Isolation of leaf peroxisomes from Spinacia oleracea L

In the present study, for preparative purposes, the purity of leaf peroxisomes obtained from an initial Percoll density gradient was further improved, by an additional purification step involving a sucrose density gradient centrifugation. For analytical puposes, a new method was developed which is described elsewhere (2.2.1.2).

2.2.1.1. Preparative method

Eight to twelve weeks old apparently healthy spinach plants with fully expanded leaves were harvested on the day of preparation shortly after the end of the dark period.

They were kept covered with aluminium foil with their roots dipping in water at 4 °C (for about 3 h) until the beginning of the preparation to reduce the amount of starch in chloroplasts.

All steps were carried out at 4 °C. About one kilogram of fresh deribbed mature leaves was finely chopped using a kitchen knife and ground in 2 L semi-frozen grinding buffer using a mortar and pestle. The homogenate was filtered through eight layers of gauze and one layer of Mira cloth. The filtered ‘brei’ was centrifuged (in polyallomer bottles) at 5,000 xg using a GS3 rotor in a Sorvall high speed centrifiuge (Sorvall RC-5B) for one min to remove cell debris and most of the plastids. The supernatant was again centrifuged using the same rotor at 12,000 rpm for 18 min to sediment the remaining organelles. The sediments were resuspended using a brush in Wash medium A and resedimented at 25,000 xg using SS34 tubes (SS34 rotor, Sorvall RC-5B) to reduce the volume. Protease inhibitors (0.5 mM PMSF in isopropanol, 1 mM benzamidine in ethanol, 1 µg/mL each leupeptin, antipain and pepstatin) were added to the 25,000 xg sediments followed by gentle resuspension in Wash medium A using a soft paint brush and

homogenization in a tight fitting Potter-Elvehjem homogenizer (capacity: 50 mL) for 20 strokes of the plunger.

The suspension was layered over 8 preformed Percoll step gradients chilled on ice.

The composition of the gradient was adapted from the original method as follows: a linear gradient of seven mL each 18 and 27% (v/v) Percoll was used instead of the discontinuous 15 + 27 % layers, the 14 mL 45% (v/v) Percoll layer was replaced with 16 mL 48% (v/v) Percoll and the volume of the 60% (v/v) Percoll cushion was reduced to three mL (earlier:

five mL); all Percoll solutions were prepared in 0.25 M sucrose. The gradients were spun in an SS34 rotor for 12 min at 10,000 xg with slow acceleration and deceleration. The peroxisome band above the 60% Percoll cushion was pooled from all the tubes, diluted with freshly prepared Wash medium B and centrifuged to sediment the organelles at 12,000 xg for 12 min. The washing step was repeated (10 min, 5,000 xg) to remove Percoll. Protease inhibitors (concentrations as above) were added to the final peroxisome pellet. This pellet was gently homogenized in a tight fitting (capacity: 5 mL) Potter-Elvehjem homogenizer and layered on a discontinuous sucrose density gradient comprised of small layers of 2 mL each 18, 25 and 35% (w/w) sucrose over a linear 40-60% (w/w) sucrose density gradient. The gradients were spun for 2 h at 83,000 xg in a Beckman Coulter ultracentrifuge using an SW28 rotor. The gradient was fractionated into 2 mL fractions from the top and saved as ready-to-use aliquots for the determination of the marker enzyme activities. For preparative purposes the gradient was slightly modified (40-50% w/w sucrose over a 60% cushion) and the peroxisome fraction above the sucrose cushion was saved after the addition of protease inhibitors. The activities of marker enzymes hydroxypyruvate reductase (peroxisomes), NADP-dependent GAPDH (plastids) and fumarase (mitochondria) and the chlorophyll content were determined as described (2.5). The typical yield of peroxisomes as determined by the activity of hydroxypyruvate reductase (HPR, marker enzyme peroxisomes, 2.5.1.1) was between 6 and 11% with respect to the activity in the crude extract. The leaf peroxisomes contained less than 0.5%

mitochondria (determined by activity of fumarase, 2.5.1.2) and almost no detectable chlorophyll (2.5.2).

