ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 217, No. 1, August, pp. 312-323,1982

Purification of Plant Mitochondria by lsopycnic Centrifugation in

Density Gradients of Percoll’
MICHEL NEUBURGER, ETIENNE-PASCAL JOURNET, RICHARD BLIGNY,
JEAN-PIERRE CARDE: AND ROLAND DOUCE3

Physiologic Gel&lake V6g&ale-DRF/BV, Centre d’Etudm Nuctiaires et Universit6 Scimtifique et M&Scale

de Grenoble, 85 X-88041 Grenoble-Cedex, France
Received December 21, 1981, and in revised form March 19, 1982

Mitochondria from potato tubers have been separated from contaminating organelles
and membrane vesicles on self-generated Percoll gradients and in a relatively short
time. The Percoll-purified mitochondria devoid of carotenoids and galactolipids showed
no contamination with intact plastids, microbodies, or vacuolar enzymes. Percoll-pu-
rified mitochondria exhibited intact membranes and a dense matrix. The intactness
of purified mitochondrial preparations was ascertained by the measurement of KCN-
sensitive ascorbate cyt c-dependent O2 uptake. When compared with washed mito-
chondria, Percoll-purified mitochondria showed improved rates of substrate oxidation,
respiratory control, and ADP:O ratios. The recovery of the cyt oxidase was 70-90s and
on a cyt oxidase basis the rate of succinate oxidation by unpurified mitochondria was
equal to that recorded for Percoll-purified mitochondria. The great flexibility of pu-
rification procedure involving silica sols was extended from mitochondria to the iso-
lation of intact peroxisomes.

Mitochondria able to sustain rapid res-
piration rates may be isolated from a large
variety of plant tissues provided that care
is taken to avoid large-scale rupture of the
two mitochondrial membranes (1, 2). In-
evitably a few ruptures occur during ex-
traction and metabolically active prepa-
rations containing large numbers of intact
mitochondria always contain a variable
proportion of damaged or envelope-free
mitochondria. The ruptured mitochondria
exhibit a low rate of respiration inasmuch
as cyt c is rapidly released into the me-
dium when the outer membrane is dam-
aged (3). In the past, the fact that both
i Supported in part by Research Grant from the
“Centre National de la Recherche Scientifique”
(ERA 847: Interactions Plastes-Cytoplasme-Mito-
chondries).
’ Permanent address: Physiologie Cellulaire Vege-
tale, Universite de Bordeaux I, Avenue des Facultes,
33405 Talence Cedex, France.
3 To whom correspondence should be addressed.
0003-9861/82/090312-12!$02.00/0
Copyright Q 1982 by Academic Press. Inc.
All rights of reproduction in any form reserved.
types of mitochondria occur in the same
preparation has not always received the
attention it deserves and has occasionally
led to erroneous conclusions concerning
the permeability of the outer mitochon-
drial membrane to cyt c. In addition, scru-
tiny by electron microscopy of the washed
mitochondrial pellets obtained by differ-
ential centrifugation shows that the mi-
tochondria are contaminated to varying
extents by smooth vesicles of unknown
origins, proplastids, and microbodies. Cen-
trifugation in sucrose density gradients
has been widely used for the purification
of plant mitochondria (4-6). However, al-
though plant mitochondria survive the
purification procedure, osmotic damage
caused by high sucrose concentrations (6)
is a severe limitation of this method. As
a matter of fact, dilution to isoosmolar
conditions must proceed very slowly to
avoid damage resulting from too rapid in-
ner membrane expansion (6).
312
 PURIFICATION OF PLANT MITOCHONDRIA 313

