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2.9.1 Subcellular fractionation and preparation of microsomes

A Beckman XL-90 Ultracentrifuge with SW 50.1 and 70 Ti rotors was used to prepare microsomes from fibroblasts. The Sepharose CL-2B column was purchased from Pharmacia and the in vitro translation kit from Promega. The [^^S]-methionine was obtained from ICN. All other reagents were from Sigma.

2.9.1.1 Buffers

B u ffe r A : 50 mM TEA, 250 mM sucrose, 50 mM potassium acetate, 6 mM magnesium acetate, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, pH 7.5 B u ffe r B: 50 mM TEA, 250 mM sucrose, 1 mM DTT, pH 7.5

B u ffe r C: 50 mM TEA, 1.5 mM magnesium acetate, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, pH 7.5

Three different methods of subcellular fractionation were used to prepare microsomes from fibroblasts. The organelles present in each o f the fractions obtained were identified by assaying the activity of marker enzymes. The translation activity and glycosylation capacity was determined by in vitro translation of a-factor in the presence o f the microsomal fraction.

2.9.1.2 Homogenisation of fibroblasts

Cells were disrupted either by shearing in an homogeniser or by nitrogen cavitation. Cells were lysed by 40 strokes in a 1 ml Dounce homogeniser. For nitrogen cavitation, a suspension of the cells was placed in the nitrogen cavitation bomb and nitrogen was applied at 50 psi for 10 min. The pressure was released gently and the cells collected. The cavitation bomb was then disconnected from the nitrogen gas supply and flushed out with 1 ml of buffer A (section 2.9.1.1). Samples of the lysed and the unbroken cells were spotted onto microscope slides and a drop of trypan blue was added to each sample. The slides were then visualised under a phase contrast microscope and the percentage of broken cells was estimated by counting the number of cells that had taken up the trypan blue dye.

2.9.1.3 Method 1: Preparation o f microsomes by gel filtration followed by ultra­ centrifugation

This method was based on that described by W alter and Blobel (1983). Fibroblasts were harvested, washed twice in buffer A, re-suspended in buffer A and homogenised as in section 2.9.1.2. The homogenate was centrifuged in the Ti 70 fixed angle rotor at 12,000 X g (12800 rpm) for 10 min at 4°C. The void volumes of two gel filtration (Sephadex CL-2B) columns were measured using Blue dextran (10 mg/ml) and measuring the absorbance of the effluent at 625 nm. The supernatants were then applied to the gel filtration columns which had been equilibrated with buffer C and the microsomes were eluted overnight at 25 ml/hr. The void volume was collected (fractions 4-15 inclusive) and centrifuged on a 1.3M sucrose cushion in buffer A at 140,000 x g for 2 hours at 4°C. The resulting pellets were re-suspended in 1.5 ml of buffer B and centrifuged on the Ti 70 fixed angle rotor for 30 min at 40,000 x g. The pellets were re-suspended in 100 pi of buffer B and stored at -20°C.

2.9.1.4 Method 2: Preparation o f microsomes by differential centrifugation

As the samples were found to be diluted too much by gel filtration it was decided that differential centrifugation should be investigated as an alternative method for the purification o f microsomes from fibroblasts. Lysed cells were centrifuged at 12,000 x g (12,800 rpm) in the Ti 70 fixed angle rotor for 10 min at 4°C. The supernatant was collected, and the pellet re-suspended in buffer A. This suspension was then re-applied to the nitrogen cavitation bomb for a second time and centrifuged as above. The pooled supernatants were added to 8 ml of buffer A and loaded on top of a 5 ml sucrose cushion (1.3M sucrose in buffer A). The tubes were centrifuged at 140,(X)0 x g (28k rpm) for 2 hours at 4°C. The resulting pellet was re-suspended in 200 pi of buffer B and stored at -70°C in 50 p i aliquots.

2.9.1.5 Method 3: Preparation o f crude microsomes by centrifugation

This method was based on that described by Knauer et al. (1994). After nitrogen

cavitation of the cell pellets and removal of cell debris by centrifugation, the supernatant was centrifuged in the SW 50.1 swing out rotor for 30 min at 66,000 x g. The resulting

pellet was re-suspended in 200 pi of buffer B and stored at -70°C in 50 pi aliquots. These microsomes were active and so this method was used to prepare microsomes from both normal and PMM-deficient CDGS type 1 fibroblasts (Chapter 6).

2.9.1.6 Assay o f marker enzymes for organelles

For each marker enzyme 50 pi of each fraction (supernatant or re-suspended pellet) was assayed in a microtitre well plate. 50 pi of the appropriate substrate was added and the plate was incubated for 2 to 4 hours at 37®C with shaking. The reaction was stopped by the addition o f 150 pi of 0.25M glycine/NaOH, pH 10.4 containing 0.01% (v/v) Triton X-100. The fluorescence was measured using an excitation wavelength of 365 nm and emission wavelength of 450 nm on a Perkin Elmer LS 50.

Substrates '

Lysosomal marker; ^hexosaminidase

3 mM 4-methylumbelliferyl-2-acetamido-2-deoxy-p-D-glucopyranoside in phosphate- citrate buffer pH 4.5

Endoplasmic reticulum marker; neutral (X-glucosidase

0.375 mM 4-methylumbelliferyl-a-glucopyranoside in 0.25M sodium cacodylate buffer, pH 7

Golgi marker; a -mannosidase