)-cleaving enzyme (cathepsin-D) were rapidly lost upon purification by DEAE-Sephacel and pepstatin-Sepharose. Differences in relative specific activity among tissues as well as differential patterns of purification suggest that the rhodopsin-cleaving enzyme and the BSA-cleaving enzyme of the RPE may not be the same. Using N-retinylopsin as a substrate for the rhodopsin-cleaving enzyme, it was shown that the regions of the cleavage products containing vitamin A were not associated with the glycopeptide. Some of the properties of the product were examined. Its response to ion exchange resins, susceptibility to further cleavage by pronase, amino acid analysis, solubility properties, isoelectric point, and binding by concanavalin A (conA)-Sepharose, were consistent with the product being described as an acidic glycopeptide. The glycopeptide was reactive with the antibody prepared against bovine rhodopsin, and retained the full antigenicity of the intact molecule. This enzyme system may be one of the means whereby the RPE participates in maintaining the physiological concentration of rhodopsin which is synthesized in the retina and catabolized in the RPE.

Amino Acids↗
PubMed1981

Isoelectric forms of bacteriorhodopsin from Halobacterium halobium.

Isoelectric focusing was performed on bacteiorhodopsin isolated from Halobacterium halobium, R1 strain, solubilized in Nonidet P-40, on sucrose density gradient stabilized columns. When 560 nm absorbance was monitored, four forms of bacteriorhodopsin were observed, having isoelectric points (pI) of: A. 3.93; B, 4.43; C, 5.03; D, 5.49. Instability of some of the isoelectric forms during the very process of electrofocusing was observed. When focused over a period of 6 days, the relative abundance of the forms changed although their pI values remains constant. Refocusing of the isolated forms A or B led to the production f form C. The latter species was stable to refocusing. Form B was unstable to storage either as an aqueous suspension of the purple membrane or as a detergent extract. Each of the forms had the absorption spectrum typical for bacteriorhodopsin and each showed identical patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Bacteriorhodopsins↗
PubMed1976

Carbohydrate composition of bovine rhodopsin.

The carbohydrate content of bovine rhodopsin was investigated and found to be different from previously reported values. Rod outer segments were isolated from dark-adapted bovine retinas by sucrose flotation and purified by sucrose density contrifugation. Rhodopsin was extracted with detergents and purified by chromatographic procedures involving calcium phosphate/celite chromatography followed by affinity chromatograpy on concanavalin A-Sepharose (or in some cases, gel filtration on agarose). Purified preparations of rhodopsin had A278/A498 ratios of 1.6 to 2.0. After treatment of the rhodopsin with chloroform/methanol (2/1) to remove lipids and detergents, the carbohydrate content was measured by gas-liquid chromatography, colorimetric and enzymatic analyses, paper chromatography, and electrophoresis. Rhodopsin was found to have about 9 mol of mannose and 5 mol of glucosamine per mol of visual pigment. A molar ratio of mannose/glucosamine of about 2 was also found in samples of rhodopsin obtained from two other laboratories. The amino acid analysis was similar to previously published values.

Amino Acids↗