Binding and hydrolysis of meperidine by human liver carboxylesterase hCE-1. (17/1703)

Human liver carboxylesterases catalyze the hydrolysis of apolar drug or xenobiotic esters into more soluble acid and alcohol products for elimination. Two carboxylesterases, hCE-1 and hCE-2, have been purified and characterized with respect to their role in cocaine and heroin hydrolysis. The binding of meperidine (Demerol) and propoxyphene (Darvon) was examined in a competitive binding, spectrophotometric assay. The hCE-1 and hCE-2 bound both drugs, with Ki values in the 0.4- to 1.3-mM range. Meperidine was hydrolyzed to meperidinic acid and ethanol by hCE-1 but not hCE-2. The Km of hCE-1 for meperidine was 1.9 mM and the kcat (catalytic rate constant) was 0.67 min-1. Hydrolysis of meperidine by hCE-1 was consistent with its specificity for hydrolysis of esters containing simple aliphatic alcohol substituents. Hence, hCE-1 in human liver microsomes may play an important role in meperidine elimination. Propoxyphene was not hydrolyzed by hCE-1 or hCE-2. This observation is consistent with the absence of a major hydrolytic pathway for propoxyphene metabolism in humans.  (+info)

A non-AUG-defined alternative open reading frame of the intestinal carboxyl esterase mRNA generates an epitope recognized by renal cell carcinoma-reactive tumor-infiltrating lymphocytes in situ. (18/1703)

A number of Ags recognized by tumor-reactive T cells have been characterized, including nonmutated gene products and a variety of epitopes shown to arise from either mutated or alternatively processed transcripts. Here, we report that the screening of a cDNA library with an HLA-B7-restricted renal cell carcinoma-reactive T cell clone derived from tumor-infiltrating lymphocytes (TILs) that were clonally amplified in vivo (as assessed by TCRBV complementarity determining region-3 length distribution analysis) resulted in the isolation of a nonamer encoded by an alternative open reading frame (ORF) (a +1 frameshift) of the intestinal carboxyl esterase gene. This peptide binds HLA-B*0702-presenting molecules as assessed in an immunofluorescence-based peptide binding assay using transfected T2 cells. Constitutive expression of this alternative ORF protein was observed in all transformed HLA-B7+ renal cell lines that were recognized in cytotoxicity assays by the TILs. The intestinal carboxyl esterase gene is transcribed in renal cell carcinoma tumors as well as in normal liver, intestinal, or renal tissues. Mutation of the natural ATG translation initiation site did not alter recognition, indicating that frameshifting (i.e., slippage of the ribosome forward) and recoding are not involved. In addition, a point mutation of the three AUG codons that may be used as alternative translation initiation sites in the +1 ORF did not abolish recognition, whereas mutation of an upstream ACG codon did, indicating that the latter codon initiates the translation of the alternative ORF. These results further extend the types of Ags that can be recognized by tumor-reactive TILs in situ (i.e., leading to clonal T cell expansion).  (+info)

Consequences of breaking the Asp-His hydrogen bond of the catalytic triad: effects on the structure and dynamics of the serine esterase cutinase. (19/1703)

The objective of this study has been to investigate the effects on the structure and dynamics that take place with the breaking of the Asp-His hydrogen bond in the catalytic triad Asp175-His188-Ser120 of the serine esterase cutinase in the ground state. Four molecular dynamics simulations were performed on this enzyme in solution. The starting structures in two simulations had the Asp175-His188 hydrogen bond intact, and in two simulations the Asp175-His188 hydrogen bond was broken. Conformations of the residues comprising the catalytic triad are well behaved during both simulations containing the intact Asp175-His188 hydrogen bond. Short contacts of less than 2.6 A were observed in 1.2% of the sampled distances between the carboxylate oxygens of Asp175 and the NE2 of His188. The simulations showed that the active site residues exhibit a great deal of mobility when the Asp175-His188 hydrogen bond is broken. In the two simulations in which the Asp175-His188 hydrogen bond is not present, the final geometries for the residues in the catalytic triad are not in catalytically productive conformations. In both simulations, Asp175 and His188 are more than 6 A apart in the final structure from dynamics, and the side chains of Ser120 and Asp175 are in closer proximity to the NE2 of His188 than to ND1. Nonlocal effects on the structure of cutinase were observed. A loop formed by residues 26-31, which is on the opposite end of the protein relative to the active site, was greatly affected. Further changes in the dynamics of cutinase were determined from quasiharmonic mode analysis. The frequency of the second lowest mode was greatly reduced when the Asp175-His188 hydrogen bond was broken, and several higher modes showed lower frequencies. All four simulations showed that the oxyanion hole, composed of residues Ser42 and Gln121, is stable. Only one of the hydrogen bonds (Ser42 OG to Gln121 NE2) observed in the crystal structure that stabilize the conformation of Ser42 OG persisted throughout the simulations. This hydrogen bond appears to be enough for the oxyanion hole to retain its structural integrity.  (+info)

Microsomal long-chain acyl-CoA thioesterase (carboxylesterase ES-4) is regulated by thyroxine. (20/1703)

