The objective of this proposal is to investigate the enzymatic mechanism of anhydroretinol synthesis. Based on the observation that proliferation of lymphocytes as well as of many other cell lines is dependent on an external supply of retinol and the hypothesis that retinol is converted intracellularly to active metabolites, we have identified two new intracellular messenger pathways, the growth supportive 14-hydroxy-retro- retinol (14-HRR) pathway and the growth suppressive anhydroretinol (AR) pathway. 14-HRR is induced in lymphocytes subsequent to activation and supports the proliferation of lymphocytes. 14-HRR is also found in most other dividing cells. AR is a competitive inhibitor of 14-HRR and prevents the activation of T lymphocytes and the proliferation of activated B and T lymphocytes. AR is also able to block antibody responses in vivo. The two retro- retinoids, 140HRR and AR, may be the first example of an agonist/antagonist pair of small lipophilic messenger molecules that are produced and used by the same cell. The observation that AR is overproduced in the insect cell line SF-21 provided the opportunity to characterize the AR producing enzyme system (ARase). AR is produced in the cytosol of SF-21 cells by a UTP dependent lyase which we purified to apparent homogeneity. The k m of the purified lyase for retinol bound to BSA or CRBP has been determined to be 0.9 x 10-9 M with Vmax of 1.8muM AR/min/mg enzyme. The molecular size on SDS/PAGE gels under reduced and nonreduced conditions is 41 kD. Trypsin digestion of the purified enzyme gave several peptides, which enabled us to get two internal amino acid sequences with a size of 19 and 22 amino acids, respectively. Arase is the first retinol metabolizing enzyme purified to homogeneity and the prototype of a physiological cytosolic retinoid metabolizing enzyme. In this proposal, we request support for the further characterization of the enzymatic pathway leading to AR. We propose to proceed as follows:
In Aim 1 we clone and express ARase from insect cells and produce antisera against ARase.
In Aim 2 we plan to purify and clone mammalian ARase using a biochemical and/or a molecular biological approach.
In Aim 3 we characterize and compare the enzyme kinetics of mammalian and insect ARase, determine the substrate specificity, and determine by site directed mutagenesis the UTP binding pocket and the enzymatic retinol/anhydroretinol binding site. ARase expression in different organs and at different developmental stages will be studied using in situ hybridization and immunohistochemistry.
In Aim 4 we determine the structure and function of the retinol derivative P25, an intermediate in AR synthesis.
| Sinclair, M L; Wang, X Y; Mattia, M et al. (2000) Specific expression of soluble adenylyl cyclase in male germ cells. Mol Reprod Dev 56:11-Jun |
| Buck, J; Sinclair, M L; Schapal, L et al. (1999) Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals. Proc Natl Acad Sci U S A 96:79-84 |
| Chen, Y; Buck, J; Derguini, F (1999) Anhydroretinol induces oxidative stress and cell death. Cancer Res 59:3985-90 |
| Vakiani, E; Luz, J G; Buck, J (1998) Substrate specificity and kinetic mechanism of the insect sulfotransferase, retinol dehydratase. J Biol Chem 273:35381-7 |
| Blumberg, B; Bolado Jr, J; Derguini, F et al. (1996) Novel retinoic acid receptor ligands in Xenopus embryos. Proc Natl Acad Sci U S A 93:4873-8 |
| Grun, F; Noy, N; Hammerling, U et al. (1996) Purification, cloning, and bacterial expression of retinol dehydratase from Spodoptera frugiperda. J Biol Chem 271:16135-8 |
