Characterization of rat testes mitochondrial retinoylating system and its partial purification.
Retinoylation (retinoic acid acylation), a posttranslational modification of proteins occurring in a variety of eukariotic cell lines both in vivo and in vitro, was studied in rat testes mitochondria. all-trans-Retinoic acid, a highly active form of vitamin A in inducing cellular differentiation, is incorporated covalently into proteins of rat testes mitochondria. The maximum retinoylation activity of rat testes mitochondrial proteins was 21.6 pmoles mg protein(-1) 90 min(-1) at 37 degrees C. The activation energy was 44 kJ mol(-1) from 5 to 37 degrees C. The retinoylation activity had a pH optimum of 7.5. The retinoylation process was specific for the presence of ATP, ADP, and GTP (even if only 30% of the control). The half saturation constant (Km) was 0.69 microM for all-trans-retinoic acid, while the inhibition constant (Ki) was 1.5 microM for 13-cis-retinoic acid. Retinoylation was not inhibited by high concentrations of myristic acid (MA) and palmitic acid (PA), indicating that retinoylation and acylation reactions involved different rat testes mitochondrial proteins. The ATP or CoASH saturation curves of retinoylation reaction showed sigmoidal behavior with apparent half saturation constants (K0.5) of 6.5 mM ATP and 40.6 microM CoASH. On SDS-gel electrophoresis, the hydroxylapaptite/celite eluate showed various protein bands between 25 and 80 kDa. This retinoylated protein was purified 17-fold with respect to the mitochondrial extract.
Cione
Erika
E
Dipartimento Farmaco-Biologico, Edificio Polifunzionale, Università degli Studi della Calabria, Cosenza, Italy.
Genchi
Giuseppe
G
D000215:Acylation; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D002478:Cells, Cultured; D008297:Male; D008928:Mitochondria / Q000378:metabolism*; D008970:Molecular Weight; D051381:Rats; D017207:Rats, Sprague-Dawley; D013737:Testis / Q000378:metabolism*; D014212:Tretinoin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D014212:Tretinoin; D000255:Adenosine Triphosphate