in vitro TGase activity assay

1. In-vitro TGase activity was measured by the conjugation of biotinylated cadaverine to exogenous substrates N,N′-dimethylcasein as previously described. Specific activity was determined as a change in A450 of 0.1 per hour per mg of pollen after subtraction of the value of the controls treated with 20 mM EGTA.
Temperature-Dependent Compatible and Incompatible Pollen-Style Interactions in Citrus clementina Hort. ex Tan. Show Different Transglutaminase Features and Polyamine Pattern.

2. Relative TGase activity was also determined using a new fluorescence procedure. The reaction mixture (final volume of 0.2 mL in a small, closed Eppendorf tube) consisted of 10 mM DTT, 10 mM CaCl₂, 100 μg of N,N-dimethylcasein, 25 μM (or in some cases 50 μM) fluorescein cadaverine, and 100 mM HEPES-NaOH at pH 8.0. The tube was heated to 37 °C, and the reaction was started by the addition of the enzyme source. After 2 h (or 16 h), the reaction was stopped by the addition of 0.4 mL of 0.4 M perchloric acid. The samples were then centrifuged at 10000g for 10 min at 4 °C to pellet coagulated protein. The supernatant was carefully discarded, and the pellet was washed with 0.4 mL of acetone/ethanol (1:1, v/v) brought to 0.1 M HCl with 12 M HCl. The pellet was again collected at 10000g and washed a further 2 times with acetone. The final pellet was air-dried and dissolved in 0.1 mL of 0.1 M NaOH followed by addition of 0.1 mL of 0.7 M boric acid (final pH ∼7.2). The fluorescence of the samples (λ_ex, 488 nm; λ_em, 521 nm) was measured using Microfluor 2 Black “U” bottom microtiter 96-well plates (Thermolabsystems, Franklin, MA) and a Spectra MAX Gemini Plate Reader (Molecular Devices, Sunnyvale, CA). One set of controls consisted of the complete reaction mixture plus the enzyme source carried through the incubation and extraction procedures except that 20 mM EGTA or EDTA was included (to chelate Ca²⁺). In another set of controls, boiled enzyme source was used in place of the active enzyme. The fluorescent method is sensitive, as shown by results obtained with commercial TGase 2 as a positive control. In this experiment, the relative fluorescence of the precipitated protein in the complete reaction mixture plus TGase 2 (2.5 μg incubated at 37 °C for 2 h) minus EDTA (100 000 RFU) was much greater than that of the control (complete reaction mixture plus TGase 2 plus EDTA) (∼800 RFU). In comparison, a 10-min incubation using the [¹⁴C]putrescine-binding assay (above) with this amount of enzyme generates a blank (complete reaction mixture plus enzyme plus EDTA) of about 200 cpm and a signal of about 40 000 cpm in the complete reaction mixture plus enzyme minus EDTA. Thus, the new fluorescence procedure with purified TGase 2 is of similar sensitivity to that of the radiochemical assay.
Transglutaminase Activity Is Present in Highly Purified Nonsynaptosomal Mouse Brain and Liver Mitochondria

Immunostaining of GTase

Compatible and self-incompatible pistils were directly thawed ina buffer solution (100 mM Pipes pH 6.8, 10 mM EGTA, 10 mM MgCl₂, 0.1% NaN₃) plus detergent and fixative (0.05% Triton X-100, 1.5% paraformaldehyde, 0.05% glutaraldehyde) for 30 min on ice and then at 4°C for an additional 30 min. For the localization of TGase,fixed pistils were cut along their length and placed in the buffer solution containing 0.75% cellulysin and 0.75% pectinase for7 min. For immunofluorescence microscopy, samples were washed in the above buffer and incubated with the anti-TGase antibody Ab3(Neomarker, Fremont, CA, USA) diluted 1:20 in the buffer;incubation was 1 h at 37°C according to previous literature (DelDuca et al., 2009). After washing with buffer, pistils were incubated with the Alexa-Fluor 488-conjugated goat anti-mouse (ThermoFisher Scientific) secondary antibody diluted 1:50 in the buffer solution, for 45 min at 37°C in the dark. Samples were observed with a Zeiss Axiophot fluorescence microscope (Ex-Max 490 nm/Em-Max 525 nm) equipped with a MRm video camera and a 63× oil-immersion objective.
Temperature-Dependent Compatible and Incompatible Pollen-Style Interactions in Citrus clementina Hort. ex Tan. Show Different Transglutaminase Features and Polyamine Pattern