9. DESARROLLO DE LA INVESTIGACIÓN
9.1. PLANEACIÓN EXTENSIVA DEL DISEÑO EXPERIMENTAL
2.1; MATERIALS:
Cell stocks came from ECACC (European Collection of Cell Cultures, Salisbury, UK). Radioactively labelled BQ123, ET-1 and ET-3, and a-^^P-ddATP were from Amersham (Little Chalfont, Bucks., UK). All other chemicals were from Sigma (Gillingham, Dorset, UK) or Fluka (Gillingham, Dorset, UK) unless otherwise stated, and were of the best grade available. Anti-ETAR and anti-ETgR antisera was a gift from Prof. Hagiwara, Tokyo Institute of Technology.
2.2: GENERAL METHODS:
2.2.1: BUFFERS:
Solutions commonly used throughout the work are listed below, along with their compositions:
Solution Preparation
Equilibration buffer 30% glycerol v/v, 2%SDS w/v, 6M Urea, 0.5mM Tris-HCl, pH 6.8 lEF buffer 8M Urea, 2M Thiourea, 4%CHAPS, 1% Triton X-100, lOmM Tris
base, 0.8% pharmalyte v/v (4-7 or 3-10 according to the strip used) (Amersham, UK), 1 ‘complete mini’ protease inhibitor cocktail tablet (Boehringer, Mannheim) per 100ml
lOX Laemmli Running Buffer
1.98M Glycine, 250mM Tris Base, 1% SDS w/v.
Loading buffer (DNA gels)
95% formamide v/v, 20mM potassium EDTA pH 8, 0.05% bromphenol blue, 0.05% xylene cyanol v/v.
Lysis buffer (WB) 20mM CHAPS, 50mM Tris base, 125mM NaCl, 5mM EGTA, 0.5mM EDTA, 1% Triton X-100 v/v, 2mM DTT, lOOmM NaF, 15mM NazPzO?, ImM Na3V0 4, lOmM (3-glycerophosphate, 1
‘complete mini’ protease inhibitor tablet per 100ml
Lysis buffer (MAP) 25mM Tris-HCl pH 7.4, 1% Nonident NP40 v/v, 1% Triton X-100 v/v, ImM Na3V0 4, 5mM NaF, 5mM p-glycerophosphate, 5mM
EDTA, 1 ‘complete mini’ protease inhibitor cocktail tablet per 100ml
lOX PBS 1.37M NaCl, 27mM KCl, 43mM Na2HP0 4.7H2 0, 14mM KH2PO4
PBS/PI IX PBS, 1 ‘complete mini’ protease inhibitor cocktail tablet per 100ml.
Solution Preparation
4X Separating gel buffer
1.5M Tris base, 0.4% SDS w/v. pH 8.3
Solubilisation buffer 20mM potassium phosphate pH 7.4, 0.4% digitonin, 0.25% CHAPS w/v, 500mM NaCl, 20mM EDTA, ImM Na3V0 4, Ipg/ml
RNase, 2mg/ml leupeptin, 2mg/ml pepstatin, 2mg/ml trypsin inhibitor S, 2mg/ml trypsin inhibitor L, 2mg/ml antipain
4X Stacking gel buffer
0.5M Tris base, 0.4% SDS w/v, pH 6.8
5XTBE 54g/l Tris base, 27.5g/l boric acid, 20ml/l 0.5M EDTA pH 8 Transfer buffer 0.2M Glycine, 25mM Tris Base, 0.05% SDS w/v, 20% Methanol
(BDH) v/v. 5 OX Tris-acetate
buffer
242g/l Tris base, 57ml/l acetic acid (BDH (Merck), Dorset, UK)
Tris-N-Tween lOmM Tris-HCl, pH 7.4, lOOmM NaCl, 0.1% Tween-20 v/v.
2.2.2: GEL ELECTROPHORESIS OF PROTEINS:
Polyacrylamide gels are used in several sets of experiments. The details of their preparation are given below:
i) ID mini-gel electrophoresis:
Gel preparation:
Mini-gels were cast using the Biorad MiniProtean system. Gels were 8x10cm, and 1.5mm thick.
