MATERIALS and METHODS
1. MATERIALS
The materials used in the experiments are listed below specifying, in square brackets, the name of the suppliers.
• Bovine kidneys and spleens were obtained from freshly slaughtered
animals at the local slaughterhouse “Consorzio Macelli San Miniato” and kept frozen at -80 ° C until use.
• Tris, hydrochloric acid, acetic acid [J.T. Baker]
• Manganese chloride, magnesium chloride [Carlo Erba Reagenti]
• Calcium chloride [Merck]
• L-γ-glutamyl-p-nitroanilide, glutathione, glycylglycine, ethanol,
methyl-α-D-mannopyranoside[Sigma]
• Ditiothreitol [Inalco S.P.A.]
• Triton X [Aldrich]
• Ammonium sulfate [Panreac]
• Ninhydrin [Fluka]
• Buthyl-Sepharose Fast Flow, Sephacril S-300 [Amersham Pharmacia
Biotech,]
• Concanavalin A [Ge Healtcare]
2. METHODS
2.1 Assay with chromogenic substrate L-γ-glutamyl-p-nitroanilide
This assay is based on the spectrophotometric measurement at 405 nm of 4-nitroaniline that is released from L-γ-glutamyl-p-nitroanilide (G-pNA).
Assay mixture contained, in a final volume of 700 µl, 224 µl of Tris HCl
400 mM pH 8.5, 28 µl of MgCl2 100 mM, 140 µl of GlyGly 200 mM, 175 µl
of G-pNA 200 mM and the appropriated amount of sample.
The assay was started by the addition of the sample and performed at 37 ° C after a preincubation of the assay mixture devoided of the sample for 10 min at 4° C.
2.2 Assay with GSH as substrate
This assay is a coupled enzymatic assay (Cappiello M. et al, 2012). Cys-Gly, produced by γ-GT and/or GT rel is hydrolized by the ancillary enzyme leucyl aminopeptidase (LAP) to Cys and Gly. The amount of Cys formed is
measured spectrophotometrically by the method of Gaitonde (GAITONDE
M. K.,1967). Assay mixture contained, in a final volume of 255 µl, 50 µl of
Tris HCl 50 mM pH 8.5, 10 µl of MgCl2 250 mM, 5 µl of MnCl2 10mM, 50
µl of GlyGly 200 mM, 25 µl of GSH 25 mM and the appropriated amount of
sample. The incubation is started by the addition of the sample. After the
incubation, the reaction is stopped by the addition of 12.5 µl of TCA 100 % (w/v) and the incubation mixture is centrifuiged at 12,000 g for 1 min in a
Beckman Microfuge E. Supernatant (100 µl) is added to 100 µl of acetic
acid and 100 µl of a reagent prepared dissolving 125mg of ninhydrin in a mixture containing 3 ml of acetic acid and 2 ml of 4N HCl. The mixture is placed in a boiling bath for 4 min and then cooled on ice. After cooling, 300
µl of the mixture is diluted with 500 µl of ethanol and used for
spectrophotometric measurement at 560 nm. The concentration of cysteine
produced is measured using a calibration curve (Fig. 7) after subtraction of a blank obtained by terminating the reaction at time 0.
Fig 7. Calibration curve for the determination of cysteine concentration.
2.3 Determination of protein concentration
The protein concentration was measured with a Micro BCA Protein Assay kit. This method utilizes bicinchoninic acid (BCA) as the detection reagent
for Cu+1, which is formed when Cu+2 is reduced by protein in an alkaline
environment. A purple-colored reaction product is formed by the chelation
of two molecules of BCA with one cuprous ion (Cu+1). This water-soluble
complex exhibits a strong absorbance at 562nm that is linear with increasing protein concentrations. The test was performed by adding 500 µl of the protein sample to 500 µl of the kit’s working solution obtained mixing
together the three Micro BCA Reagents following the kit information. The mixture was incubated for 1 hour at 60°C and read at 562 nm in a spectrophotometer Beckman DU7. The protein concentration in samples was evaluated using a calibration curve obtained with known bovine serum albumin concentrations (Fig. 8).
Fig 8. Calibration curve for the determination of proteins concentration
2.4 Preparation of crude extracts
A portion of bovine kidney or spleen was weighed and, after thawing, 5 volumes of Tris / HCl 50 mM, pH 8 (purification buffer), were added. After homogenization with a blender, the suspension was continuously stirred on ice for 30 min.
Cellular debris were removed by centrifugation at 3000 xg for 15 min.
0 0,05 0,1 0,15 0,2 0,25 0,3 0 0,5 1 1,5 2 2,5 3 3,5 µg BSA Abs
2.5 Ultracentrifugation
The supernatant obtained from the first centrifugation was subjected to ultracentrifugation for 1 hour at 123000 xg at 4° C.
After ultracentrifugation , the precipitate was resuspended in purification buffer containing 1% Triton X-100 and stirred overnight at 4° C.
2.6 Ammonium sulphate precipitation
After stirring overnight, the suspension was subjected to ultracentrifugation for 45 min at 123,000 xg and the supernatant was subjected to fractional precipitation with ammonium sulphate. The suspension was brought to 50%
of salt saturation. After 30 min of stirring at 4°C, the sample was subjected
to centrifugation at 3000 xg for 15 min.
The precipitate was then resuspended in the purification buffer.
2.7 Hydrophobic interaction chromatography
The resuspended precipitate was subjected to hydrophobic interaction chromatography (HIC) on a 1.5x20cm column packed with Buthyl-Sepharose Fast Flow resin. The solution was applied onto the column equilibrated with purification buffer supplemented with 1.5 M ammonium sulphate. After washing the column with purification buffer supplemented with 1.5 M ammonium sulphate, the elution was performed stepwise with purification buffer and then with milliQ water supplemented with 2% Triton X100.
2.8 Gel filtration
Pooled fractions were applied on a 1.5x90cm column packed with Sephacryl S-300 resin and equilibrated with the purification buffer. The active fractions were pooled and used for the subsequent affinity chromatography step.
2.9 Affinity chromatography
A 1.5x20cm column was packed with Concanavalin A (ConA) resin and
equilibrated with the purification buffer supplemented with 1mM MgCl2,
1mM MnCl2, 1mM CaCl2 and 0.1M NaCl. The sample to be loaded was
brought to the same salt concentrations of the equilibration buffer and loaded onto the column. After loading and washing with the purification buffer
supplemented with 1mM MgCl2, 1mM MnCl2, 1mM CaCl2 and 0.1M NaCl,
the elution was performed with a 0-100mM linear gradient of methyl-α-D-mannopyranoside.
