2 MATERIALS AND METHODS
2.8 ASSAY PROCEDURES
2.8.1 Spectrophotometric enzyme assays 2.8.1.1 Aldehyde oxidase in vitro assay
Aldehyde oxidase activity was determined spectrophotometrically by monitoring the decrease in absorbance of DCIP at 600 nm (Courtright 1967) in an Anthelie Advanced
Spectrophotometer. The reaction mixture consisted of 0.2 mL enzyme extract and 1.8 mL 0.1 mM PMS and either 2 mM I-aid (for XAN oxidase activity) or lA-aid (for lA-aid oxidase activity) in 50 mM potassium phosphate buffer (pH 7.4). The reaction was initiated by the addition of 0.002% (w/v) DCIP in 50 mM potassium phosphate buffer (pH 7.4) and allowed to proceed for a maximum of 10 min. The final concentration of reduced OCIP was detennined by applying Beer's lawEA600
=
2.2 x 104 L mor1 cm-1 and specific activity of AD is expressed as nmol OCIP reduced mg-1protein min-1(Sagi etal. 1998).2.8.1.2 Xanthine dehydrogenase in vitro assay
For the detennination of XOH activity, the reaction mixture included 1 mM hypoxanthine and 1 mM OTT in 50 mM Tris-HCI buffer (pH 6.5). The reaction was initiated by the addition of either 2.5 mM NAO+ or 0.002% (w/v) OCIP in 50 mM Tris-HCI buffer (pH 6.5) followed by incubation at 30°C (Sagi et al. 1998). Xanthine dehydrogenase activity was monitored spectrophotometrically, in an Anthelie Advanced Spectrophotometer, by following the production of NAOH at 340 nm (Triplett etal. 1982) or the decrease in absorbance of OCIP at 600 nm (Perez-Vicente et al. 1988), for a maximum of 10 min. The final concentration of NAOH or OCIP wasdetennined by applying Beers law EA340
=
6.333 X 103 L mor1 cm-1 forNAOH and EA600
=
2.2 X 104 L mor1 cm-1for OCIP and is expressed as nmol OCIP reduced (NAOH) mg-1protein min-1.For the detennination of the kinetic properties of AD and XOH, assays were conducted with 1.0 to 5.0 mM substrate for 1 to 3 min, during which time the reaction rate remained constant, according to the methods of Courtright (1967) for AD and Triplett etal. (1982) for XOH.
2.8.1.3 Cytokinin oxidase spectrophotometric assay
Cytokinin oxidase activity was assayed using the method of Liberos-Minotta ,and Tipton (1995). The reaction mixture in 0.2 M imidazole-HCI buffer (pH 7.5), contained 80 ~MiP and 1 mM CuCI2,and was initiated by the addition of enzyme and incubated at 37°C for 30 min. The reaction was tenninated using 40 % (w/v) trichloroacetic acid. Following the addition of p- aminophenol reagent (see section 2.3.2 for preparation) and development of colour for 10 min at room temperature, the absorbance at 352 nm was detennined in an Anthelie Advanced Spectrophotometer. Cytokinin oxidase activity is expressed as ~mol 3-methyl-2-butenal produced mg-1 protein, interpolated from a standard curve prepared by reacting 3-methyl-2- butenal with p-aminophenol reagent.