A 10% acrylamide separating gel mixture was prepared as follows:
Reagent / solution Quantity (for 1 mini gel)
4X separating buffer 1.88ml
acrylamide, 30% stock (Duracryl, Genomic solutions)
2.5ml
water 3.13ml
APS, 10% stock 25|li1
TEMED 5|il
Separating buffer, acrylamide and water were mixed before adding the TEMED and APS. The gel was then stirred briefly, poured to 2/3 of the height of the plates, and left to polymerise under a layer of water-saturated n-butanol.
When this had polymerised, a layer of stacking gel (4.5%) was added. Stacking gel was prepared as follows:
Reagent/solution Quantity (for 1 mini gel)
4X Stacking gel buffer 1.25ml
acrylamide, 30%stock (Duracryl, Genomic Solutions)
0.75ml
water 2.5ml
APS, 10% stock 25^il
TEMED 5 lull
The gel was allowed to polymerise with a 10 well comb inserted.
Sample preparation:
Samples were dissolved in Laemmli Sample Buffer (Sigma), containing bromphenol blue and warmed for 2 mins in a 95°C heating block. Markers were Sigma Low Molecular Weight (66, 48.5, 29, 18.4 and 14.2kD, cat no. M5630), and lOjxl of marker, prepared according to the instructions, were loaded for each gel. Markers were already in sample buffer, but were also boiled.
Electrophoresis:
Gels were run at 120V until the blue line produced by the bromphenol blue reached the bottom of the gel (generally about 1 hour). The electrophoresis buffer was Laemmli Running Buffer.
ii) 2°^ Dimension gel electrophoresis:
Gel Preparation:
Gels were cast using the Hoefer-Dalt system (AP Biotech). The gels were 13 x 16 cm and 1 mm thick.
11% acrylamide gel mixture was prepared as follows:
Reagent / solution Quantity (for 1 gel)
water 11.65ml
1.5M Tris-HCl, pH 8.8 7.45ml
acrylamide, 30% stock (Duracryl, Genomic Solutions)
10.45ml
10% SDS 310^11
TEMED I5\i\
10% APS 90|Li1
The mixture was stirred, poured, and left to polymerise under a layer of water saturated n-butanol.
lEF strip loading:
The equilibrated lEF strip (see section 2.6.1 for details of sample preparation and first dimension electrophoresis) was washed with Laemmli running buffer to remove excess iodoacetamide and trimmed to fit the 13cm gel leaving a small space for the marker lane
against the glass of the back plate. A piece of drinking straw was used to make a marker well, and the strip was sealed in place with 0.5% agarose in Laemmli Running Buffer, containing bromphenol blue. When the agarose had set, the straw was removed and a small piece of Whatman no.l filter paper soaked in 7p.l of lOkD protein ladder markers (Biorad (Hemel Hempstead, UK)) was inserted into the well. The well was then also sealed with agarose.
Electrophoresis:
Gels were run vertically at 300V until the bromphenol line was at the bottom of the gel, (between 3 and 4 hours). The electrophoresis buffer was Laemmli Running Buffer, cooled to 8°C.
2.2.3: SILVER STAINING:
The gel was fixed overnight in 50% methanol, 12% acetic acid (both BDH). It was then washed in 50% ethanol (BDH) three times for 20 mins each. The gel was pre treated with a solution of 0.2g/l sodium thiosulphate for 1 min, rinsed three times with water for 20s each, and then stained with 0.1% silver nitrate for 20 mins. After rinsing twice in water for 20s, the gel was developed using a 3% w/v solution of sodium carbonate containing 250pl/l 37% formaldehyde and 2mg/l sodium thiosulphate. The time taken for this step varies and the time to stop is chosen subjectively - the longer a gel develops the more likely it is that low abundance bands/spots will become visible, but this is countered by increasing levels of background staining as other parts of the gel become overexposed. The gel was then rinsed twice for 2 mins in water, and development stopped by shaking for lOmins in 50% methanol, 12% acetic acid. The gel
was washed in 50% methanol for 20mins, then finally reswelled in 5% methanol, also used for storing the gel.
2.2.4: CELL CULTURE:
Two cell lines were used during the course of this work, CCD19Lu and CCD33Lu. The cell line used for the binding studies, and for the receptor isolation was CCD19Lu. This is a cell line, developed from the normal lung tissue. The cell line used for the mapping work was CCD33Lu, also from normal human lung tissue, which was used due to failure of supply for CCD19Lu.
Cells were acquired as frozen ampoules from ECACC and plated into a 75cm^ vented flask (Sarstedt (Numbrecht, Germany)) with 10ml of culture medium.
