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Ultrastructural localization of rhodopsin in the vertebrate retina

Jan, Lily Yeh and Revel, Jean-Paul (1974) Ultrastructural localization of rhodopsin in the vertebrate retina. Journal of Cell Biology, 62 (2). pp. 257-273. ISSN 0021-9525.

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Early work by Dewey and collaborators has shown the distribution of rhodopsin in the frog retina. We have repeated these experiments on cow and mouse eyes using antibodies specific to rhodopsin alone. Bovine rhodopsin in emulphogene was purified on an hydroxyapatite column. The purity of this reagent was established by spectrophotometric criteria, by sodium dodecyl sulfate (SDS) gel electrophoresis, and by isoelectric focusing. This rhodopsin was used as an immunoadsorbent to isolate specific antibodies from the antisera of rabbits immunized with bovine rod outer segments solubilized in 2% digitonin. The antibody so prepared was shown by immunoelectrophoresis to be in the IgG class and did not cross-react with lipid extracts of bovine rod outer segments. Papain-digested univalent antibodies (Fab) coupled with peroxidase were used to label rhodopsin in formaldehyde-fixed bovine and murine retinas. In addition to the disk membranes, the plasma membrane of the outer segment, the connecting cilium, and part of the rod inner segment membrane were labeled. We observed staining on both sides of the rod outer segment plasma membrane and the disk membrane. Discrepancies were observed between results of immunolabeling experiments and observations of membrane particles seen in freeze-cleaved specimens. Our experiments indicate that the distribution of membrane particles in freeze cleaving experiments reflects the distribution of membrane proteins. Immunolabeling, on the other hand, can introduce several different types of artifact, unless controlled with extreme care.

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Additional Information:© 1974 The Rockefeller University Press. Received for publication 24 October 1973, and in revised form 4 March 1974. We are grateful for the indispensable technical assistance of Mr. Patrick F. Koen and Mr. Micheal Rees. We would like to thank Professor Max Delbriick for his inspiration and advice, Dr. Meredithe L. Applebury for the very helpful consultations on the purification of bovine rhodopsin before the publication of her work, Elizabeth P. Blankenhorn and Suzanne O. Rosenberg for their professional help on immunology, and Henry V. Huang and Dr. Robert S. Molday for carrying out the isoelectric focusing experiments and giving us access to unpublished material. We also wish to thank Professor W. Stockenius for his useful comments on our manuscript. The work was supported in part by grants GM 06965 and GM 19224 of the United States Public Health Service. Lily Yeh Jan was partially supported by a generous grant from the McCallum Fund.
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Public Health Service (PHS)GM 06965
Public Health Service (PHS)GM 19224
Record Number:CaltechAUTHORS:20120725-114816635
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:32719
Deposited By: Jason Perez
Deposited On:25 Jul 2012 20:15
Last Modified:26 Dec 2012 15:44

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