2.8.2 Radioactive enzyme assays 2.8.2.1 Cytokinin oxidase assay
Alternatively, CKOX activity was assayed by monitoring the conversion of [2-3
H]iP to [3H]adenine according to the method of Motyka and Kaminek (1994). The assay mixture (50
~Lfinal volume) contained 50 mM Tris-HCI buffer (pH 8), 10 ~M substrate ([2-3H]iP, 1.25 TBq mmor1) and 25 ~L of enzyme preparation. After incubating the reaction mixture for 4 h at 37 oC, the reaction was terminated by the addition of 10~L 200 mM Na2EDTA and 120~Lof cold 95 % (v/v) ethanol, containing 0.75 mM unlabelled iP and adenine. Residual substrate and labelled product were subsequently separated on thin layers of silica gel (GF2504), developed once to 10 cm in chloroform/methanol/ammonium hydroxide (25 %, w/v) (9:2:0.1, v/v/v) (Redig etal. 1997). Zones corresponding to authentic iP (R, 0.7-0.8) and Ade (R,O.3-0.4) standards were eluted with 2 mL 80 % ethanol, to which was added 5 mL Pico-Fluor™ 40 (Universal LSC cocktail). Radioactivity was determined by liquid scintillation spectrometry using a Packard Tri-Carb~ 1500 Scintillation Counter (Packard, Downers Grove, ILL, USA) programmed for automatic quench correction. Cytokinin oxidase activity is expressed as Jjmol [3H]adenine produced mg-1protein h-1. For substrate specificity analysis trans-[2-3H]zeatin was used as a substrate.
2.8.3 Native-PAGE assays
2.8.3.1 Polyacrylamide gel electrophoresis
Native-PAGE was performed with a 10 % acrylamide gel in a Laemmli buffer system (Laemmli 1970), in the absence of sodium dodecyl sulfate (SDS) at 4 °C. A CBS Scientific Vertical Mini- Gel System (Model MGV-202; CBS Scientific Company Inc., Del Mar, CA, USA) was assembled as described in the manufacturer's manual. Before use the glass plates, spacers, combs and Gel WrapTMgaskets were washed with soap water and cleaned with alcohol. The two glass plates (inner plate 8.5 cm x 11 cm, outer plate 9.5 cm x 11 cm) were positioned in the clamp assembly and separated by 1.0 mm polyethylene spacers. Removable silicone Gel WrapTM gaskets ensure that the acrylamide solution does not leak from the sandwich assembly.
The separating gel solution (Table 2.1) was run into the space between the two glass plates, to a depth of 2.0 cm from the top of the inner glass plate and was overJayered with distilled H20, to exclude atmospheric oxygen, which inhibits polymerisation. Polymerisation involves the production of an acrylamide monomer chain which is cross-linked by the bifunctional compound N, N'-methylenebisacrylamide. N, N, N', N'-tetramethylethylenediamine catalyses the formation of free radicals from ammonium persuIfate, which in turn initiates polymerisation (Hames and Rickwood 1981). Once the gel had set (approximately 1 h, evidenced by the
re-appearance of an interface between the gel solution and the over-layered distilled H20) the water was removed. Stacking gel solution (Table 2.1) was poured into the sandwich to the top of the inner glass plate and a 1o-well comb inserted to form the sample application wells.
Once the stacking gel had set, the comb was removed and the wells were washed with distilled H
20. The gel sandwiches were then transferred and clamped onto the electrophoresis unit.
Table2~1 Reagents for two Laemmli gels (Laemmli 1970) for the CBS SCientific Vertical Minl-Gel System. (For reagent preparation see appendix I)
Separating Gel Stacking Gel Reagent
(10 %) (3.5 %)
3.75 2.25
Gel Buffer (mL)
5.0 1.05
Monomer (mL)
75 50
Ammonium persulfate (IJL)
15 7.5
TEMED (IJL)
6.25 5.7
Dist. H20 (mL)
Tank buffer was poured into the upper and lower chamber of the electrophoresis unit. The voltage was set at 150 V for 30 min, to remove free radicals formed during the polymerisation process. Sample was mixed with non-reducing treatment buffer and bromophenol blue marker dye (10-12 IJL), which migrates with the buffer front and monitors the progress of electrophoresis. The samples were centrifuged at 5,500 g for 5 min, in a Hitachi Himac Miniature Centrifuge (model SCT15B; Hitachi Koki Co. Ltd, Tokyo, Japan), to remove any insoluble material. The sample was loaded onto the gel and the voltage was set at 100 V (maximum current) until the bromophenol blue front had migrated through the stacking gel.