Cell Culture Medium:
Culture medium was prepared as follows for the fibroblast cell lines: 2mM L-Glutamine (Gibco (Paisley, Scotland, UK))
1% NE A A (Gibco)
10% FBS (PerBioscience, batch no.AOl 126-536) 0.8% antibiotic/antimycotic mixture (Gibco)
MEM with Earle’s salts, w/o L-glutamine to volume (Gibco)
The antibiotic mixture contains lOu/ml penicillin G, 10|ig/ml streptomycin sulphate, 2.5pg/ml amphotericin B.
Medium was replaced every 2-3 days.
Subculture:
Cells were split when 80-90% confluent. Cells were split 1:4, either into 75cm^ flasks, when only to be used for further subculture, or into plOO dishes (Falcon), when harvesting was intended. Cells were split according to the following procedure:
The cells were initially washed with 10ml, and then 5ml of HBSS w/o calcium and magnesium (Gibco) to prepare for trypsinisation. 5ml of trypsin/EDTA (porcine, 0.5g/l trypsin, 0.2g/l EDTA in HBSS w/o calcium and magnesium, Gibco) was added to the flask or dish, and gently agitated. Cells were incubated at 37°C for 2 mins, then checked under the microscope to ensure they had detached from the plates.
5ml of medium was added to halt the action of the trypsin, and the cells were spun at 4°C for 5 mins at lOOOrpm. The washing medium was aspirated, the cells were resuspended in fresh medium, washed again, and plated into 4 flasks or dishes in fresh medium. Medium was changed the day after splitting.
Attempts were made to freeze CCD19Lu, using freezing media containing glycerol (Gibco), DMSG (Sigma), in FBS containing 10% DMSG, and in preconditioned media taken from their own culture. However, all attempts were unsuccessful, and we were informed by the suppliers that they were unlikely to be successful under any feasible circumstances, so the cells were continually cultured until they reached the end of their lifespan. The cells were generally able to undergo 10 splittings before showing signs of ill-health, when they were destroyed, and a new vial started. CCD33Lu cells were
frozen for storage in liquid nitrogen in FBS containing 10% DMSO. There is apparently no limit on the number of passages they will undergo.
2.2.5: MASS SPECTROMETRY:
i) In-gel digestion:
Small pieces of gel approximately Imm^ were cut with a scalpel from the bands or spots of interest. They were initially reswelled in lOOpl of 50mM NH4HCO3 for 15 mins, and this step was repeated 3 times, until the pH was found to be 7-8 using universal indicator paper. As the gel had been silver stained, it was necessary to destain it using reducing agents. The gel was incubated at RT in 30pl of a 1:1 mixture of lOOmM sodium thiosulphate, and 30mM potassium ferricyanate until the brown colour had disappeared. The gel, now yellow, was washed in 3 x lOOpl of ddH20 until colourless. The destained gel was cut into smaller pieces and left for lOmins in lOOjxl of acetonitrile (AcN). The liquid was removed, and this step was repeated 3 times. The gel was then dried in the speedvac for 30mins.
The gel was rehydrated in 50pl lOOmM NH4HCO3 containing lOmM DTT for 30 mins at 56°C, then dehydrated in AcN as described above, and dried down in the speedvac for 30 mins. The dry gel pieces were incubated overnight at 30°C in 5|il of 75ng/pl trypsin (Promega) (50mM NH4HCO3). For the receptor isolation experiments, 5pi of 70% DMF was added to the trypsin, to assist in the extraction of hydrophobic membrane protein.
ii) MALDI-TOF Mass Spectrometry:
0.5|xl of the tryptic digest was spotted on the target plate and allowed to dry. 0.5pl of matrix solution (a saturated solution of a-cyano-4-hydroxysuccinnamic acid in 50% acetonitrile containing 0.1% TFA) was then added over the top and allowed to dry. The spectrometer (Biflex
in,
Bruker Daltonik (Bremen, Germany)) was first calibrated using a mixture of 5 known peptides ranging in mass between 1046 and 3495 Daltons, then MALDI-TOF spectra were taken of the samples. The spectra were recalibrated using the peaks derived from trypsin (and therefore present and predictable in all tryptic digest spectra) as an internal standard and identified manually using Bruker ‘DataAnalysis’ software. The machine was used in reflector mode. The detector voltage was varied between 1.6 and 1.85kV, and the laser attenuation between 60 and 85%.For the receptor isolation experiments, the peaks identified were compared with a theoretical digest (using MS-Digest tool, http://jspl.ludwig.edu.au) of the endothelin receptors A and B to establish the identity of the gel band.
