Human carboxylesterases in term placentae: enzymatic characterization, molecular cloning and evidence for the existence of multiple forms.
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Yan B, Matoney L, Yang D
Human carboxylesterases in term placentae: enzymatic characterization, molecular cloning and evidence for the existence of multiple forms.
Placenta. 1999 Sep;20(7):599-607.
- PubMed ID
- 10452915 [ View in PubMed]
- Abstract
The placenta is a temporary organ that is known to metabolize numerous endogenous and xenobiotic compounds. Carboxylesterases represent a family of enzymes which hydrolyse a variety of esters, amides and thioesters. Many studies have demonstrated that carboxylesterases are widely distributed among mammalian tissues, but little is known about these enzymes in the placenta. The present study was conducted to establish the kinetic parameters of placental carboxylesterases toward several p -nitrophenol and 1-naphthol esters, and to establish the molecular basis for these enzymes in the placenta. The enzymatic rate of the hydrolysis of 1-naphthylacetate and carboxylic esters of p -nitrophenol as a function of substrate concentration (0.01-1.00 m m) was examined with human placental microsomes pooled from six placentae. Data from these studies yielded a linear Lineweaver-Burk plot with each substrate examined. K(m)values for these substrates ranged from 92 to 370 microm, and V(max)values ranged from 85 to 170 nmol/mg/min. These results suggest that each substrate is hydrolysed by a single enzyme, or enzymes that are kinetically indistinguishable, or that one of them is dominant. Microsomes from all individual placentae contained esterase activity toward all four substrates, and exhibited a one- to three-fold variation. The activity toward p -nitrophenylacetate correlated well with the activity toward 1-naphthylacetate (r(2)=0.957). In contrast, the activity toward p -nitrophenylbutyrate correlated poorly with the activity toward 1-naphthylacetate (r(2)=0.121). These results suggest that placental microsomes have more than one carboxylesterase activity. Screening of a placental cDNA library with gene-trapping hybridization resulted in the isolation of three distinct cDNAs, designated PCE-1, PCE-2 and PCE-3. PCE-1 and PCE-2 have a significant sequence identity (approx 99 per cent) with liver carboxylesterases hCE and hCE-2, respectively. PCE-3 has a 96 per cent sequence identity with hCE but only at the first 874 nucleotide of the 5' end. The rest of the 1396 nucleotides of the 3' end exhibit no significant sequence identity with any known mammalian carboxylesterases. A probe derived from the 3' end of PCE-3 detected an approx 2.2 kb messenger transcript, the size of a regular carboxylesterase. However, the entire PCE-3 cDNA has multiple internal stop codons and encodes only 269 amino acids; half the size of a regular carboxylesterase. Northern blotting experiments detected the transcripts coding for PCE-1, PCE-2 or PCE-3 in all placentae, and the levels of these messengers showed an approx six-fold individual variation. Placenta 6 had the highest activity toward all four substrates, and highest levels of the messengers for PCE-1, PCE-2 and PCE-3. In contrast, placenta 1 had relatively high levels of messengers for PCE-1 and PCE-2, but the activity toward these four substrates was only moderate. These results suggest that a discrepancy between the messenger level and the enzyme protein exists or that there are other as yet unidentified carboxylesterase(s) in the placenta which contribute to the hydrolytic activity. Carboxylesterases are known to involve the detoxication and metabolic activation of various drugs, environmental toxicants and carcinogens. Therefore, placental carboxylesterases have both pharmacological and toxicological significance in the development of the fetus.