Supporting Information
Table S1 The inhibition of mitochondrial and chloroplastic carnitine palmitoyltransferase by
etomoxir
Palmitoyl carnitine formed
% of control
(pmol min-1 mg-1 protein)
Mitochondrial CPT
0 etomoxir
123.9 ± 14.2
50M etomoxir
55.7 ± 6.1
250M etomoxir
100
44.9
0
0
Chloroplastic CPT
0 etomoxir
96.0 ± 7.4
100
50M etomoxir
91.1 ± 6.8
94.9
250M etomoxir
99.1 ± 8.5
103.2
Isolation of mitochondria
Pea seeds were germinated in the dark for 48 h at 25oC as previously described (Thomas &
McNeil, 1976). The mitochondria were isolated as described for “washed preparations”
(Wood et al., 1984) and the 5 ml final resuspended pellet was divided into two equal aliquots.
Each aliquot was incubated for 1 h at 220C with gentle shaking in an incubation mixture of
0.4 M sucrose, 2 mM MgSO4, 2 mM ATP, 200 M CoASH, 0.5% (w/v) defatted BSA
(Thomas et al., 1982) and 200 mM Tris-HCl pH 7.2. To one aliquot 50 or 250 M etomoxir
was added with a corresponding volume of distilled water added to the control. This
incubation mixture allows the long-chain acyl CoA synthetase situated on the outer
mitochondrial membrane to convert the etomoxir to its CoA ester.
Following the incubation period the two aliquots were diluted to 50 ml with 0.4 M sucrose, 1
mM EDTA (disodium salt) containing 0.25% (w/v) defatted BSA and centrifuged at 8000 x g
for 10 min. The resulting pellets were resuspended in 3 ml 0.4 M sucrose and 0.5% (w/v)
defatted BSA and the mitochondria were purified on sucrose density gradients (Burgess et
al., 1985). This step concentrated the mitochondria essentially free of other contaminating
organelles and also removed any residual CoASH from the preparations as CoASH
competitively inhibits carnitine palmitoyltransferase (Wood et al., 1984).
Isolation of chloroplasts
Chloroplasts were prepared from 2- to 3-week-old pea leaves by the method of Miflin &
Beevers (1974) and purified on sucrose density gradients as described previously (Thomas et
al., 1982). Fresh, intact chloroplasts were collected from the gradients as described by
Masterson & Wood, 2000b and the resulting pellet was resuspended in 5 ml of a medium
consisting of 0.33 M sorbitol, 50 mM HEPES pH 7.5, 0.1% (w/v) Bovine serum albumin
(BSA). This 5 ml final resuspended pellet was divided into two equal aliquots. Each aliquot
was incubated for 1 h at 220C with gentle shaking in an incubation mixture of 0.33 M
sorbitol, 2 mM MgSO4, 2 mM ATP, 200 M CoASH, 0.5% (w/v) defatted BSA (Thomas et
al., 1982) and 50 mM HEPES pH 7.5. To one aliquot 50 or 250 M etomoxir was added with
a corresponding volume of distilled water added to the control. This incubation mixture
allows the chloroplastic long-chain acyl CoA synthetase (Stumpf, 1980) to convert the
etomoxir to its CoA ester.
Following the incubation period the two aliquots were diluted to 50 ml with 0.33 M sorbitol,
50 mM HEPES pH 7.5 and 0.1% (w/v) defatted BSA and centrifuged at 3000 x g for 5 min.
The resulting pellets were resuspended in 3 ml 0.33 M sorbitol, 50 mM HEPES pH 7.5 and
0.1% (w/v) defatted BSA (Masterson & Wood, 2000b). This step removed any residual
CoASH from the preparations as CoASH competitively inhibits carnitine
palmitoyltransferase (Wood et al., 1984).
The purified mitochondrial and chloroplast preparations were assayed for carnitine
palmitoyltransferase activity using the direct method of Wood et al., (1984) with palmitoyl
CoA as substrate. The resulting palmitoylcarnitine was extracted by the method of Bremer
(1963) as used by Wood et al., (1984).