Browsing by Author "García-Huidobro, T"

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    A major portion of synaptic basal lamina acetylcholinesterase is detached by high salt- and heparin-containing buffers from rat diaphragm muscle and Torpedo electric organ
    (1998) Casanueva, OI; García-Huidobro, T; Campos, EO; Aldunate, R; Garrido, J; Inestrosa, NC
    Collagen-tailed asymmetric acetylcholinesterase (AChE) forms are believed to be anchored to the synaptic basal lamina via electrostatic: interactions involving proteoglycans. However, it was recently found that in avian and rat muscles, high ionic strength or polyanionic buffers could not detach AChE: from cell-surface clusters and that these buffers solubilized intracellular non-junctional asymmetric AChE rather than synaptic: forms of the enzyme. In the present study, asymmetric AChE forms were specifically solubilized by ionic buffers from sg synaptic basal! lamina-enriched fractions, largely devoid of intracellular material, obtained from the electric organ of Torpedo californica and the end plate regions of rat diaphragm muscle, Furthermore, foci of AChE activity were seen to diminish in size, number, and staining intensity when the rat synaptic basal lamina-enriched preparations were treated with the extraction buffers, Pn the case of Torpedo, almost all the AChE activity was removed from the pure basal lamina sheets. We therefore conclude that a major portion of extracellular collagen-tailed AChE is extractable from rat and Torpedo synaptic basal lamina by high ionic strength and heparin buffers,;although some non-extractable AChE activity remains associated with the junctional regions.
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    At least two receptors of asymmetric acetylcholinesterase are present at the synaptic basal lamina of Torpedo electric organ
    (1998) Casanueva, OI; Deprez, P; García-Huidobro, T; Inestrosa, NC
    Asymmetric acetylcholinesterase (AChE) is anchored to the basal lamina (BL) of cholinergic synapses via its collagenic tail, yet the complement of matrix receptors involved in its attachment remains unknown. The development of a novel overlay technique has allowed us to identify two Torpedo BL components that bind asymmetric AChE: a polypeptide of similar to 140kDa and a doublet of 195-215kDa. These were found to stain metachromatically with Coomassie blue R-250, mere solubilized by acetic acid, and were sensitive to collagenase treatment. Upon sequence analysis, the 140kDa polypeptide yielded a characteristic collagenous motif. Another AChE-binding BL constituent, identified by overlay, corresponded to a heparan sulfate proteoglycan. Lastly, we established that this proteoglycan, but not the collagenous proteins, interacted with at least one heparin binding domain of the collagenic tail of AChE. Our results indicate that at least two BL receptors are likely to exist for asymmetric AChE in Torpedo electric organ. (C) 1998 Academic Press.
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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.