Solutions

Grinding buffer A: 0.3 M sucrose, 60 mM N-tris [hydroxymethyl]- methyl-2

aminoethanesulphonic acid (TES), 10 mM ethylene diamine tetra-acetic acid (EDTA), 10 mM
KH2PO4, 25mM tetrasodium pyrophosphate, 1 mM glycine, 1% (w/v) polyvinylpyrrolidone-40,
1% (w/v) defatted bovine serum albumin (BSA), pH-(KOH) 8.0, 50 mM sodium ascorbate, plus
20 mM cysteine added just prior to grinding, and readjustment of the pH to 8.0 with KOH.
Grinding buffer B: as grinding buffer A, but with 2 mM EDTA. Wash buffer A: 0.3 M sucrose,
10 mM TES, 2 mM EDTA, 10 mM KH2PO4, pH-(KOH) 7.5. Washing buffer B: as Washing
Buffer A, but without EDTA. [Olivier Keech, 2005]

Protein determination

Protein determination was carried out Lowry and Bradford technique [Lowry et al. 1951]
[Bradford 1976]. Bovine serum albumin was used as the calibration standard.

Isolation of Mitochondria

About 5 g of leaves were cut up with scissors and placed in a mortar. They were then ground,
using a pestle, in 20 ml of grinding buffer A with a small amount (about 0.5 g) of quartz. The
extract was filtered through a 20-mm nylon mesh and centrifuged at 2500 g for 5 min to remove
most of the intact chloroplasts and thylakoid membranes. The supernatant was transferred to a
new tube and centrifuged at 15 000 g for 15 min. The pellet obtained was suspended in wash
buffer A and centrifuged at 15 000 g for 15 min. The resulting supernatant was discarded and the
pellet containing the crude mitochondria was resuspended in 200 ml of wash buffer A, giving a
final volume of approximately 400 ml. All steps of the two methods presented below were
Performed at 4 0 C. [Olivier Keech, 2005]

Fractionation of Mitochondrial Membranes

Both the interface and pellet fractions were dialyzed for 16-18 h in
the cold against 1,000 vol of buffer T.
After a thorough dialysis against 10 mm KH2PO4-KOH
buffer, pH 8.0, containing 0.5 mm EDTA and 10% (w/v)
glycerol, the whole 40% to 60% ammonium sulfate fraction
was applied to a DEAE-Sepharose CL-6B column (2
3 30 cm, Pharmacia), which had been equilibrated with
200 mm KH2PO4-KOH buffer, pH 7.0, and then with 10 mm
KH2PO4-KOH buffer, pH 8.0, containing 0.5 mm EDTA
and 10% (w/v) glycerol. Elution was carried out at 0.75 mL
min21 with a linear gradient of NaCl from 0 to 600 mm in
10 mm KH2PO4-KOH buffer, pH 8.0, containing 0.5 mm
EDTA and 10% (w/v) glycerol.
The fractions containing ACAD activity were concentrated
by osmotic dehydration against Suc and dialyzed
against 10 mm KH2PO4-KOH buffer, pH 7.0, containing
10% (w/v) glycerol. The resulting preparation was applied
to a hydroxylapatite BIO-GEL HT column (1 3 15 cm,
Bio-Rad), which had been equilibrated with 200 mm
KH2PO4-KOH buffer, pH 7.0, and then with 10 mm
KH2PO4-KOH buffer, pH 7, containing 10% (w/v) glycerol.
Elution was carried out at 0.75 mL min21 with a linear
gradient of phosphate from 10 to 500 mm, pH 7.0, in the
presence of 10% (w/v) glycerol. All chromatographic steps
were carried out at 0°C to 4°C and driven by an Econo
System (Bio-Rad).

Gel Electrophoresis

Mitochondrial membrane specimens were electrophoresed on 1.5-mm-thick polyacrylamide slab

gels consisting of a 6% acrylamide stacking gel (2-cm long) and a 12% acrylamide separation
gel (20-cm long), prepared and stained (with Coomassie Brilliant Blue G) as described by [Chua
and Bennoun]. The buffer systems [Neville] or [Laemmli] were used with 0.1% SDS in the
upper reservoir buffer and in the gels themselves. Mitochondrial membrane specimens
containing 10 to 50 ~g of protein were prepared in 60-td mixtures containing 2% SDS, 25%
glycerol, 1.5%/3-mercaptoethanol, and 0.04% brnmophenol blue. These were heated to 100°C
for 1 min before they were loaded onto the gels. For molecular weight calibration protein
standards BSA Molecular weight marker 66399 Da were electrophoresed on the same gels. The
gels were run at 30 m/~ for 7.5 h (Neville buffers) or at 50 mA for 6 h (Laemmli buffers).

Reference

Solutions

Oliver Keech, Pierre Dizengremel and Per Gardestrom 2005. Preparation of leaf mitochondria
from Arabidopsis thaliana. Physiologia Plantarum 124:403-409.

Protein determination

Lowry O, Rosebrough N, Farr A, Randall R (1951) Protein measurement with the folin phenol
reagent. J Biol Chem 193: 265–275.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities
of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254

Gel Electrophoresis

ChuB, N.-H,, and P. Bennoun. 1975. Tbylakoid membrane polypeptides of Chlamydomonas

reinhardtii: wild-type and mutant strains deficient in photosystem I1 reaction center. Proc. NaiL
Acad. Sci. Ur S. A. 72:2175 2179.

Neville. D. M., Jr 1971. Molecular weight determinations of protein-dodecyl sulfate complexes

by gel electrophoresis in a discontinuous buffer system. J. BioL Chem. 246:6328-6334.

Laemmti, U. K. 1970. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature (Land.). 227:680-685.