The vertebrate visual cycle is comprised of biochemical reactions involved in processing all-trans retinal produced by the photoisomerisation of 11-cis retinal in rhodopsin. The visual cycle is essential for vision and visual adaptation. The goal of this project is to characterize on a molecular level essential components of the visual cycle and to learn how the cycle is regulated. Two of the key reactions in the visual cycle are catalyzed by the retinal pigment epithelium membrane bound enzymes, lecithin retinol acyl transferase (LRAT)and the isomerohydrolase. LRAT transfers an acyl group from lecithin to vitamin A to generate all-trans retinyl esters and the omerohydrolase processes the esters to produce 11-cis retinol. Both enzymes are essential for vision. An understanding of LRAT at the molecular level is of major interest in this grant proposal. LRAT has a unique sequence which does not reveal the mechanistic class to which it belongs. Biochemical studies including chemical mapping studies using the novel technique of biotin affinity labeling and site-specific mutagenic studies are proposed to both map elements of the active-site structure of LRAT and define its molecular mechanism of action. Chemical mapping studies are also proposed to begin to elucidate the structure of LRAT in the membrane and to reveal nearest neighbor proteins in RPE membranes. One of the LRAT associated proteins may be the isomerohydrolase. Identification and characterization of isomerohydrolase is another important aspect of this proposal. Approaches to the identification of this enzyme system will involve both exploiting interactions with LRAT to either affinity purify or cross-link isomerohydrolase and photoaffinity labeling approaches to label the enzyme. When isomerohydrolase is identified it will be coned sequenced and expressed in LRAT transfected HEK cells. The structure, mechanism of action and regulation of the isomerohydrolase will be explored as will possible relationships to diseases of vision caused by mutations in the enzyme.