Preparation and characterization of bovine retinal pigment epithelial cell plasma membrane
department or school: Medicine, Faculty of
department or school: Biochemistry and Molecular Biology, Department of
data provider: DSpace
degree campus: UBCV
creator: Laird, Dale W.
issued: 2010-05-19T13:18:20Z
issued: 1984
related degree: Master of Science - MSc
degree grantor: University of British Columbia
description: A 7-9 fold enriched preparation of bovine retinal pigment epithelial cell plasma membrane was prepared and characterized by enzymatic analysis. SDS-polyacrylamide gel electrophoresis and transmission electron microscopy revealed a large rod outer segment contamination in the preparation due to a tight retinal pigment epithelial cellular adhesion to the rod photoreceptor cells. The contaminating rhodopsin was partially removed by anti-rhodopsin immunoaffinity chromatography as determined by SDS-polyacrylamide gels stained with coomassie blue or silver. Monoclonal antibodies raised against the retinal pigment epithelial plasma membrane preparation cross reacted with rod outer segment preparations. A monoclonal antibody, designated Rho-5A3, was classified as an IgG₃ kappa light chain immunoglobulin. It was shown to be specific for rhodopsin as determined by radioimmune labeling of bovine rod outer segment membrane proteins electrophoretically transferred to CNBr-activated paper. Limited proteolytic digestion of rhodopsin followed by electrophoretic transfer to CNBr- activated paper localized the binding site of this antibody to the N-terminal two-thirds of the rhodopsin molecule. Competition assays with rhodopsin polypeptides further defined the antigenic site to be within the 17-39 amino-acid segment of rhodopsin. The Rho-5A3 antibody did not bind to sealed ROS discs or frozen-thawed ROS discs but did bind to Triton X-100 solubilised discs indicating a detergent solubilisation dependence for antigenic site accessibility. Cultures of bovine retinal pigment epithelial cells were started by initial enzymatic isolation followed by recovery in RPMI-1640 culture medium. The retinal pigment epithelial cells established a doubling time of 52 hours until the cells reached culture confluency . The cells also maintained many of their in vivo characteristics such as a high degree of pigmentation and an abundance of microvilli. Cell surface glycoproteins labelled with FITC-Con A, …
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/24835
note: PREPARATION AND CHARACTERIZATION OF BOVINE RETINAL PIGMENT E P I T H E L I A L CELL PLASMA MEMBRANE by DALE W.LAIRD B . S c . , U n i v e r s i t y of P r i n c e Edward I s l a n d , C h a r l o t t e t o w n , P r i n c e Edward I s l a n d , 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE UNIVERSITY OF BRITISH COLUMBIA We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA O c t o b e r , 1 9 8 4 © D a l e W. L a i r d , 1984 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department/, or by his or her representatives. I t i s under s tood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. k o '/A. Department of (^^>^^d^^ The University of B r i t i s h CoMimbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6 (3/81) - i i -ABSTRACT A 7-9 fold enriched preparation of bovine r e t i n a l pigment e p i t h e l i a l c e l l plasma membrane was prepared and characterized by enzymatic analysis. SDS-polyacrylamide gel electrophoresis and transmission electron microscopy revealed a large rod outer segment contamination in the preparation due to a tight r e t i n a l pigment e p i t h e l i a l c e l l u l a r adhesion to the rod photoreceptor c e l l s . The contaminating rhodopsin was p a r t i a l l y removed by a n t i -rhodopsin immunoaffinity chromatography as determined by SDS-polyacrylamide gels stained with coomassie blue or s i l v e r . Monoclona…
has type: Thesis/Dissertation
is shown at: 10.14288/1.0096095
language: eng
degree discipline: Biochemistry and Molecular Biology
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
scholar level: Graduate
type: Text
identifier URI: http://hdl.handle.net/2429/24835