Spatial/temporal concentrations of all-trans-retinoic acid (RA), and therefore of enzymes that catalyze RA biosynthesis, are crucial during the life span of mammals, from development throughout adulthood, because RA exerts global control over differentiation and apoptosis in numerous cell types. The long-term goals of this project are to characterize the second and committed step in RA biosynthesis, the conversion of retinal into RA, at the biochemical, molecular and endocrine levels. The immediate goal is to test the hypothesis that physiologically-significant retinal dehydrogenases (RalDH) recognize retinal bound to cellular retinol binding-protein, type I, as substrate. Working from this hypothesis, at least three RalDH isozymes have been identified in cytosol of rat liver. Each responds to vitamin A deficiency with an increase in activity and/or amount. The quantitatively major isoform in adult liver has been purified and partial CDNA clones have been obtained for one of the testis isoforms.
The specific aims are to continue these investigations by: 1) obtaining full-length CDNAS of the RalDHs isoforms that occur in rat tissues; 2) expressing the CDNAS and characterizing the RalDHs physically and kinetically; 3) quantifying whether physical interactions occur between RalDHs and cellular retinol-binding protein; 4) determining the temporal-spatial expression patterns of RalDHs during embryonic development with immunocytochemistry; 5) determining the mechanism(s) of regulation of RalDHs by vitamin A status and whether aging affects RalDH expression or activity. This work will provide further insight into pathways of RA biosynthesis and their regulation, and will generate reagents (antibodies, CDNA) necessary for extensive study of endocrine and aging effects on RA biosynthesis. These reagents will be useful for identifying disease and/or age-related degenerative processes that may be caused or exacerbated by impaired RA biosynthesis or vitamin A nutritional inadequacy or excess.
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