The studies described here are concerned with elucidating the molecular and physiological basis of rhodopsin regeneration. Specifically, the studies are focused on understanding the process by which 11-cis-retinol, the ultimate precursor of the visual chromophore, is synthesized in the vertebrate pigment epithelium. We have shown that the membranes of the pigment epithelium contain a novel biosynthetic enzyme system capable of transforming free all-trans-retinol into its 11-cis congener. This thermodynamically uphill process derives its energy from the phospholipids of the membranes in a novel process which utilizes the free energy of hydrolysis of the ester bonds to drive the isomerization. The mechanism employed involves the transesterification of all-trans-retinol with a phospholipid followed by enzymatic processing of the retinyl ester directly to 11-cis-retinol. We have solubilized and partially purified the enzymes involved. In this grant request, we propose to completely purify the enzyme components of this isomerization process, namely, the retinyl ester synthetase and isomerase. We propose to study the mechanisms of action of these enzymes and prepare mechanism-based enzyme inhibitors of them to be used to study their physiological roles. Furthermore, monoclonal and monospecific polyclonal antibodies will be prepared against the enzymes. These antibodies will be used to determine the steady state levels, the turnover rates, and the possible post-translational modification of the enzymes. The mechanisms by which the enzymes might be regulated as well as the mechanism by which the enzymes interact with the membrane are of exceptional interest here. The availability of sequence information on the enzymes and the availability of specific antibodies to the enzymes should enable us to clone them and fully sequence them. This information is important for determining what these enzymes are related to, what their major structural motifs might be, and how the enzymes interact with the membrane. Sequence information will also be of interest clinically for determining whether or not these enzymes, which are the sine qua non of vertebrate vision, are altered in major diseases of vision.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
4R37EY004096-14
Application #
2158994
Study Section
Special Emphasis Panel (NSS)
Project Start
1982-04-01
Project End
2000-03-31
Budget Start
1995-04-01
Budget End
1996-03-31
Support Year
14
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Harvard University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Fishkin, Nathan; Yefidoff, Revital; Gollipalli, Deviprasad R et al. (2005) On the mechanism of isomerization of all-trans-retinol esters to 11-cis-retinol in retinal pigment epithelial cells: 11-fluoro-all-trans-retinol as substrate/inhibitor in the visual cycle. Bioorg Med Chem 13:5189-94
Xue, Linlong; Rando, Robert R (2004) Roles of cysteine 161 and tyrosine 154 in the lecithin-retinol acyltransferase mechanism. Biochemistry 43:6120-6
Xue, Linlong; Gollapalli, Deviprasad R; Maiti, Pranab et al. (2004) A palmitoylation switch mechanism in the regulation of the visual cycle. Cell 117:761-71
Gollapalli, Deviprasad R; Rando, Robert R (2004) The specific binding of retinoic acid to RPE65 and approaches to the treatment of macular degeneration. Proc Natl Acad Sci U S A 101:10030-5
Gollapalli, Deviprasad R; Rando, Robert R (2003) Molecular logic of 11-cis-retinoid biosynthesis in a cone-dominated species. Biochemistry 42:14921-9
Krosky, Paula M; Baek, Moon-Chang; Jahng, Wan Jin et al. (2003) The human cytomegalovirus UL44 protein is a substrate for the UL97 protein kinase. J Virol 77:7720-7
Jahng, Wan Jin; Xue, Linlong; Rando, Robert R (2003) Lecithin retinol acyltransferase is a founder member of a novel family of enzymes. Biochemistry 42:12805-12
Gollapalli, Deviprasad R; Maiti, Pranab; Rando, Robert R (2003) RPE65 operates in the vertebrate visual cycle by stereospecifically binding all-trans-retinyl esters. Biochemistry 42:11824-30
Gollapalli, Deviprasad R; Rando, Robert R (2003) All-trans-retinyl esters are the substrates for isomerization in the vertebrate visual cycle. Biochemistry 42:5809-18
Bok, Dean; Ruiz, Alberto; Yaron, Orna et al. (2003) Purification and characterization of a transmembrane domain-deleted form of lecithin retinol acyltransferase. Biochemistry 42:6090-8

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