For identification of unknown proteins, the peaks appearing in the spectra were run through the internet databases Mascot (http://www.matrixscience.com), Peptident (http://www.expasy.ch) and MS-Fit (http://jspl.ludwig.edu.au).
iii) ESI-Ion Trap Mass Spectrometrv:
Where DMF had been used in the extraction, the tryptic digest samples were diluted 1:7 with ddH20 to reduce the DMF concentration to 10%, They were then diluted 1:10
in a 50:50 mixture of methanol and 1% acetic acid. The samples were vortexed, then loaded into the micro-capillary for nanospray analysis. Nanospray mass spectrometry was performed using a Finnigan-Matt LCQ (San Jose, CA, USA), in positive ion mode for the most part. Negative ion mode was occasionally used to enhance the signal from very negatively charged peptides. Collision energies ranged between 20 and 50%. Peaks previously identified using MALDI-TOF MS (Bruker Biflex IB) were chosen for sequencing, both to confirm the identity of the protein, and the phosphorylation status of the tryptic peptides. Fragmentation spectra were compared to theoretical fragmentations of the likely sequences obtained from MS-Product (http://jspl.ludwig.edu.au ), and the location of post-translational modifications ascertained where possible.
2.3: W ESTERN BLOTTING:
In order to check for receptor expression in CCD19Lu, a Western blot was performed using antisera against endothelin receptors A and B:
i) Cell preparation:
Eight plOO dishes of cells at passage 18 were harvested in lysis buffer (WB). All dishes were washed on ice with 6ml of ice-cold PBS/PI. This was aspirated and the wash repeated twice with 3ml PBS/PI. The cells were then scraped on ice in the PBS/PI, divided into two equal volumes, and spun at 4°C, 1000 rpm for 5 mins. The buffer was aspirated, and the pellets resuspended in 60p.l each of lyses buffer (WB).
ii) Electrophoresis and blotting:
Samples were mixed with an equal volume of 2X Laemmli Sample Buffer (Sigma), and heated at 95°C for 5 mins. SDS-7B molecular weight markers (Sigma) were boiled for the same time. Samples were then loaded on Biorad 8 x 10cm precast gels with a gradient of 4-15% Tris-HCl. The gels were run at 120V for 90 mins in Laemmli Running Buffer.
6 pieces of blotting paper (Whatman (Maidstone, Kent, UK) approximately 20 x 16cm were soaked in transfer buffer, as were the gels and a 20 x 16cm piece of nitrocellulose membrane (Schleicher and Schuell (Dassel, Germany)). The gel was placed on top of the membrane, on top of three pieces of soaked blotting paper, with the remaining pieces on top. The sandwich was placed in a semi-dry blotter (Biorad), and run at 20V for 1 hr.
Transfer was checked by Ponceau-S staining. The membrane was incubated at RT for 15 mins in a solution of 10% acetic acid containing 0.2% Ponceau-S. Molecular weight markers not coupled to coomassie were marked with biro, and the membrane was destained in frequent changes of PBS for 40 mins.
iii) Probing of the membrane:
The membrane was left to block overnight in Tris-N-Tween containing 7.5% fat-free milk powder (Carnation) and 1% BSA (Sigma), shaking at 4°C.
The following day, the membrane was divided up, and incubated in a solution of 1:500 rabbit œ-ETaR serum
1:250 rabbit (X-ETgR serum
in Tris-N-Tween containing 2% milk powder. The membranes were left shaking at RT for 2 hrs.
The membranes were washed in Tris-N-Tween containing 2% milk powder for three periods of 15 mins, changing the wash solution. They were then incubated in this solution for a further hour with the addition of secondary antibody (goat a-rabbit, horseradish peroxidase-conjugated. Sigma).
The washing step was repeated then the membrane was shaken in developer solution (ECL kit, Amersham Pharmacia) for two mins, and flurographs taken over the next 15 mins (Kodak film (Hemel Hempstead, UK)).
2.4; CHARACTERISATION OF ENDOTHELIN RECEPTOR BINDING IN