Monoclonal antibodies to crayfish rhodopsin. I. Biochemical characterization and cross-reactivity.
Five antibody secreting cell lines were selected on the basis of specific binding to photoreceptive structures from a fusion of myeloma cells with spleen cells from BALB/c mice immunized with photoreceptor membrane from crayfish compound eyes. On Western blots derived from one- and two-dimensional polyacrylamide gels of purified photoreceptor membrane the antibodies bound strongly to the major 35 kDa peptide and are therefore specific for the visual pigment, rhodopsin. Four antibodies also recognized a minor 24 kDa peptide probably representing a breakdown product generated in vivo by the action of lysosomal hydrolases. Epitope characterization of the antibodies using peptide maps of opsin after protease treatment revealed three grossly different specificities. Three antibodies recognize a major antigenic site located within the large proteolytic fragment of about 24 kDa, possibly derived from the aminoterminus of the molecule. Antibodies applied to lightly fixed frozen sections or semi-thin sections of crayfish retina embedded in Lowicryl or polyethyleneglycol specifically bound to the rhabdomeral structure formed by receptor cells R1-R7, but failed to show significant cross-reaction with R8, the blue receptor, proving significant differences in the primary structure of the apoproteins of visual pigments involved in crayfish colour vision. None of the antibodies revealed any cross-reactivity with Drosophila or squid rhodopsin, corroborating this finding. The antibodies also recognized granular material in the vicinity of the rhabdoms at sites occupied by secondary lysosomes containing degraded rhabdomeral membrane. No significant binding was observed to the outer plasma membrane of the retinula cells, or in any other part of the retina.
H G
HG
de Couet
C
C
Sigmund
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D003400:Astacoidea; D003429:Cross Reactions; D000939:Epitopes; D005136:Eye Proteins / Q000378:metabolism; D008970:Molecular Weight; D012160:Retina / Q000378:metabolism / Q000648:ultrastructure; D012168:Retinal Pigments / Q000276:immunology*; D012243:Rhodopsin / Q000276:immunology*; D017299:Rod Opsins
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005136:Eye Proteins; D012168:Retinal Pigments; D017299:Rod Opsins; D012243:Rhodopsin