Arabidopsis

5.9. Isolation of Arabidopsis microsomes and P450 assay

15 g of rosette leaves of 28 dag Arabidopsis plants were ground in 200 mL extraction buffer (100 mM ascorbic acid, 1 mM EDTA, 100 mM Tris, 20% v/v glycerol, 20% w/v Sucrose, 5 mM Dithiothreitol) with 4.5 g Polyklar AT (Merck) and sea sand. The raw extract was filtered through cloth and centrifuged twice at 15 000 g for 10 min. Microsomes were isolated from the supernatant by centrifugation at 120 000 g for 40 min and resuspended in 1 mL suspension buffer (50 mM potassium phosphate buffer pH 7.5, 20% v/v Glycerol, 1 mM Dithiothreitol). The integrity of the microsomes was tested by measuring the cytochrome C reductase activity as described by Urban et al. (1990).

The in vitro activity of the BX P450 enzymes was tested by incubation of 1 mg total microsomal protein with the respective substrate (2 mM Indole, 1 mM ION, 250 μM HION, 200 μM HBOA) in 100 mM potassium phosphate buffer pH 7.5 and 1 mM NADPH at room temperature. The reaction was stopped after 30 min by addition of 1 vol methanol and precipitated protein was pelleted by centrifugation. 2.5 vol of 100 mM acetic acid were added to the supernatant and the products were extracted three times with 2 vol of ethyl acetate. The solvent was evaporated in a vacuum centrifuge, the remaining products were resolved in methanol and analysed by HPLC.