Retinoylation of proteins in cell-free fractions of rat tissues in vitro.
all-trans-Retinoic acid, a highly active form of vitamin A in inducing cellular differentiation, is incorporated covalently into proteins both in vivo and in vitro. The relative rates of incorporation of all-trans-11,12-(3)H-retinoic acid into rat tissue homogenates in the presence of ATP and coenzyme A were testes>>lung> or =brain> or =kidney>liver. Although all studied cellular organelles of the testes incorporated (3)H-retinoic acid into protein, mitochondria were by far the most active; indeed, up to 25% of the added tritiated retinoic acid (RA) became covalently bound to protein in a 90 min incubation period. In the absence of ATP, coenzyme A, or both cofactors, the amount of RA incorporated into the proteins of testes mitochondria fell to 37%, 16%, and 11%, respectively, of that incorporated in their presence. N-Ethylmaleimide (5 mM) strongly inhibited the reaction. Boiled mitochondria were inactive. After extensive extraction with CHCl(3)-CH(3)OH, the protein-bound radioactivity, which proved largely to be retinoic acid, was released by treatment with proteinase K, hydroxylamine, and dilute base. Thus, retinoic acid is most probably linked to protein as a thiol ester. By SDS-polyacrylamide gel electrophoresis, four protein fractions with molecular masses of approx. 20, 24, 29, and 45 kDa, as well as smaller amounts of larger entities, were labeled in testes mitochondria. The possible identities and roles of these retinoylated proteins are currently being explored.
Department of Pharmacobiology, University of Calabria, Cosenza, Italy. ggenchi@farmbiol.uniba.it
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Genchi
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Olson
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D002458:Cell Fractionation; D003065:Coenzyme A / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D008297:Male; D008928:Mitochondria / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013010:Sonication; D013737:Testis / Q000378:metabolism*; D014212:Tretinoin / Q000378:metabolism*; D014316:Tritium
D011506:Proteins; D014316:Tritium; D014212:Tretinoin; D000244:Adenosine Diphosphate; D004072:Digitonin; D005033:Ethylmaleimide; D003065:Coenzyme A