Arabidopsis
Creators
- 1. * & Chair of Plant Breeding, Technical University of Munich, Liesel-Beckman Str. 2, 85354, Freising, Germany
Description
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.
Notes
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Linked records
Additional details
Identifiers
Biodiversity
- Family
- Brassicaceae
- Genus
- Arabidopsis
- Kingdom
- Plantae
- Order
- Brassicales
- Phylum
- Tracheophyta
- Taxon rank
- genus
References
- Urban, P., Cullin, C., Pompon, D., 1990. Maximizing the expression of mammalian cytochrome P- 450 monooxygenase activities in yeast cells. Biochimie 72, 463 - 472. https: // doi. org / 10.1016 / 0300 - 9084 (90) 90070 - w.