A simple and rapid procedure for the isolation of intact bovine rod outer segments (ROS).
A method is described which allows the rapid isolation and purification of intact rod outer segments (ROS) from cattle eyes. It requires very fresh retinal material and can be completed within less than 2 h of the death of the animals. Cattle eyes are dissected in the usual manner, the retinae are isolated and the ROS are separated from the rest of the retina by gentle vortexing and filtration through a nylon mesh. The resulting crude ROS suspension is purified on a discontinuous sucrose density gradient. Two fractions are obtained, the major one consisting of mostly intact ROS, the minor one of RIS-ROS, i.e. of ROS which are still connected to part of their inner segment. The ROS are washed once and can be stored on ice for several days without loosing their intact plasma membrane. They can be transformed to leaky ROS by a quick freeze/thawing cycle or, if one wants unobstructed access to the interdiskal space, they can be subjected to a mild lysis treatment. The resulting ROS material is characterised using light microscopy, electron microscopy, light scattering, gel electrophoresis and absorption spectroscopy. It contains unusually low levels of 48k-protein and very high levels of G-protein. The latter cannot be washed out in the presence of GTP-gamma-S, even in the case of leaky ROS.
Uhl
R
R
Max Planck Institut für biophysikalische Chemie, Am Fassberg, Göttingen, F.R.G.
Desel
H
H
Ryba
N
N
Wagner
R
R
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000648:ultrastructure; D002469:Cell Separation; D002501:Centrifugation, Zonal / Q000379:methods; D005615:Freezing; D008027:Light; D008854:Microscopy, Electron; D010786:Photoreceptor Cells / Q000166:cytology*; D012374:Rod Cell Outer Segment / Q000166:cytology* / Q000648:ultrastructure; D012542:Scattering, Radiation; D013053:Spectrophotometry