Paraoxonasa-1 y arteriosclerosis: al disminuir la oxidación se reduce la respuesta inflamatoria / Paraoxanase-1 and arteriosclerosis: reducing oxidation reduces inflammatory response

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Effects of chronic administration of the peroxisome proliferator, clofibrate, on cytosolic acetyl-CoA hydrolase in rat liver.

The hypolipidemic drug ethyl chlorophenoxyisobutyrate (clofibrate) is known to induce peroxisome proliferation and to be carcinogenic after long term administration to rats and mice. We examined the effects of treatment with this drug for periods of up to 18 months on cytosolic ATP-stimulated and ADP-inhibited acetyl-CoA hydrolase in rat liver. In male Donryu albino rats on a diet containing 0.5% clofibrate, the enzyme activity increased to about 2- and 3-fold the initial level per milligram liver protein and cytosolic protein, respectively, and 2-fold per milligram DNA in 3 days, and then remained at this level for up to 18 months. The increased activity in rats receiving clofibrate for 3 months returned to control level within a week when clofibrate was withdrawn. The change in enzyme activity paralleled the change in the amount of enzyme protein determined by immunoblotting with antibody against purified acetyl-CoA hydrolase from rat liver cytosol. No liver tumors were detected macroscopically after administration of clofibrate for 18 months. However, our results suggest that cytosolic acetyl-CoA hydrolase could be an extraperoxisomal marker enzyme induced by this type of drug.

Sulfhydryl groups of an extramitochondrial acetyl-CoA hydrolase from rat liver.

An extramitochondrial acetyl-CoA hydrolase (EC 3.1.2.1) purified from rat liver was inactivated by heavy metal cations (Hg2+, Cu2+, Cd2+ and Zn2+), which are known to be highly reactive with sulfhydryl groups. Their order of potency for enzyme inactivation was Hg2+ greater than Cu2+ greater than Cd2+ greater than Zn2+. This enzyme was also inactivated by various sulfhydryl-blocking reagents such as p-hydroxymercuribenzoate (PHMB), N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and iodoacetate (IAA). DL-Dithiothreitol (DTT) reversed the inactivation of this enzyme by DTNB markedly, and that by PHMB slightly, but did not reverse the inactivations by NEM, DTNB and IAA. Benzoyl-CoA (a substrate-like competitive inhibitor) and ATP (an activator) greatly protected acetyl-CoA hydrolase from inactivation by PHMB, NEM, DTNB and IAA. These results suggest that the essential sulfhydryl groups are on or near the substrate binding site and nucleotide binding site. The enzyme contained about four sulfhydryl groups per mol of monomer, as estimated with DTNB. When the enzyme was denatured by 4 M guanidine-HCl, about seven sulfhydryl groups per mol of monomer reacted with DTNB. Two of the four sulfhydryl groups of the subunit of the native enzyme reacted with DTNB first without any significant inactivation of the enzyme, but its subsequent reaction with the other two sulfhydryl groups seemed to be involved in the inactivation process.

A cold-labile acetyl-coenzyme-A hydrolase from the supernatant fraction of rat liver. Reactivation and reconstitution of the active species from the inactive monomer.

An acetyl-coenzyme-A hydrolase from the supernatant fraction of rat liver is known to be rapidly inactivated at low temperature. Loss of catalytic activity is accompanied by apparent dissociation of tetrameric and dimeric forms of the enzyme into monomers. It was found that rewarming under appropriate conditions almost completely reversed the cold-induced inactivation and dissociation of the enzyme: At a protein concentration of 14 micrograms/ml, simple rewarming only partially restored the enzyme activity (less than 3% of the original activity), but at a higher concentration of the enzyme or in the presence of 1 mg/ml bovine serum albumin, the reactivation by warming was greater. Warming at 37 degrees C appeared to be optimal for reactivation; warming at 25 degrees C or at 43 degrees C was less effective. Longer exposure to cold did not affect reactivation on rewarming, but on repeated inactivation and reactivation the reactivation decreased to some extent, especially at lower concentrations of enzyme protein. Among various nucleotides tested, ATP greatly enhanced the restoration of the activity, while ITP, UTP and ADP were less effective and AMP, GTP, TTP and CTP had little effect. At an enzyme-protein concentration of 14 micrograms/ml, 2 mM ATP restored the enzyme activity to about 70% of that before cold treatment, while acetyl-CoA (0.5 mM) restored the activity about 50%. High concentrations of phosphate (0.92 M) and pyrophosphate (0.45 M) restored about 80% and 95%, respectively, of the original activity. Sucrose density gradient centrifugation of the active dimer at high enzyme concentration at 4 degrees C for 20 h produced a monomeric form without catalytic activity. Gel filtration showed that simple rewarming mostly converted the monomeric enzyme obtained in this way to the dimeric form, whereas on rewarming with ATP the monomer was mostly converted to a tetrameric form. The dimeric and tetrameric forms both had catalytic activity.