Long chain acyl-CoA thioesterase activity is mainly located in microsomes after subcellular fractionation of liver from untreated rats. The physiological function and regulation of expression of this activity is not known. In the present study we have investigated the effect of thyroxine on expression of carboxylesterase ES-4, the major acyl-CoA thioesterase of liver microsomes. Thyroidectomy of rats decreased the palmitoyl-CoA thioesterase activity to about 25% of normal activity. This decrease was accompanied by similar decreases at the protein and mRNA levels (31% and 57%, respectively, of controls). Treatment with thyroxine completely reversed the effect of thyroidectomy and resulted in elevated levels in both thyroidectomized and control rats. For reasons of comparison we also studied the possibility that ES-10 and ES-2, two other members of the same gene family, are affected by thyroxine. ES-10 was not changed at the protein or mRNA level by any of the treatments, while ES-2 expression in liver was decreased by thyroxine treatment. The data shows that changes in activity and expression of ES-4 correlate to thyroxine status in the rat suggesting a physiological regulatory role by this hormone. Since thyroxine regulates the expression of lipogenic enzymes, these results are consistent with a function for this microsomal acyl-CoA thioesterase in fatty acid synthesis and/or secretion, rather than in oxidative degradation of fatty acids.  (+info)

CPT-11 converting carboxylesterase and topoisomerase activities in tumour and normal colon and liver tissues. (21/1703)

CPT-11 is a prodrug activated by carboxylesterases to the active metabolite SN-38 which is a potent inhibitor of topoisomerase I. CPT-11 is of clinical interest in the treatment of colorectal cancer. We evaluated the activities of CPT-11 converting carboxylesterase (CPT-CE) and topoisomerase I (topo I) in 53 colorectal tumours, in eight liver metastases and in normal tissue adjacent to the tumours. Both CPT-CE and topo I activities were widely variable in the malignant and the normal tissue of patients with colorectal carcinomas. CPT-CE was only two to threefold lower in primary tumours compared to normal liver, suggesting that a local conversion to SN-38 might occur in tumour cells. CPT-CE was similar in liver and in normal colon tissues. Levels of topo I in tumour ranged from 580 to 84 900 U mg protein(-1) and was above 40 000 U mg protein(-1) in 11 of 53 patients. Similarly, a very high ratio (> 5) between tumour and normal tissues were observed in 12 of 53 patients. An inverse correlation was observed between the topo I activity and the clinical stage of disease. Clinical studies are in progress in our institution to explore a possible relationship between CPT-CE and topo I activities in tumour cells and the response to CPT-11-based chemotherapy in patients with colorectal cancer.  (+info)

Paraoxonase polymorphisms are not associated with cardiovascular risk in renal transplant recipients. (22/1703)

BACKGROUND: Paraoxonase (PON1) gene variants have been identified as risk factors for cardiovascular disease (CVD). There are two common PON1 polymorphisms at position 55 (Leu-Met change) and 192 (Gln-Arg change) of the amino acid chain. Leucine at position 55 and arginine at position 192 have been associated with increased cardiovascular risk. The increased prevalence of CVD in renal transplant recipients can be only partly explained by the increased prevalence of conventional risk factors. METHODS: We therefore investigated PON1 polymorphisms in renal transplant recipients (N = 491) with (N = 103) and without CVD (N = 388) using polymerase chain reaction-restriction fragment length analysis. PON1 polymorphisms and their associated PON1/arylesterase activities were also assessed in a subgroup of patients (N = 165). RESULTS: The genotype distribution and allele frequencies for both polymorphisms were similar in both groups. The frequencies for LL, LM, and MM genotypes for the 55 position in patients with CVD were 0.39, 0.51, and 0.10, respectively, compared with 0.43, 0.43, and 0.14 in patients without CVD (P = 0.31). The distribution for the QQ, QR, and RR genotypes at the 192 position were 0.48, 0.43, and 0.09, respectively, in patients with CVD compared with 0.46, 0.46, and 0.08 in patients without CVD (P = 0.8). There were highly significant differences in serum activities of PON1/arylesterase between genotypes defined by 55 and 192 polymorphisms. Leucine at position 55 and arginine at position 192 were associated with higher activities. CONCLUSION: These data indicate that there is no association between the PON1 gene variants, conferring higher enzyme activity, and the increased cardiovascular risk in renal transplant recipients.  (+info)

Tryptophan mediated photoreduction of disulfide bond causes unusual fluorescence behaviour of Fusarium solani pisi cutinase. (23/1703)

The fluorescence signal of the single tryptophan residue (Trp69) of Fusarium solani pisi cutinase is highly quenched. However, prolonged irradiation of the enzyme in the tryptophan absorption band causes an increase of the tryptophan fluorescence quantum yield by an order of magnitude. By using a combination of NMR spectroscopy and chemical detection of free thiol groups with a sulfhydryl reagent we could unambiguously show that the unusual fluorescence behaviour of Trp69 in cutinase is caused by the breaking of the disulfide bond between Cys31 and Cys109 upon irradiation, while the amide-aromatic hydrogen bond between Ala32 and Trp69 remains intact. This is the first example of tryptophan mediated photoreduction of a disulfide bond in proteins.  (+info)

Clustering of the chemoreceptor complex in Escherichia coli is independent of the methyltransferase CheR and the methylesterase CheB. (24/1703)

The Escherichia coli chemoreceptors and their associated cytoplasmic proteins, CheA and CheW, cluster predominantly at the cell poles. The nature of the clustering remains a mystery. Recent studies suggest that CheR binding to and/or methylation of the chemoreceptors may play a role in chemoreceptor complex aggregation. In this study, we examined the intracellular distribution of the chemoreceptors by immunoelectron microscopy in strains lacking either the methyltransferase CheR or the methylesterase CheB. The localization data revealed that, in vivo, aggregation of the chemoreceptor complex was independent of either CheR or CheB.  (+info)