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Type of Document	Dissertation
Author	Jan, Lily Kung-Chung Yeh
URN	etd-05182006-080145
Persistent URL	http://resolver.caltech.edu/CaltechETD:etd-05182006-080145
Title	Investigations on rhodopsin and bacteriorhodopsin. I. Ultrastructural localization of rhodopsin in vertebrate retina. II. The isomeric configuration of the bacteriorhodopsin chromophore
Degree	PhD
Option	Biology
Advisory Committee	Advisor Name Title Max Delbruck Committee Chair Jean-Paul Revel Committee Member
Keywords	none
Date of Defense	1974-05-30
Availability	unrestricted
Abstract Chapter I: 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 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 disc 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 disc 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 types of artifact, unless controlled with extreme care. Chapter II: A method for cutting thin sections of frozen glutaraldehyde-fixed tissue developed by Tokuyasu and collaborators has made it possible to label antigens on thin sections of a tissue. Thin sections of frozen retina obtained by this method were treated first with rabbit antibodies specific for bovine rhodopsin, then with conjugates of ferritin and goat antibodies specific for rabbit antibody. Both mouse and bovine retina were labeled specifically by this method. The ferritin label on mouse retina was less heavy than that on bovine retina. Chicken retina was not labeled at all by these reagents. Ferritin label was formed on the disc membranes, on rod outer and inner segment cell membranes, on the inside of the rod inner segment, on the outer membrane of the connecting cilium and in the cytoplasmic bridge of the mouse retina. Chapter III: Oesterhelt and Stoeckenius in 1971 found a pigment in the cell membrane of Halobacterium halobium which they called bacteriorhodopsin because it resembled rhodopsin in many aspects. We have studied the isomeric configurations of its chromophore by thin layer chromatography of the retinal itself and of the retinal oxime derivatives. Under physiological conditions the dark-adapted bacteriorhodopsin contains 13-cis retinal, in contrast to the 11-cis retinal of rhodopsin. Upon illumination the 13-cis retinal is converted to all-trans retinal. Substantial thermal isomerization of retinal occurs if the extraction procedure is performed at room temperature. Implications of the different isomeric forms found in bacteriorhodopsin and rhodopsin are discussed.
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