Human esterase D (carboxylesterase; carboxylic-ester hydrolase, EC 3.1.1.1), a genetic marker of retinoblastoma, was purified to biochemical homogeneity from erythrocytes. The purification scheme including carboxymethylcellulose, phenyl-Sepharose, chromatofocusing, and hydroxylapatite chromatographies resulted in a 10,000-fold purification of the enzyme with 15% recovery of total activity. The Km of esterase D was estimated to be 10 x 10-6 M using 4-methylumbelliferyl acetate as substrate. (8,9). In addition, the defective gene of Wilson disease was found to be linked to the esterase D gene (10). Thus, it is important to investigate the nature of the esterase D protein and its gene so as to understand these inherited disorders.Esterase D has been found in most tissues, but not much is known about its structure and function. A protocol for partial purification of this enzyme has been described (11).However, it remains difficult to obtain a sufficient amount of the homogeneous enzyme and to generate specific antibodies. In this communication, we describe the purification and characterization of this enzyme as well as the preparation of esterase D-specific polyclonal and monoclonal antibodies. Furthermore, we observed that this enzyme was mainly distributed in liver and kidney and could be induced by treatment with phenobarbital but not with phorbol ester. MATERIALS AND METHODSEsterase D Enzymatic Assays. (i) Quantitative assay. Esterase D activity was determined essentially as described (11). The reaction was started by adding the enzyme into 1 ml of reaction buffer containing 50 mM potassium phosphate, 1 mM EDTA (pH 6.0), with 0.1 mM 4-methylumbelliferyl acetate as substrate. The increase in absorbance at 340 nm was recorded. A unit of activity was defined as the amount of enzyme that hydrolyzed 1 /.kmol of substrate per min at 230C using 7.27 mM-1 cm-1 as the coefficient of absorption.(ii) Spot test. A rapid semiquantitative test was developed by us for assaying enzyme activity in column fractions.Briefly, 5 jul of 4-methylumbelliferyl acetate (20 Ag/ml) in sodium acetate (pH 5.2) was spotted onto a piece of SaranWrap over a UV transilluminator together with 5 A.l of enzyme samples from each column fraction. The intensity of fluorescence emitted corresponded with the enzyme activity.(iii) Qualitative assay. Electrophoresis in 1% agarose gels was performed as described (12) with slight modifications. The agarose gel buffer was a 1:5 dilution ofrunning buffer that contained 62 mM Tris, 15.5 mM citric acid, 18 mM boric acid, and 1.65 mM lithium hydroxide (pH 7.5). Electrophoresis was performed at 40C for 90 min at 20 V/cm. The gel was stained in 50 ml of 50 mM sodium acetate (pH 5.2) containing 1.0 ml of 4-methylumbelliferyl acetate (1 mg/ml in acetone) for 5 min and then photographed under UV light.Purification of Human Esterase D.Step 1. Outdated human erythrocytes were washed once in phosphate-buffered saline (PBS) with 1 mM EDTA. A precooled (-20°C) mixture of chloroform and butanol was added to the pa...
The percent of protein-free and protein-bound methadone were separated in methadone-spiked bank and artificial plasma, and in plasma samples taken from methadone-maintained patients using the Amicon MPS-1 ultrafiltration device. Following the separation procedure, protein-bound and protein-free methadone were extracted from the protein-bound and free fractions, and their respective concentrations were determined by gas chromatography and nitrogen-phosphorus detection. Eighty-five patient samples from 38 men and 10 women receiving methadone maintenance were collected and subjected to the ultrafiltration methodology. Two independent procedures demonstrated that, following the ultrafiltration process, no proteins were measurable in the filtrate. In addition, the ultrafiltration process was found to function independently of the concentration of methadone and the volume of sample, assuming the amount filtered never exceeded 40% of the original volume. In the patient samples, the %-free methadone varied sixfold across all patients. Female patients were found to have a mean +/- SD %-free methadone of 11.9 +/- 3.8% vs. 10.1 +/- 3.4% for men. Pearson correlation values suggest that steady-state protein-free methadone levels (r = 0.521) and total methadone levels (r = 0.491) rise as methadone dose is increased. Corresponding to these results, free methadone levels are highly correlated with total methadone levels (Pearson r = 0.85). The Amicon MPS-1 ultrafiltration device appears to be a reliable and relatively easy system to use for separating protein-free from protein-bound methadone, though further study is required to clarify the clinical applications of free methadone levels.
The development of homozygosity or hemizygosity in the 13q14 region by deletion, mitotic recombination, or chromosomal loss has been interpreted as a primary event in retinoblastoma. This finding is consistent with the hypothesis that inactivation of both alleles of a gene located at 13q14.11 is required for tumorigenesis. Observations reported by Benedict and colleagues in one case of bilateral retinoblastoma, LA-RB 69, provided early evidence in favor of this hypothesis. By examining levels of esterase D, an enzyme also mapping to 13q14.11, it was previously inferred that one chromosome 13 was lost. Using a rabbit anti-esterase D antibody and the esterase D cDNA probe, we have found that low but detectable quantities of esterase D protein and enzymatic activity are present in tumor cells from LA-RB 69; fibroblasts from this patient contain two copies of the esterase D gene, indicated by heterozygosity at an Apa I polymorphic site within this gene; and tumor cells from the same patient are homozygous at this site, indicating loss and reduplication of the esterase D locus. These results demonstrate that one of the two esterase D alleles in this patient acted as a "null" or silent allele--that is, was present in the genome with markedly decreased protein expression. This mutant allele acted as a marker for tumor-associated loss of chromosome 13 heterozygosity, in concordance with previous proposals.
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