Claus-Peter Witte, Edwige Isidore, Sarah A. Tiller, Howard V. Davies and Mark A. Taylor (2001)
Plant Molecular Biology 45, 169-179
Abstract of this publication
Extraction
Using a mortar and pestle, 3 g of fresh plant material is homogenised with 9 ml of extraction buffer (50 mM sodium phosphate buffer (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1.5% (w/v) polyvinylpolypyrollidone (PVPP), 10 mM dithiothreithol (DTT) and 0.1 mM phenylmethylsulfonyl fluoride (PMSF)). DTT and PMSF have to be added shortly before the extraction.
The plant material should be fresh, since urease is may be partially inactivated in the thawing process!
Centrifugation
The homogenised tissue is transferred to a 15-ml centrifuge tube and the total extract volume is determined (for example, by comparing the filling height with to a scale on a similar tube). The tube is centrifuged at 10000 g for 10 minutes to remove coarse debris, the supernatant transferred into a new tube and centrifuged at 40000 g for 20 minutes. The clarified supernatant is stored on ice.
Gel filtration
In order to be able to use the phenol hypochloride reagents for urease quantification, low molecular substances that interfere with the assay chemistry usually have to be removed. The DTT added during urease extraction is not compatible with the phenol hypochloride chemistry and plant extracts might contain further low molecular substances that interfere.
A 5-ml High Trap G25 desalting column (Pharmacia) is used to eliminate interfering low molecular weight substances. The column is equilibrated with four column volumes of gel filtration buffer (25 mM sodium phosphate buffer, pH 7.5; 25 mM NaCl; 0.5 mM EDTA) at the beginning and between individual runs. A flow rate of 6 ml/min is applied with a peristaltic pump. For sample processing, the pump is disconnected, a sample of 1.5 ml is applied with a 2.0 ml syringe, the pump is reconnected, and 2.0 ml are immediately recovered from the column outlet in a 2 ml microfuge tube. Two microliters 100 mM DTT is added to the gel-filtered extract which was then stored on ice.
If you determine the urease activity per unit fresh weight on an extract prepared in this way, you may consider the dilution caused by the gel filtration step with a factor 1.35 (2.0 ml / 1.5 ml). You can also test your real dilution by measuring the total protein content before and after the gel filtration step. In my experience the real dilution is usually between 1.3 and 1.5.
A larger volume (e.g. 0.5 ml) of extract may be incubated, eliminating the need to spin down the extract for the collection of condensation water before each time point.
Samples of up to 50 microliters may be taken (into 950 microliters water). A further increase in sample volume might cause interferences.
