Bovine retinal outer segment fragments were isolated by density gradient centrifugation in a high centrifugal field. Assays of the final preparation for enzymes of the mitochondrial respiratory chain indicated mitochondrial contamination not in excess of 1 per cent. Glucose-6-phosphatase and TPNH-cytochrome c reductase activities, presumably diagnostic for microsomes, were also absent. Electron micrographs did not disclose the presence of significant numbers of particles other than fragments of the outer segment discs. The red flagments were characterized by an ascorbate-oxidizing system and a high lipid content.Electron microscopic observations by numerous investigators (1-6) have established fundamental similarities between the ultrastructure of the outer segment of the retinal receptor and that of other membranous structures such as the mitochondrion and chloroplast. Wald (7-9), MacNichol (10, 11), and others have presented unequivocal evidence that the outer segment houses the carotenoid visual pigments required for vision in bright or dim light. Futterman (12-14) has undertaken to elaborate the mechanism which governs carotenoid maintenance and metabolism. Nonetheless, investigation of the device by which the energy of light absorbed by the carotenoid is ultimately transduced into an electrical impulse has been very meagre. The starting assumption of our studies has been that this device must have certain enzymic principles in common with energy-conserving mechanisms found in organelles possessing similar membranous ultrastructure.While all investigators may not share this assumption, all must agree that the serious study of the enzymology of this unique organelle demands its physical isolation from contaminating particles. It now appears inescapable that such isolation has not been achieved in the past. The standard sucrose suspensions of outer segments, from which extracts of the visual pigments are classically derived (8, 9, 15-18), are almost certainly contaminated by mitochondria. Our own observation of the light sensitivity of the ATPase found in digitonin extracts of rhodopsin (19) must in due course be reexamined on this account. The purpose of the present communication is to describe the methodology we have used to isolate the outer segments, and to present the criteria used in evaluation of the completeness of the separation.Many investigators have used one or more modifications of the method of Saito (20), which entails breaking off large numbers of outer segments, or their distal fragments, by shaking the retina in dilute sucrose and then filtering the suspension through gauze. Despite claims made for this method, we have found that the outer segment does not invariably break free of the mitochondrialaden inner segment. This appendage, complete with connecting cilium and enclosed mitochondria, can be seen with the electron microscope in suspensions of outer segments prepared in this way. The inner segments are not removed by gauze filtration, and are not easily removed by sedimentation.
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α-Tocopherol was identified in lipid extracts of bovine retinal outer segment (ROS) preparations. Positive identification was obtained by the thin layer chromatographic characteristics of the tocopherol form and its oxidation product α-tocopherylquinone, and by the ultraviolet spectrum of the oxidized and KBH4-reduced form of the tocopherylquinone. In the ROS preparations used, α-tocopherol chromanol was the predominant species, the quinone form accounting for 25% or less of the total. The concentration of α-tocopherol in the ROS preparations was about 0.1 mole α-tocopherol per mole rhodopsin, or about 1 nmole/mg, protein. Mitochondria from bovine retina contained about 0.4 nmole α-tocopherol per mg protein.
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