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Supplementary material:

Online Resource 1: COMT activity assay of MB-COMT isolated from the kidneys of S-COMT mutant and wild-type mice

The strategy used for the construction of a mouse strain which only produced membrane bound (Käenmäki et al. 2009) required the methionine 44 to be mutated to threonine in order to assure no translation of the soluble form. There has been some concern that this mutation would affect enzyme activity. To support our theoretical considerations, we isolated and assayed MB-COMT from the kidneys of S-COMT mutated and wild-type mice.

Methods

Shortly, kidney tissues (20-50 mg from 6 mice) were homogenized in 10 volumes of homogenisation buffer (50 mM phosphate, 50 mM NaCl, 1 mM EDTA, 0.32 M sucrose supplemented with protease inhibitors) and after removal of debris and unbroken cells, the crude homogenate was diluted 5 times with 10 mM phosphate and centrifuged at 100000 g for 45 min. The pellet, containing the membrane fraction, was washed with 50 mM phosphate, 300 mM NaCl, 1 mM EDTA. The membranes were resuspended in 10 mM phosphate, 1 mM EDTA, 0.5 mM DTT and frozen at -80°C.

For COMT activity, 10 μl of 10 fold dilution of the membrane preparation were incubated with 5 mM dihydroxybenzoic acid (DHBA) in a buffer containing 50 mM phosphate 50 mM MgCl2, and 2 mM SAM, for 30 min at 37°C. After the proteins were precipitated with perchloric acid and the precipitate was removed, samples were subjected to HPLC detection of the vanillic and isovanillic acid. Meta/para ratios were calculated of the vanillic and isovanillic acid concentrations in the samples. Kidney membranes obtained from full COMT knockout mice were used as a negative control having no COMT activity.

Results

Both in tissue homogenates and isolated membrane fractions, meta/para ratios were close to 20, a landmark of MB-COMT activity (WT 19 ± 6, S-COMT deficient 25 ± 4) and there was no difference in the overall COMT activity between the membrane fractions. A summary figure of the activities in the kidney is given below (Figure 1) for S-COMT mutant and wild-type mice. The situation was very similar also in the liver (not shown). We are somewhat surprised about the large difference between the sexes in both cases. This particular matter needs to be studied further, but in any case, there was no genotype-related difference.

Conclusion

We do not have any reason to believe that the mutation Met44Thr has introduced any secondary effects on MB-COMT activity.

Figure 1 COMT activity (pmol of vanillic acid formed/min/mg protein) of the isolated kidney membrane associated COMT of the wild-type and S-COMT mutated mice in male and female mice. Total number of mice is 6.

Reference

Käenmäki M, Tammimäki A, Garcia-Horsman JA, Myöhänen T, Schendzielorz N, Karayiorgou M, Gogos JA, Männistö PT (2009) Importance of the two forms of catechol-O-methyltransferase (COMT) in L-dopa metabolism: a pharmacokinetic study in two types of Comt gene modified mice. Brit J Pharmacol 158:1884-1894