Browsing by Author "Loyola, G"

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    High-affinity binding of fatty acyl-CoAs and peroxisome proliferator-CoA esters to glutathione S-transferases -: Effect on enzymatic activity
    (1999) Silva, C; Loyola, G; Valenzuela, R; García-Huidobro, T; Monasterio, O; Bronfman, M
    Acyl-CoAs an present at high concentrations within the cell, yet are strongly buffered by specific binding proteins in order to maintain a low intracellular unbound acyl-CoA concentration, compatible with their metabolic role? their importance in cell signaling, and as protection from their detergent properties. This intracellular regulation may be disrupted by nonmetabolizables acyl-CoA esters of xenobiotics, such as peroxisome proliferators, which are formed at relatively high concentration within the liver cell. The low molecular mass acyl-CoA binding protein (ACBP) and fatty acyl-CoA binding protein (FABP) have been proposed as the buffering system for fatty acyl-CoAs. Whether these proteins also bind xenobiotic-CoA is not known. Here we have identified new liver cytosolic fatty acyl-CoA and xenobiotic-CoA binding sites as glutathione S-transferase (GST), using fluorescent polarization and a acyl-etheno-CoA derivative of the peroxisome proliferator nafenopin as ligand. Rat liver GST and human liver recombinant GSTA1-1, GSTP1-1 and GSTM1-1 were used. Only class alpha rat liver GST and human GSTA1-1 bind xenobiotic-CoAs and fatty acyl-CoAs, with K-d values ranging from 200 nM to 5 mu M. One mol of acyl-CoA is bound per mol of dimeric enzyme, and no metabolization or hydrolysis was observed. Binding results in strong inhibition of rat Liver GST and human recombinant GSTA1-1 (IC50 at the nanomolar level for palmitoyl-CoA) but not GSTP1-1 and GSTM1-1. Acyl-CoAs do not interact with the GSTA1-1 substrate binding site, but probably with a different domain. Results suggest that under increased acyl-CoA concentration, as occurs after exposure to peroxisome proliferators, acyl-CoA binding to the abundant class alpha GSTs may result in strong inhibition of xenobiotic detoxification, Analysis of the binding properties of GSTs and other acyl-CoA binding proteins suggest that under increased acyl-CoA concentration GSTs would be responsible for xenobiotic-CoA binding whereas ACBP would preferentially bind fatty acyl-CoAs.
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    The hypolipidemic drug metabolites nafenopin-CoA and ciprofibroyl-CoA are competitive P2Y1 receptor antagonists
    (2003) Coddou, C; Loyola, G; Boyer, JL; Bronfman, M; Huidobro-Toro, JP
    Coenzyme A (CoA-SH), endogenous and drug-derived CoA-derivatives were tested as putative antagonists of P2Y receptors expressed in Xenopus laevis oocytes, a method used to determine calcium-activated chloride current, an indicator of the activation of these receptors. CoA-SH antagonized reversibly and in a concentration-dependent manner the ATP-gated currents evoked by the human P2Y(1) but not the P2Y(2) receptor. Palmitoyl-CoA was four-fold more potent than CoASH as an antagonist while palmitoyl-carnitine was inactive, highlighting the role of the CoA-SH moiety in the antagonism. The CoA derivatives of nafenopin and ciprofibrate, two clinically relevant hypolipidemic drugs, increased 13 and three-fold the potency of CoA-SH, respectively. The K(B)s of nafenopin-CoA and ciprofibroyl-CoA were 58 and 148 nM, respectively; the slopes of the Schild plots were unitary. Neither 100 muM nafenopin nor ciprofibrate alone altered the P2Y, receptor activity. Neither CoA-SH nor ciprofibroyl-CoA antagonized the rat P2X(2) or the P2X(4) nucleotide receptors nor interacted with the 5-HT2A/C receptors. The bulky drug CoA-SH derivatives identify a hydrophobic pocket, which may serve as a potential target for novel selective P2Y(1) antagonists. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.