UBC Theses and Dissertations
Characterization of a high-molecular weight glycoprotein in bovine rod photoreceptor outer segment by a monoclonal antibody Wong, Simon Yuk Chun
A monoclonal antibody designated as 4B2 was used to investigate the molecular properties and structural organization of a 220,000 M[sub r] glycoprotein, referred to as ROS 1.2, in bovine retinal rod photoreceptor outer segment disk membranes. The approaches taken were first to produce, characterize, and purify the 4B2 antibody, then to use the purified 4B2 antibody as a ligand for the purification of ROS 1.2 by affinity chromatography, and finally to determine the amino acid composition of ROS 1.2 by amino acid analysis of purified ROS 1.2. The 4B2 antibody was produced by growing the 4B2 hybridoma cell line as ascites tumors in mice or as mass culture. The former method proved to be easier and more economical. Also, the antibody concentration in ascites fluid was shown by solid-phase radioimmune assay to be 30-100 fold greater than in culture supernatant. The 4B2 antibody was shown by radioimmune assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be an IgM molecule with μ heavy chains of apparent M[sub r]=55,000 and k light chains of apparent M[sub r]=22,000. The 4B2 antibody was purified from ascites fluid by DEAE-Sephacel ion exchange chromatography. The yield of this 4B2 antibody was low (0.5 mg/ml ascites fluid) compared to the yield of an anti-rhodopsin antibody (7 mg/ml ascites fluid) obtained from 1D4 hybridoma cell line. ROS 1.2 could only be partially purified by affinity chromatography on a 4B2 antibody-agarose column. As shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a residual amount of rhodopsin, the major rod outer segment (ROS) protein, copurified with ROS 1.2. Therefore, the amino acid composition of ROS 1.2 could not be determined. The 4B2 antibody was used to probe the structural and functional properties of ROS 1.2 by determining whether immunological cross-reactivity exists between ROS 1.2 and proteins of similar M[sub r] in other cell types, namely, myosin in muscle, spectrin in red blood cells, and brain spectrin (fodrin, or calspectin ) in brain cells. The 4B2 monoclonal antibody was shown by radio immune competition assays to cross-react with rabbit skeletal muscle myosin, bovine brain homogenate, and human red blood cell membranes. Radioimmune labeling studies indicated that the cross-reactivity was due to a 160,000 M[sub r] protein in bovine brain homogenate. Similar radioimmune labeling studies were unable to demonstrate which protein in red blood cell membranes cross-react with the 4B2 monoclonal antibody. These studies also indicated that the degraded fragments of ROS 1.2 and myosin with apparent M[sub r] of 140,000 retained 4B2 antigenic sites. In addition, actin was shown by radioimmune assay to be present in rod outer segment preparations. On the basis of these and other results, it is concluded that ROS 1.2 is a myosin-like protein, and in conjunction with other cytoskeletal elements such as actin, serves to link ROS disks to each other or to ROS plasma membrane for the maintenance of the highly-ordered ROS structure. It is speculated that the loss of this ordered structure during the process of disk renewal involves the proteolytic degradation of ROS 1.2.
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