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Study of the orientation of retinal in bovine rhodopsin: the use of a photoactivatable retinal analog

Conference · · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
OSTI ID:6180117

Rhodopsin is the major transmembrane protein in the photoreceptor cells of vertebrate and invertebrate retina. Bovine rhodopsin consists of a polypeptide chain of 348 amino acids of known sequence in which the chromophore, 11-cis-retinal, is linked to Lys-296 as a Schiff base. To investigate the orientation of retinal in the protein and to study the interactions between retinal and the protein, the authors have developed a crosslinking approach using a /sup 3/H-labeled photoactivatable analog of retinal. Bleached rhodopsin in rod outer segments was reconstituted with the analog to give a pigment with lambda/sub max/ at 460nm. Reduction of the Schiff base with borane dimenthylamine, followed by degradation with CNBr and sequencing of the radioactive fragment showed that the analog is attached to Lys-296, as in the native rhodopsin. Further, the reconstitute protein after photolysis was phosphorylated by rhodopsin kinase. Photolysis of the reconstituted pigment at -15/sup 0/C resulted in crosslinking of the analog to the opsin to the extent of 30% as analyzed by SDS electrophoresis. The site(s) of crosslinking in the protein are under investigation.

Research Organization:
Massachusetts Institute of Technology, Cambridge
OSTI ID:
6180117
Report Number(s):
CONF-870644-
Journal Information:
Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 46:6; ISSN FEPRA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACID SEQUENCE
ANIMALS
BODY
BODY AREAS
CARBOXYLIC ACID ESTERS
CATTLE
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL REACTIONS
CROSS-LINKING
DOMESTIC ANIMALS
ELECTROPHORESIS
ESTERS
EYES
FACE
HEAD
IMINES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MAMMALS
MEMBRANES
MOLECULAR STRUCTURE
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PHOSPHORYLATION
PIGMENTS
POLYMERIZATION
PROTEINS
RETINA
RETINOIC ACID
RHODOPSIN
RUMINANTS
SCHIFF BASES
SENSE ORGANS
STRUCTURE-ACTIVITY RELATIONSHIPS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES