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.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D007525:Isoelectric Focusing; D010743:Phospholipids / Q000378:metabolism; D012168:Retinal Pigments / Q000032:analysis*; D012243:Rhodopsin / Q000032:analysis* / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000032:analysis; D012995:Solubility; D014810:Vitamin E / Q000378:metabolism
D000596:Amino Acids; D010743:Phospholipids; D012168:Retinal Pigments; D014810:Vitamin E; D012243:Rhodopsin