Grinding buffer:

Mannitol 350 mM

Mops - NaOH pH 7.5 30 mM EDTA (Na²⁺) 1 mM

BSA 0.2 % (w/v)

PVP – 40 0.6 % (w/v)

Cystein 4 mM

Wash medium A:

Mannitol 300 mM

MOPS- NaOH pH 7.2 20 mM

EDTA (Na²⁺) 1 mM

BSA 0.2 % (w/v)

Percoll density gradient medium:

MOPS- NaOH pH 7.2 10 mM

Sucrose 250 mM

BSA 0.2 % (w/v)

Percoll 18, 27,

48, 60

% (v/v) Wash medium B:

Sucrose 250 mM

Hepes –NaOH pH 7.5 2 mM Sucrose density gradient medium:

Hepes- NaOH pH 7.5 10 mM EDTA (Na²⁺) 1 mM

Sucrose 18, 25,

35, 40, 50, 60

% (w/v)

2.2.1.2. Analytical method

A new analytical method was developed in the present study that facilitated isolation of leaf peroxisomes from spinach within a very short time. This method was suitable for the comparison of peroxisomal proteomes from differently treated tissues. In this method, the ‘post-plastidic’ supernatant obtained after centrifugation of the filtered crude extract was directly applied on a Percoll density gradient.

• the leaf tissue was homogenized using a limited volume of grinding buffer (the ratio of leaf tissue to the grinding buffer was 1.5 g/ mL)

• the peroxisomes were not subjected to any kind of manual handling by way of sedimentation and resuspension

• the separation and purification of peroxisomes occurred within a single Percoll density gradient which was shortened to accommodate a larger volume of sample.

It constituted a linear gradient of 18-27% (v/v) (2 x 6 mL) over a 55% (v/v) Percoll layer (12 mL) that formed a density barrier and prevented further migration of contaminants, yet, allowed the peroxisomes to sediment. The former (18 and 27%

(v/v) Percoll) was prepared in 250 mM sucrose and the latter (55% (v/v)) in 250 mM raffinose. (Propane 1, 2-diol was omitted from the Percoll density gradient solutions).

All steps were carried out at 4 °C. Freshly harvested leaves were washed, deribbed, cut into small pieces and homogenized in a limited volume of grinding buffer using a mortar and pestle. The protease inhibitor 0.5 mM PMSF was included during homogenization. The homogenate was filtered through a layer of Mira cloth and centrifuged at 5,000 xg for 1 min. About 10 to 12 mL of the supernatant was applied on preformed Percoll density gradients and spun for 12 min at 10,000 xg in a Sorvall SS34 rotor. After centrifugation, the upper layers were aspirated away and the pale white peroxisome pellets were quickly aliquoted after transferring then to clean tubes. Aliquots were immediately frozen for later determination of enzyme activities and protease inhibitors (see 2.2.1.1) were added to the remaining peroxisome fraction which was saved as larger aliquots for 2-DE. It was not possible to concentrate the peroxisome fraction by

washing away the Percoll because of a significant loss of protein (<10% recovered after washing). Peroxisomes obtained by this method were of high purity with no detectable chlorophyll. Fumarase activity could not be detected. The contamination with pro-plastid like organelles was about 0.2 to 0.6%.

Grinding buffer:

The composition of the grinding buffer was the same as that used for the preparative method.

Percoll density gradient medium:

MOPS- NaOH pH 7.2 10 mM

Osmoticum* 250 mM

Percoll 18, 27, 55 % (v/v)

* The 18 and 27% (v/v) Percoll solutions were prepared with sucrose and the 55% (v/v) Percoll solution was prepared with raffinose as the osmoticum