Heterogeneity of carboxylesterases in rat liver cells.

Rat liver cells were separated into parenchymal cells (PC), Kupffer cells (KC) and endothelial cells (EC). The distribution of carboxylesterases (EC 3.1.1.1) between these cell types was investigated by PAGE and chromatogenic substrate staining, and compared with the results for total liver preparation and individual isoenzymes isolated by chromatofocusing. All of the liver carboxylesterase isoenzymes could be detected in the PC, whereas in both KC and EC only those with isoelectric point (pI) 6.4/6.2 could be detected. Use of carboxylesterase inhibitors like bis-(4-nitrophenyl)phosphate and paraoxon, and organophosphorus compound hydrolase inhibitors like 4-hydroxymercuribenzoate and EDTA confirmed that these esterases were of the carboxylesterase type.

Monoclonal antibodies distinguish between carboxylesterase isoenzymes in different tissues of rat and guinea pig.

The carboxylesterase (CarbE) activity in the main tissues (lung, liver, plasma and small intestine) of both the rat and guinea pig was separated by chromatofocusing. The three CarbE isoenzymes in the small intestine from both species showed nearly identical pI values. Monoclonal antibodies (MAbs) raised against rat lung CarbE (pI 5.8) were used in enzyme-linked immunosorbent assays to distinguish between these closely related CarbE isoenzymes. None of the MAbs did bind to the active site as no inhibition of the enzyme was seen when the MAbs were added. The immunological study showed a strong relationship between lung CarbE (pI 5.8) and the rat liver CarbE (pI 6.0). The MAbs were also strongly bound to the high pI forms of the CarbE isoenzymes in plasma and small intestine from both rat and guinea pig, but not with the low pI forms. These results indicate that two immunochemically distinct categories of CarbE isoenzymes are present in the plasma and small intestine.

Carboxylesterases in guinea pig. A comparison of the different isoenzymes with regard to inhibition by organophosphorus compounds in vivo and in vitro.

The different isoenzymes of carboxylesterase (CarbE) from guinea pig liver, lung and plasma were separated by gel filtration and chromatofocusing. The isoenzymes were characterized by inhibition with several different organophosphorus compounds. The bimolecular rate constants showed the same tendency of decreased inhibition for all of the isoenzymes in the order; paraoxon greater than soman greater than diisopropylphosphofluoridate (DFP) greater than bis(p-nitrophenyl)phosphate. With two exceptions the inhibition constants for the different isoenzymes differed little. Subcutaneous and intraperitoneal administration of DFP and paraoxon rapidly inhibited the CarbE activity in guinea pig plasma. Much higher doses were necessary to obtain a marked inhibition in lung and liver. About 25% of CarbE activity in lung was resistant to paraoxon and DFP inhibition. Gel filtration of lung homogenate after treatment with the organophosphorus compounds showed that the CarbE activity of the medium molecular mass fractions was inhibited only weakly. This could be due to reduced accessibility to some of the lung CarbE isoenzymes.

Purification and characterization of carboxylesterases from rat lung.

Carboxylesterase (EC 3.1.1.1) has played an important part in our understanding of the toxicokinetic behaviour of the organophosphorus cholinesterase inhibitors. Carboxylesterases are a heterogeneous group of enzymes that can be separated on the basis of their isoelectric points and by their substrate-specificity. We have purified the isoenzyme (pI 5.8) present in greatest activity in rat lung to near homogeneity. The enzyme was purified by (NH4)2SO4 precipitation, gel filtration, chromatofocusing, separation on anion- and cation-exchangers and hydrophobic-interaction chromatography. The purified enzyme has a molecular mass of approx. 180 kDa with subunits of 60 kDa. The substrate and inhibitor specificities of the enzyme have been characterized. Edman degradation revealed the first 19 amino acid residues of the enzyme. The N-terminus was found to be tyrosine. Inhibition of the enzyme by organophosphorus compounds differed greatly from that of cholinesterases, despite the partial analogy at the N-terminal region.

Studies on the binding of copper to dopamine beta-monooxygenase and other proteins using the Cu2+ ion-selective electrode.

The binding of Cu2+ to native and copper-free dopamine beta-monooxygenase has been investigated by potentiometric titrations using a Cu2+-selective electrode. Stoichiometric formation constants have been determined from regression analysis of the resulting titration curves. The results establish a stoichiometry of four high-affinity binding sites for Cu/+ (log Kf approximately 11) per enzyme tetramer, and more binding sites of lower affinity (log Kf approximately 5-7). The data for binding of the first four Cu2+ to the enzyme tetramer indicate interactions in the binding to the sites. Bovine serum albumin, metal-free carbonic anhydrase, and ovotransferrin have also been titrated with Cu2+, and the formation constants of both high-affinity binding sites and other sites have been determined. The stoichiometry of one high-affinity binding site of Cu2+ for carbonic anhydrase (log Kf approximately 10-12) and two sites for ovotransferrin (log Kf approximately 11) agree with the reported metal binding properties of these proteins. The number of high-affinity binding sites for bovine serum albumin was pH dependent.