Rabbit Liver Carboxylesterase Research Articles

Purification and Cloning of a Broad Substrate Specificity Human Liver Carboxylesterase That Catalyzes the Hydrolysis of Cocaine and Heroin

A human liver carboxylesterase (hCE-2) that catalyzes the hydrolysis of the benzoyl group of cocaine and the acetyl groups of 4-methylumbelliferyl acetate, heroin, and 6-monoacetylmorphine was purified from human liver. The purified enzyme exhibited a single band on SDS-polyacrylamide gel electrophoresis with a subunit mass of approximately 60 kDa. The native enzyme was monomeric. The isoelectric point of hCE-2 was approximately 4.9. Treatment with endoglycosidase H caused an increase in electrophoretic mobility indicating that the liver carboxylesterase was a glycoprotein of the high mannose type. The complete cDNA nucleotide sequence was determined. The authenticity of the cDNA was confirmed by a perfect sequence match of 78 amino acids derived from the hCE-2 purified from human liver. The mature 533-amino acid enzyme encoded by this cDNA shared highest sequence identity with the rabbit liver carboxylesterase form 2 (73%) and the hamster liver carboxylesterase AT51p (67%). Carboxylesterases with high sequence identity to hCE-2 have not been reported in mouse and rat liver. hCE-2 exhibited different drug ester substrate specificity from the human liver carboxylesterase called hCE-1, which hydrolyzes the methyl ester of cocaine. hCE-2 had higher catalytic efficiencies for hydrolysis of 4-methylumbelliferyl acetate, heroin, and 6-monoacetylmorphine and greater inhibition by eserine than hCE-1. hCE-2 may play an important role in the degradation of cocaine and heroin in human tissues.

Purification of human monocyte‐specific esterase (MSE): molecular and kinetic characteristics

Human monocyte-specific esterase (MSE) derived from leukaemic AMoL-M5 blast cells was purified to homogeneity by the sequential application of anion-exchange, hydrophobic interaction, affinity and gel filtration chromatographic procedures. The resulting enzymatically active MSE primarily existed as an apparent trimer which, under both reducing and non-reducing conditions, dissociated to an inactive 63.4 kD glycoprotein monomer. Electrophoretic studies further confirmed that purified MSE comprised a narrow series of pI (5.5-6.1) forms and one main charge species. Neuraminidase failed to modify observed pI values for individual MSE isoenzymes, and endoglycosidase H treatment revealed that the deglycosylated form of MSE had an apparent molecular weight of 60.1 kD. In support of the known cytochemical characteristics of human MSE, substrate kinetic studies demonstrated that purified enzyme hydrolysed esters of higher acyl chain length (butyrate > propionate > acetate) but did not show peptidase activity. Amino acid sequencing of the MSE N-terminus further revealed that there was almost complete identity with human alveolar macrophage esterase and close similarities with rat and rabbit liver carboxylesterases. These kinetic and molecular studies are particularly important in elucidating the biological and functional role(s) of one of the few haemopoietic cell enzymes that can be considered truly lineage-specific.

Cloning and sequencing of a human liver carboxylesterase isoenzyme

A human liver lambda gtll library was screened with antibodies raised to a purified rat liver carboxylesterase, and several clones were isolated and sequenced. The longest cDNA contained an open reading frame of 507 amino acids that represented 92% of the sequence of a mature carboxylesterase protein. This sequence possessed many structural features that are highly conserved among rabbit and rat liver carboxylesterase proteins, including Ser, His, and Asp residues that comprise the active site, two pairs of Cys residues that may participate in disulfide bond formation, and one Asn-Xxx-Thr site for N-linked carbohydrate addition. When the clone was used to probe human liver genomic DNA that had been digested with various restriction enzymes, many hybridizing bands of differing intensities were observed. The results suggest that the carboxyl-esterases exist as several isoenzymes in humans, and that they are encoded by multiple genes.

Studies on organophosphorus impurities in technical malathion: Inhibition of carboxylesterases and the stability of isomalathion

Four organophosphorus esters found as impurities in technical malathion were synthesized, and their abilities to inhibit monomeric and oligomeric carboxylesterases from rabbit liver, as well as type I and type II esterases from porcine liver, were studied. The equilibrium dissociation constant ( K d ), phosphorylation constant ( k 2), and bimolecular rate constant ( k i ) were determined in the presence of substrate. Inhibition, as judged by the k i , was in the following order: isomalathion > O,S,S-trimethyl phosphorodithioate > O,O,S-trimethyl phosphorothioate > O,O,S-trimethyl phosphorodithioate. All of the k 2 values were relatively small, indicating that the k d values contribute most to the overall inhibitory power. Rabbit liver carboxylesterases are more sensitive to inhibition by isomalathion than porcine liver esterases, reflecting a species difference. Isomalathion undergoes nonenzymatic degradation in the presence of fluoride ions, producing diethyl thiomalate. A kinetic investigation of the nonenzymatic degradation of isomalathion was conducted, and reaction rate constants were determined in phosphate buffer at various molarities, temperatures, and pH values.