A spectrin-like protein in bovine retinal rod photoreceptor outer segments as defined by monoclonal antibodies

Biochemical and immunological studies indicate that rod outer segments (ROS) of bovine photoreceptor cells contain a Mr 240,000 polypeptide related to the ∝-subunit of red blood cell (RBC) spectrin. With the use of sodium dodecyl sulfate gel electrophoresis in conjunction with the immunoblotting technique, monoclonal antibody 4B2 was found to bind to a Mr 240,000 polypeptide in ROS that is distinct from the prominent Mr 220,000 concanavalin A binding glycoprotein. The Mr 240,000 polypeptide is highly susceptible to degradation by endogenous proteases. It does not appear to be an integral membrane protein but is tightly membrane associated since it can be partially extracted from ROS membranes with urea in the absence of detergent. The 4B2 antibody cross-reacted with RBC ghost membranes and bovine brain microsomal membranes. Radioimmune assays and immunoblotting analysis of purified bovine RBC spectrin further revealed that the 4B2 antibody predominantly labelled the ∝-chain of RBC spectrin having an apparent Mr of 240,000. Monoclonal antibody 3A6 was found to bind to a polypeptide with a slightly lower Mr than the 4B2-specific polypeptide. It is also highly susceptible to degradation by endogenous proteases, but unlike the 4B2 antibody, it predominantly labelled the β-chain of RBC spectrin having an apparent M of 220,000. Polyclonal anti-spectrin antibodies that bound to both the ∝ - and β-chain of RBC spectrin predominantly labelled a Mr 240,000 polypeptide of ROS membranes. Two faintly labelled bands in the Mr range of 210,000-220,000 were also observed. These components may represent variants of the β -chain of spectrin that are weakly cross-reacting or present in smaller quantities than the ∝-chain. Immunocytochemical labelling studies using the 4B2 antibody and immunogold-dextran markers indicated that the ROS spectrin-like protein is preferentially localized in the region where the discs come in close contact to the plasma membrane of ROS. Immunoblotting analysis indicated that rhodopsin and peripherin which constitute over 90% of total disc membrane proteins were selectively solubilized in Triton X-100, whereas a set of polypeptides including the 4B2-specific polypeptide and the Mr 220,000 concanavalin A-binding glycoprotein was only partially soluble. Electron microscopy of a negatively stained Triton-extracted ROS pellet revealed a filamentous network. These studies indicate that ROS contain a protein related to RBC spectrin, which may constitute a major component of a filamentous network lining the inner surface of the ROS plasma membrane as previously seen by electron microscopy. This membrane skeletal system may serve to stabilize the ordered ROS structure and maintain a constant distance between the rim region of the discs and the plasma membrane.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat