RESUMO
Acyl-CoAs are 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 Kd values ranging from 200 nM to 5 microM. 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.
Assuntos
Acil Coenzima A/metabolismo , Glutationa Transferase/metabolismo , Proliferadores de Peroxissomos/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Humanos , Fígado/enzimologia , Ligação Proteica , Ratos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
A quick subcellular fractionation procedure using differential centrifugation, which is applicable to isolated and cultured cells, is presented. This technique was developed for studying the subcellular localization of phosphorylated proteins in isolated liver cells after various stimuli, but is also applicable to many other situations. The main difference with the usual techniques is that by including digitonin in the homogenization buffer, the procedure is greatly shortened. Furthermore, because the soluble fraction is separated from the particulate fraction very early in the fractionation procedure, subcellular organelles are not exposed to phosphatases and other soluble enzymes such as esterases and proteases during the fractionation. The entire procedure is carried out in an Eppendorf centrifuge, which allows isolation of the cytosolic fraction in less than 1 min, a washed nuclear fraction in about 4 min, a mitochondrial fraction in less than 10 min, and a washed light mitochondrial L fraction in about 40 min. Judging by the behavior of marker enzymes and the morphology of the fractions, the method is highly comparable to classical procedures.
Assuntos
Centrifugação/métodos , Digitonina/farmacologia , Indicadores e Reagentes/farmacologia , Fígado/efeitos dos fármacos , Fígado/ultraestrutura , Organelas , Animais , Fracionamento Celular/métodos , Células Cultivadas , Ratos , Frações SubcelularesRESUMO
We have shown that patients with previous acute pancreatitis (AP) may have an abnormal catabolism of chylomicron remnants (CMR). Because apoprotein E (Apo E) genetic polymorphism has an important influence on CMR clearance, we compared frequency distribution of Apo E phenotypes in 52 patients with AP, 109 patients with gallstones, and 110 control subjects. Apo E phenotypes were detected by isoelectric focusing and immunoblotting. After adjusting for differences in age and gender, fasting triglyceride level was comparable between the study groups. The frequency distribution of Apo E phenotypes was not different between the three study groups and it was in Hardy-Weinberg equilibrium. The gene frequency for Apo E2 was 0.212, 0.273, and 0.243 in AP, gallstone, and control group, respectively. For Apo E3 it was 0.701, 0.627, and 0.674, and for Apo E4 0.090, 0.100, and 0.083 in the same groups, respectively. Differences were not statistically significant (chi 2). In conclusion, the abnormal catabolism of CMR in patients with AP is not attributable to Apo E polymorphism. An alternative explanation may be sought in the activity of the recently identified hepatocytic Apo E receptor [LDL-related receptor protein (LRP)].