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Binding of all-trans-retinoic acid to MLTC-1 proteins

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Abstract

The covalent incorporation of [3H]all-trans-retinoic acid into proteins has been studied in tumoural Leydig (MLTC-1) cells. The maximum retinoylation activity of MLTC-1 cell proteins was 710 ± 29 mean ± SD) fmoles/8 × 104 cells at 37 °C. About 90% of [3H]retinoic acid was trichloroacetic acid-soluble after proteinase-K digestion and about 65–75% after hydrolysis with hydroxylamine. Thus, retinoic acid is most probably linked to proteins as a thiol ester. The retinoylation reaction was inhibited by 13-cis-retinoic acid and 9-cis-retinoic acid with IC50 values of 0.9 μM and 0.65 μM, respectively. Retinoylation was not inhibited by high concentrations of palmitic or myristic acids (250 μM); but there was an increase of the binding activity of about 25% and 130%, respectively. On the other hand, the retinoylation reaction was inhibited (about 40%) by 250 μM lauric acid. After pre-incubation of the cells with different concentrations of unlabeled RA, the retinoylation reaction with 100 nM [3H]RA involved first an increase at 100 nM RA and then a decrease of retinoylation activity between 200 and 600 nM RA. After cycloheximide treatment of the tumoural Leydig cells the binding activity of [3H]RA was about the same as that in the control, suggesting that the bond occurred on proteins in pre-existing cells. (Mol Cell Biochem 276: 55–60, 2005)

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Abbreviations

BSA:

bovine serum albumin

HEPES:

N-2-hydroxy-ethylpiperazine-N′-2-ethane sulphonic acid

LA:

lauric acid

MA:

myristic acid

PA:

palmitic acid

RA:

all-trans-retinoic acid

9-cis-RA:

9-cis-retinoic acid

13-cis-RA:

13-cis-retinoic acid

RAR:

retinoic acid nuclear receptor

RXR:

9-cis-retinoic acid specific nuclear receptor

StAR:

steroidogenic acute regulatory

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Authors and Affiliations

  1. Dipartimento Farmaco-Biologico, Università della Calabria, Cosenza, 87100, Italia

    Erika Cione, Paola Tucci, Valentina Senatore & Giuseppe Genchi

  2. Dipartimento di Scienze Farmaceutiche, Edificio Polifunzionale, Università della Calabria, Italia

    Giuseppina Ioele

Authors
  1. Erika Cione
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  2. Paola Tucci
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  3. Valentina Senatore
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  4. Giuseppina Ioele
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  5. Giuseppe Genchi
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Correspondence to Giuseppe Genchi.

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This paper is dedicated to the memory of Prof. E. Quagliariello.

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Cione, E., Tucci, P., Senatore, V. et al. Binding of all-trans-retinoic acid to MLTC-1 proteins. Mol Cell Biochem 276, 55–60 (2005). https://doi.org/10.1007/s11010-005-2845-2

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  • DOI: https://doi.org/10.1007/s11010-005-2845-2

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