The covalent incorporation of [3H]all-trans-retinoic acid into proteins has been studied in Leydig(TM-3) cells. The maximum retinoylation activity of Leydig cells proteins was 570 ± 27 fmoles/8 ×104 cells at 37◦C. About 95% of [3H]retinoic acid was trichloroacetic acid-soluble after proteinase-Kdigestion or after hydrolysis with hydroxylamine. Thus, retinoic acid is most probably linked toproteins as a thiol ester. The retinoylation process was inhibited by 13-cis-retinoic acid and 9-cisretinoicacidwith IC50 values of 0.6 and 1.2μMrespectively. Dibutyryl-cAMP and forskolin increasedthe retinoylation activity by 75 and 81% at 500 and 25 μM respectively. Also hCG increased theretinoylation binding activity of 110% at 250 ng/mL. After cycloheximide treatment of the Leydigcells the binding activity of [3H]RA was about the same that in the control, suggesting that the bondoccurs on proteins in pre-existing cells. Retinoylation was not inhibited by high concentrations ofpalmitic or myristic acids (500 μM); on the contrary, there was an increase of the binding activity ofabout 60 and 50% respectively.
Retinoylation reaction of proteins in Leydig (TM-3) cells
Cione E.;Tucci P.;Chimento A.;Pezzi V.;
2005-01-01
Abstract
The covalent incorporation of [3H]all-trans-retinoic acid into proteins has been studied in Leydig(TM-3) cells. The maximum retinoylation activity of Leydig cells proteins was 570 ± 27 fmoles/8 ×104 cells at 37◦C. About 95% of [3H]retinoic acid was trichloroacetic acid-soluble after proteinase-Kdigestion or after hydrolysis with hydroxylamine. Thus, retinoic acid is most probably linked toproteins as a thiol ester. The retinoylation process was inhibited by 13-cis-retinoic acid and 9-cisretinoicacidwith IC50 values of 0.6 and 1.2μMrespectively. Dibutyryl-cAMP and forskolin increasedthe retinoylation activity by 75 and 81% at 500 and 25 μM respectively. Also hCG increased theretinoylation binding activity of 110% at 250 ng/mL. After cycloheximide treatment of the Leydigcells the binding activity of [3H]RA was about the same that in the control, suggesting that the bondoccurs on proteins in pre-existing cells. Retinoylation was not inhibited by high concentrations ofpalmitic or myristic acids (500 μM); on the contrary, there was an increase of the binding activity ofabout 60 and 50% respectively.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