The voltage was then increased to 150 V for the remainder of the electrophoretic run. Equal amounts of protein were loaded per sample onto the gel, varying from 20 to 30 JJg for activity staining and 10 to 15 JJg for silver staining of mesocarp samples and 2 "to 3 JJg for silver staining of seed and seed coat samples. The temperature of the unit was maintained at 4
ac,
by attaching the unit to a Lauda Compact Low-Temperature Thermostat (Messgerate-Werk Lauda, Lauda-Konigshofen, Germany). Electrophoresis was continued until the bromophenol
blue marker dye was 0.5 cm from the bottom of the separating gel. The gels were removed and prepared for activity staining or placed in the appropriate staining solution for visualisation.
2.8.3.2 Activity staining for aldehyde oxidase and xanthine dehydrogenase
Enzyme activity staining is a technique used to locate the relative position of multiple forms of enzymes with common catalytic activity, after they have been resolved by PAGE (Tanksley and Orton 1983). This is usually achieved through the production of a non-diffusable chromogenic precipitate at the site of enzyme activity, such as the conversion of a soluble tetrazolium salt to a coloured insoluble formazan. The quantity of formazan formed is directly, proportional to enzyme activity during a given incubation time and in the presence of excess substrate and tetrazolium salt (Rothe 1974).
Following electrophoresis, gels were removed, washed with distilled H20 and immersed in 0.2 M potassium phosphate buffer (pH 8.0) for 5 min. Following this the gels were washed again with distilled H20, placed in the reaction mixture containing 1 mM substrate, 0.2 mM PMS and 1 mM MD in 0.1 MTris-HCI buffer (pH 8.0) and incubated for 45 min at 30 QC on an orbital shaker, in the dark. Following staining the gels were fixed in 5 %(v/v) acetic acid to facilitate diffusion of non-reacted substrate out of the gel, to remove background staining and to keep the bands sharp. The gels were removed from the fixing solution, washed with distilled H2
0
and scanned immediately using a Hewlett-Packard Scanjet 5300C with HP PrecisionScan Software, Version 3.01 at 300 dpi resolution.
For the assessment of AO activity the following substrates were used I-aid, lA-aid, heptaldehyde, benzaldehyde, citral, acetaldehyde and 3-methyl-2-butenal at a concentration of 1 mM. Aldehyde oxidase activity was also assessed using XAN as a substrate at a concentration 0}17 IJM. For assessment of XDH activity hypoxanthine and xanthine were used as substrates at a concentration of 1 mM.
2.8.3.3 Silver stainingofproteins in polyacrylamide gels
This method provides an attractive alternative to the traditionally used Coomassie Brilliant Blue R-250 protein stain, in that proteins may be detected in the nanogram range. The sensitivity of this technique, which is based on the reduction of silver ions to metallic silver, is only surpassed by radioactive labelling. Blum et al. (1987) modified the original silver staining procedure to avoid non-specific background staining without the loss of sensitivity or contrast.
All steps were carried out on an orbital shaker at room temperature and in scrupulously clean glass containers. Gloves were worn at all times to prevent contamination of the gels. After electrophoresis, the gels were soaked in fixing solution overnight and washed in washing solution 1 (3 x 20 min) to remove any acetic acid. Gels were placed in pretreatment solution (1 min), washed in deionised H20 (3 x 20 sec) and placed in the impregnation solution (25 min).
After washing (2 x 20 sec in deionised H20), the developer was added. As soon as bands were visible, the gels were washed in deionised H20 until the bands were fully developed. The stopping solution was added (> 20 min), the gels washed in washing solution 2 (10 min), and scanned immediately as previously described (see section 2.8.3.2). For reagent preparation see appendix I.
2.9 EXTRACTION AND ANALYSIS OF PURINES AND PLANT HORMONES