Plant materials
Three-year-old pecan seedlings, originating from seed of open pollinated 'Desirable’ cv. trees, were grown in pots within a greenhouse to produce two Ni nutritional classes of trees—'Ni sufficient’ (Ni+) vs. 'Ni deficient’ (Ni-), based on expression of morphological symptoms of Ni deficiency [11]. The Ni- trees were produced from growing in a Tifton Loamy Sand soil. This soil often causes Ni deficiency symptoms in associated commercial pecan orchards. Ni+ trees from the same soil had prior season July leaf Ni concentrations of 1–4 μg.g-1 dry weight, which is considered to be within the 'sufficiency’ range for Ni in pecan. The Ni- trees had Ni concentrations ≤ 0.8 μg.g-1 dry weight [33], which is low enough to trigger the appearance of easily visible morphology based symptoms.
Six specimens from each of the two 'Ni status classes’ were randomly chosen for study from a population of trees, based on expression of visual symptoms of Ni deficiency occurring at the time of spring bud break. Spring xylem sap was collected and analyzed from several trees exhibiting the two classes of Ni nutritional status. Collection was at bud break, with bud break defined as inner bud scale split of >50% of primary apical buds. Xylem sap was collected by vacuum extracted from stems severed at the root collar and again just below the apical tip; with the phloem and bark associated with the stem base peeled back to ensure that xylem sap was not contaminated with phloem sap. The base of severed stems was placed under vacuum and the exuding xylem sap dripped into 2 ml collection vials. Total Ni concentration of xylem sap of Ni- and Ni+ trees was determined with xylem sap collected as described above, and quantified by ICP-MS [34].
Extraction and purification of xylem sap urease
The xylem sap samples were diluted in Buffer E [25 mM 2-morpholineethanesulfonic acid (Mes), pH 6.2, 2.5 mM MnSO4, and 2.5 mM DTT] and centrifuged at 20,000 g for 1 h and 30,000 g for 1 h. This supernate was passed through a 0.22 μM membrane to remove impurities, and then twice extracted with chilled acetone (90%) to precipitate proteins. This precipitate was dissolved in Buffer E, with protein purification via ammonium sulfate fractionation, gel filtration chromatography and ion exchange chromatography according to previously reported methods [35]. Proteins were further purified by passing through ammonium sulfate fractionation (50%), filtering to exclude ≤ 10 kDa molecules (Amicon Centricon YM-10, Millipore Corporation, Bedford, Ma, USA), and finally via gel filtration (Superdex 200 10/300 GL column; controlled by ÄKTAbasic –Systems, Amersham Biosciences, Piscataway, NJ, USA) and ion exchange (Mono Q H/R 5/5 column; controlled by ÄKTAbasic –Systems) chromatography. Buffer E was used as solvents and NaCl was added into Buffer E for the salt gradient elution during purification [36]. Fractions with urease activity were pooled, desalted using a small P-10 column, and concentrated by centrifugation for 2 h at 5,000 g at 4°C with Amicon Centricon YM-10. The smaller molecules were removed because they contain phenolic substances that interfere with enzyme activity. Fresh purified enzyme was subsequently loaded on large (16 cm × 16 cm) gradient (8-16%) sodium dodecyl sulphate -polyacrylamide electrophoresis (SDS-PAGE) gels for purity analysis.
RNase A source and activity
RNase A from bovine pancreas (90 units/mg protein) came from the USIB Corporation (Cleveland, OH, USA). Its purity was examined with SDS-PAGE (10-20% gradient gel, 16 cm × 16 cm in size) while controlling temperature (at 12–15°C) during electrophoresis. Purified protein was denatured in SDS-gel sample buffer and electrophoresed on a SDS-10 to 20% polyacrylamide gradient gel. The nuclease activity of RNase A from bovine pancreas was verified by suspending RNA (ribonucleic acid from baker’s yeast, Saccharomyces cereviae; Sigma, St. Louis, Mo, USA) in Buffer E [25 mM 2-morpholineethanesulfonic acid (Mes), pH 6.2, 2.5 mM MnSO4, and 2.5 mM DTT) [35]. RNase A (10 μL) was then added to the RNA suspension and the reaction mixture (2 mg/ml) incubated at 25°C for 1 h and then centrifuged (5,500g at 4°C). The mixture was then filtered to exclude ≤ 50 kDa molecules (Amicon Centricon YM-50, Millipore Corporation, Bedford, Ma, USA). The fraction was rinsed with Buffer E, centrifuged (at 5,500g at 4°C), and again filtered to exclude ≤ 50 kDa molecules. The > 50 kDa fractionation was suspended in Buffer E (2 ml) and the reaction monitored based on absorbance at 260 nm. Buffer E served as a blank and untreated RNA (2 ml) served as a positive control.
Assay of urease activity in protein solutions with molecular mass ≥10 kD
Urease activity was described by Kaltwasser and Schlegel [36], but with slight modification [37]. The assay is completed as a coupled enzyme with Glu dehydrogenase. All chemical assay reagents were dissolved in 0.1 M potassium phosphate buffer (pH 7.6). The assay mix was 0.37 ml of 0.1 M potassium phosphate buffer (pH 7.6), 0.1 ml of 1.8 M urea, 0.1 ml of 0.025 M ADP, 0.2 ml of 0.008 M NADH, 0.1 ml of 0.025 M α-ketoglutarate before adding 0.1 ml of 50 units/ml Glu dehydrogenase and 5 μL of enzyme solution. The change in A340 at 25°C was recorded at 0.5, 1, 3, and 5 min. Urease of jack bean (Canavalia ensiformis; 29.5 units/mg; Sigma, St. Louis, Mo, USA) was used as a reference. A unit is defined as the amount of urease causing oxidation of 1 μM of NADH/min at 25°C, pH 7.6, in a coupled reaction using Glu dehydrogenase. Protein concentration was determined with the Bio-Rad protein assay with bovine serum albumin as standard.
Effect of nickel ions on urease activity of RNase A
The purified RNase A solution (10 μl), either from bovine or pecan sap, was mixed with 10 μl of Ni-nitrate solution at different concentrations (0, 333, 1,000, and 3,300 μM, respectively) in a cuvette to give a final Ni concentration of 0, 0.0033, 0.0100, and 0.0333 μM/ml. After 2 min, 998 μl of chemical substrate solution was added and mixed, with measurement of urease activity determined as described above.
Determination of the N-terminal amino acid sequence
Purified urease from pecan sap was denatured in SDS-gel sample buffer and electrophoresed on an SDS-10 to 20% polyacrylamide gradient gel and then the protein was transferred to a PVDF membrane. The amino acid sequence of N-terminals was determined by Edman degradation and performed by a Molecular Biology Resource Facility (University of Oklahoma Health Science Center, Oklahoma City, OK, USA).