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In this study, we characterized the enzymology of cytosolic catechol-O-methyltransferase (COMT)-catalyzed …


Biology Articles » Biochemistry » Enzymology » Enzymology of Methylation of Tea Catechins and Inhibition of Catechol-O-methyltransferase by ()-Epigallocatechin Gallate » Figures

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- Enzymology of Methylation of Tea Catechins and Inhibition of Catechol-O-methyltransferase by ()-Epigallocatechin Gallate

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Fig. 1.   Representative HPLC chromatograph showing the separation of EGCG, EGC, 4"-MeEGCG, 4',4"-DiMeEGCG, 4'-MeEGC, and 3'-MeEGC.

The chromatographic conditions are described under Materials and Methods. Four channels with potential settings of -100, 100, 300, and 500 mV were used to analyze catechins and methylated catechin metabolites simultaneously.

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Fig. 2.   A summary of possible sites of methylation (M) and glucuronidation (G) of EGCG and EGC.


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Fig. 3.   Time course of methylation of catechins by rat liver cytosolic COMT.

Incubations were conducted using 1 mg/ml of rat hepatic cytosolic proteins and 1 µM catechins as the substrate: EGCG (A), 4"-MeEGCG (B), EGCG or EGC (C), and EGCG-4"-Gluc, EGCG, or EGC (D), in the presence of 200 µM (A and B) or 50 µM (C and D) SAM. The formation of methylated EGCG and EGC was quantified with HPLC. The remaining substrate levels were shown (D). Values represent mean of duplicate analysis.

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Fig. 4.   Concentration-dependent methylation of EGCG and EGC by liver cytosol from humans (open circle), mice (), and rats (black-square).

Incubations were conducted for 2 min with EGCG (A) or 5 min with EGC (B) in the presence of 1 mg/ml of hepatic cytosolic proteins and 50 µM SAM. The formation of methylated EGCG and EGC was quantified with HPLC. Values represent mean of duplicate analysis.

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Fig. 5.   Methylation of EGCG and 4"-MeEGCG by rat liver cytosol.

Incubations were conducted for 20 min using 1 mg/ml of hepatic cytosolic proteins in the presence of 50 µM SAM and different concentrations of EGCG or 4"-MeEGCG. The formation of methylated products was quantified with HPLC. With EGCG, the sum of both products (4"-MeEGCG and 4',4"-DiMeEGCG) was used in the plot (A). 4"-MeEGCG and 4',4"-DiMeEGCG were also shown as percentage of the sum (B). Values represent mean of duplicate analysis.

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Fig. 6.   Methylation of EGCG and EGC by cytosol preparation from small intestine of rats () and mice (open circle).

Incubations were conducted for 20 min using 1 mg/ml of intestinal cytosolic proteins in the presence of 50 µM SAM and different concentrations of EGCG (A) or EGC (B). The formation of methylated EGCG and EGC was quantified with HPLC. Values represent mean of duplicate analysis.

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Fig. 7.   Product-ion mass spectrum of methylation products of EGCG glucuronides.

Incubations were conducted for 20 min using 1 mg/ml of rat hepatic cytosolic proteins in the presence of 50 µM SAM and 1 µM EGCG-3'-Gluc. The formation of EGCG metabolites was detected with LC/MS/MS. The spectrum of the product mono-methylated EGCG-3'-Gluc is shown in (A) with parent ion of m/z 647. The same spectrum was also observed with a mono-glucuronide of 4"-MeEGCG formed by incubation of 100 µM 4"-MeEGCG with 1 mM UDPGA in the presence of mouse hepatic microsomal protein (2 mg/ml) for 30 min (B). LC/MS/MS chromatogram of di-methylated EGCG-4"-Gluc with parent ion of m/z 661 (C); chromatogram obtained by monitoring the deprotonated product ion of m/z 485. Product-ion mass spectrum of di-methylated EGCG-4"-Gluc, P1 (D) and P2 (E).

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Fig. 8.   Concentration-dependent inhibition of COMT activities by EGCG.

Lineweaver-Burk plot of reciprocal enzymatic activity versus reciprocal SAM (B), EGC (C-E), or L-DOPA (F) concentrations. Incubations were conducted for 10 min using 1 mg/ml of rat or mouse hepatic cytosolic proteins in the presence of 10 µM EGC, 50 µM SAM, and different concentrations of EGCG (A). (B-E) Incubations were conducted for 5 min using 1 mg/ml of rat hepatic cytosolic proteins in the presence of EGCG (0, 0.5, 1.0, and 2.0 µM) and 1 mM EGC and different concentrations of SAM (in B); and 200 µM SAM, different concentrations of EGC, 0, 0.5, 1.0, and 2.0 µM of EGCG (in C), 4"-MeEGCG (in D), or 4',4"-DiMeEGCG (in E). 25 units porcine liver COMT, different concentrations (5, 10, 20, 50, and 100 µM) of L-DOPA, 0.2 mM SAM, and different concentrations of 4',4"-DiMeEGCG (0, 0.2, 0.4, and 0.8 µM) were used (F). The formation of 4'-MeEGC or 3-MeDOPA was quantified with HPLC. Values represent mean of duplicate analysis.

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