The retinal rod outer segment of the frog: Detachment, isolation, phosphorus fractions and enzyme activity

Abstract

Frog retinal rod outer segments were detached from dark adapted retinas by (1) agitation in frog Ringer's solution or (2) by crushing between two glass surfaces. The resulting suspensions were further purified by low and high speed centrifugation procedures in Ficoll density gradients. The density of the outer segments in Ficoll solutions was found to be 1.09. The large frog outer segments, unlike bovine outer segments, are not readily separated from nuclei, which were estimated to comprise 2.6–8% of the material, based on DNA analyses. The RNA/DNA ratio was 0.4–0.5, like that of neuronal nuclei. Representative enzymes of glycolysis (lactic dehydrogenase and glyceraldehyde phosphate dehydrogenase), phosphogluconate oxidation (glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase), the citric acid cycle (malic dehydrogenase) and ATP degradation (ATPase) were assayed. A major part of the malic dehydrogenase activity was probably due to inner segments attached to some of the outer segments. Glyceraldehyde phosphate dehydrogenase and glucose‐6‐phosphate dehydrogenase (but not lactic dehydrogenase) activities were lower in detached outer segments purified on Ficoll gradients than in samples of outer segment layer microdissected from freeze‐dried sections of frog retina, as well as in whole retina. The data suggest that the activities of all the enzymes studied are intrinsically low in rod outer segments.