Article Figures & Data
Figures
- Fig. 1.
CPT-11 metabolism.
CPT-11 is oxidized by cytochrome P450 3A4 (CYP3A4) isoenzyme to produce 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecin (APC) and 7-ethyl-10-[4-(1-piperidino)-1-amino]-carbonyloxycamptothecin (NPC). NPC, APC, and CPT-11 are metabolized by carboxylesterases (CES) to produce active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38). SN-38 is inactivated by UDP-glucuronosyltransferase isoform 1A1 to SN-38 glucuronide.
- Fig. 2.
SDS-PAGE of samples at various stages of purification of the recombinant human CES3 isoenzyme expressed in Sf9 insect cells.
Lane 1, 40 μg of cell lysate; lane 2, 5 μg of protein after concanavalin A chromatography; lane 3, 5 μg of protein after preparative nondenaturing gel; lane 4, molecular weight markers.
- Fig. 3.
Predicted protein sequence for CES3 gene (GI: 7019977).
This protein has several characteristics of the carboxylesterase family; the N-terminal signal peptide and the potential C-terminal endoplasmic reticulum retention signal both are shown in italics. The active-site residues Ser229, Glu347, and His460 predicted from sequence alignment with other isoenzymes are shown in bold face, and the positions of the four conserved cysteine residues are marked by *. One potential glycosylation site, Asn105, is shown in bold and underlined. The peptides that were identified by MALDI-TOF mass spectrometry of Glu-C and tryptic peptides of the recombinant protein are underlined.
- Fig. 4.
Human multi-tissue Northern blot probed for CES3.
A CES3-specific probe was generated by PCR across 919-1234 nt of CES3 gene (GI: 7019977). 32P-dCTP was used for random primed labeling of the probe, and the blot was hybridized overnight. The autoradiograph was developed after 8 days of exposure.
- Fig. 5.
The exon-intron map of CES3 gene on human chromosome 16 (Contig; GI: NT_010478) as determined by Spidey software.
One hundred percent of the cDNA sequence (GI: 7019977) matched with the genomic sequence. The cDNA was distributed over 13 exons (shown as boxes) with 100% identity and with no gaps and no mismatches. The mRNA coordinates based on GI: 7019977 and genomic coordinates based on Contig; GI: NT_010478 for each exon are as follows: exon 1, 1–100, 632,917–633,016; exon 2, 101–305, 634,806–635,010; exon 3, 306–444, 635,118–635,256; exon 4, 445–578, 635,429–635,562; exon 5, 579–732, 635,984–636,137; exon 6, 733–837, 636,250–636,354; exon 7, 838–939, 637,871–637,972; exon 8, 940-1080, 638,352–638,492; exon 9, 1081–1161, 641,306–641,386; exon 10, 1162–1309, 642,799–642,946; exon 11, 1310–1459, 643,983–644,132; exon 12, 1460–1538, 644,295–644,373; exon 13, 1539–2093, 644,481–645,035. Blasting the human genome with the GI: 7019977 also identified this gene on human chromosome 16 and adjacent to CES2 gene.
Tables
Volume
α-NA Activitya
Total Activity
Protein
Total Protein
Specific Activity
Fold Purificationml U/ml U mg/ml mg U/mg Cell Lysate 100 38 1.26 48 9.4 356 0.13 1 Concanavalin A concentrate 0.7 5.6 3.9 4.0 2.8 1.4 10 Prep Cell concentrate 0.4 9.3 3.9 1.6 0.67 5.8 44 -
↵ a The activity was monitored by α-napthylacetate (α-NA) hydrolase with Fast Violet Blue. One unit is defined as 1 μmol of product formed per minute
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Volume
4-MUAa Activity
Total Activity
Protein
Total Protein
Specific Activityml U/ml U mg/ml mg U/mg Liver homogenate 220 5.29 1164 18.8 4136 0.28 Concentrate after DE52 123 8.6 1057 16.7 2054 0.51 Concanavalin A concentrate 4 186 742 8.8 35 21.2 Prep Cell concentrate CES1A1 2 77 154 11.6 23.2 6.6 Prep Cell concentrate CES2 0.5 814 407 6.8 3.4 120 -
↵ a The 4-methylumbelliferyl acetate (MUA) activity was measured at 37°C in 90 mM KH2PO4, 40 mM KCl, pH 7.4 buffer with 0.5 mM 4-methylumbelliferyl acetate. The formation of product, 4-methylumbelliferone, was monitored spectrophotometrically at 350 nm
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- TABLE 3
Steady state kinetic studies for carboxylesterase isoenzymes with CPT-11, NPC, and APC as substrates The substrates, CPT-11 (0–200 μM), NPC (0–200 μM), or APC (0–1000 μM) were incubated in 20 mM Hepes buffer, pH 7.0, with 10% ethylene glycol at 37°C for 2–24 h in 250 μl of reaction volume. The product, SN-38, was extracted with 2 × 5 ml CHCl3 or with Oasis HLB columns and quantitated on an Agilent 1100 HPLC equipped with a fluorescence detector, with excitation set at 375 nm and emission monitored at 560 nm. The concentration of SN-38 was determined from the standard curve generated under identical conditions with each experiment. The substrate consumption was always less than 6%.
Isoenzyme
Substrate
KM a
kcat
Catalytic Efficiency
Normalization of Catalytic Efficiency to CES2 for Each SubstrateμM 10-3 min-1 min-1 mM-1 CES1A1 CPT-11 39 ± 3 4.9 ± 0.1 0.13 1 CES2 CPT-11 1.1 ± 0.1 13 ± 0.2 12 100 CES3 CPT-11 137 ± 11 0.9 ± 0.04 0.0063 0.05 CES1A1 NPC 80 ± 6 1.8 ± 0.06 0.023 0.5 CES2 NPC 3.2 ± 0.2 16 ± 0.2 5 100 CES3 NPC 460 ± 50 0.26 ± 0.01 0.00056 0.01 CES2
APC
270 ± 50
2.45
0.015
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↵ a Independent estimations of KM and kcat values were made for each enzyme and substrate. The values for one such experiment with standard error for the fit are reported. The estimations were made by nonlinear regression analysis of the data to the Michaelis-Menten equation (GraFit 4.0; Erithacus Software Ltd.)
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