The purpose of this paper is to demon- collected with a needle attached to a syringe. All the
strate that centrifugation of the mito- fractions were well mixed with 15 times their volume
chondrial preparation on a nontoxic silica of washing medium (0.3 M mannitol; 10 mM phosphate
buffer, pH ‘7.2; 1 mM EDTA; and 0.1% (w/v) BSA) to
sol gradient (Percoll, Pharmacia) which
dilute Percoll, and the organelles were sedimented
maintains isoosmotic conditions through- by centrifugation. Fraction 1 (Fig. 1) was pelleted at
out the purification procedure (7) effects 100,OOOgfor 90 min and the pellet was yellow. Frac-
a clear separation of mitochondria from tion 2 was pelleted at 12,000g for 15 min and the
the contaminating material in less than pellet was brown-reddish. Fraction 3 was pelleted at
45 min. In addition, we describe a simple 40009 for 10 min and the pellet was black. Each pellet
method for the rapid estimation of the was gently resuspended in a small volume of washing
proportion of intact mitochondria based medium. The mitochondria (fraction 2) were stored
upon reactions utilizing electron acceptors at approximately 50 mg protein per milliliter.
which do not rapidly cross the outer mi- The purification of mitochondria by centrifugation
in a discontinuous sucrose gradient was carried out
tochondrial membrane.
according to the method of Deuce et al. (6).
0, uptake measurements. 0s uptake was measured
MATERIALS AND METHODS
at 25°C using a Clark-type Oa electrode system pur-
Preparation of mitochxmdria. Potato tubers (So chased from Hansatech Ltd, Hardwick Industrial
hum tubrosum, L.) were obtained from a local Estate, King’s Lynn, Norfolk (8). The reaction me-
market. Tubers (4 kg) were cut (cubes l-2 cm on a dium (medium A) contained: 0.3~ mannitol; 5 mM
side) into 6 liters of chilled extraction medium con- MgC12; 10 mM KCl; 10 mM phosphate buffer, pH ‘7.2;
taining: 0.3 M mannitol; 4 mM cysteine; 1 mM EDTA, 0.1% (w/v) defatted BSA and known amounts of
20 mM pyrophosphate buffer, pH 7.5, and 0.1% (w/v) mitochondrial protein in a total volume of 1 ml. The
defatted BSA.’ The material was homogenized in O2 concentration in air-saturated medium A was
three successive batches in a l-gallon Waring blender taken as 240 PM.
at low speed for 2s (1.3 kg tubers for 2 liters ex- Measurement of cyt c-dependent 0, uptake by mi-
traction medium in each batch). It is desirable to tochondtia. The intactness of mitochondrial prepa-
have the extraction medium as a semifrozen slush. rations can be rapidly evaluated by cyt c oxidation,
It is also critical that the grinding (mincing) process using an Oa electrode. Mitochondria were added to
be as short as indicated. The brei was squeezed medium A (isotonic medium) in a reaction vessel and
through eight layers of muslin (Ruby, Voiron, France) to distilled water in an other. The latter was then
and a 50-hrn nylon net. The crude mitochondrial pel- stirred for 10 s before adding double-strength me-
let was prepared as fast as possible according to the dium A to restore isotonic conditions following the
method of Bonner (1). Mitochondria thus obtained osmotic shock. Under these conditions, the outer
(washed mitochondria) were purified by centrifuga- membrane bursts and exogenous cyt c has access to
tion in a Percoll gradient. Two centrifuge tubes were the outer surface of the inner membrane where it is
filled with 36 ml each of Percoll medium (28% (v/v) oxidized. The basic medium for cyt c-dependent O2
Percoll; 0.3 M sucrose; 10 mM phosphate buffer, pH
7.2; 1 mM EDTA; and 0.1% (w/v) BSA). The final
concentration of Percoll and sucrose should be care-
fully determined according to the origin of mito- input- -fraction 1
chondria and to the nature of the contaminations 60 mg O’Of d-1 054
present in the washed mitochondria which are ex-
-fraction 2
tremely variable. Aliquots (3-ml sample, 50-60 mg (mitochondria)
protein) of the washed mitochondria were then lay- d-1 067
- fraction 3
ered on the Percoll medium. The tubes were placed (peroxisomes)
in a precooled Sorvall SS 34 angle-head rotor. Cen-
trifugation at 40,OOOgfor 30 min resulted in the for-
mation of three bands in the tube. As a matter of FIG. 1. Purification of potato tuber mitochondria.
fact, under these conditions, centrifugation of Percoll Schematic representation of the Percoll gradient
in an angle-head rotor resulted in spontaneous for- fractionation of the washed mitochondria (input) in
mation of a smooth density gradient. Fractions were order to obtain intact mitochondria (fraction 2) and
microbodies (fraction 3). The rotor used is an angle-
’ Abbreviations used: BSA, bovine serum albumin; head rotor (SS 34, Sorvall). The use of an automatic
TPP, thiaminepyrophosphate; MOPS, morpholino- rate controller (Sorvall) is recommended. By using
propanesulfonic acid, cyt c, cytochrome c; PVP, po- density markers, the apparent density of mitochon-
lyvinylpyrrolidone. dria is within the range 1.054 to 1.067.
314 NEUBURGER ET AL.

FIG. 2. Electron micrographs of Percoll-purified potato tuber mitochondria (fraction 2, Fig. 1).
Fixation methods described in text. Note that mitochondria exhibit intact membranes and a dense
matrix. In addition, at high magnification, the dark-light-dark construction of both membranes
is clearly visible.

uptake contained: intact or swollen mitochondria-
i.e., without outer membrane-(0.5 mg protein); 8mM
ascorbate; 1 ml medium A. The O2 consumption was
initiated with 30 pM cyt c. After 1 min, and in order
to inhibit cyt oxidase, 200 PM KCN was added. The
cyanide insensitive cyt c-dependent O4 uptake which
is very low (in 56 experiments this rate ranged from
4 to 7 nmol Or/min) corresponds to a nonenzymatic
oxidation of reduced cyt c by molecular Oz. Conse-
quently, the KCN-sensitive cyt c-dependent Or up-
take is entirely attributable to cyt oxidase function-
ing. Therefore it should be possible to estimate very
rapidly the proportion of intact mitochondria in a
given preparation from the relative rate of KCN sen-
sitive cyt c-dependent Oc uptake by untreated and by
osmotically shocked mitochondria in the same man-
ner as for NADH or succinate-dependent cyt c re-
duction (6). The KCN sensitive cyt c-dependent Or
uptake was assayed under conditions of optimum
substrate and cofactor concentrations.
Pigment ana&.& Pigments were extracted from
the different fractions according to Jeffrey et aL (9).
Pigments were dissolved in either ethanol or chlo-
roform and absorption spectra were recorded on an
Aminco (DW2) spectrophotometer.
Mitodxmdrial phosphdipid bre&down. Phospho-
lipid breakdown was achieved by incubating mito-
chondria (10 mg protein/ml) in the following me-
 PURIFICATION OF PLANT MITOCHONDRIA 315

dium: 0.3~ mannitol; 10 mM MOPS buffer, pH 6.5;

smtdascorbate smMascorbate
FIG. 3. Ascorbate cyt c-dependent O2 uptake of
Percoll-purified potato mitochondria. (A) Intact mi-
tochondria; (B) burst mitochondria. Cyanide (0.2 mM)
is added as indicated. The numbers on the traces
refer to nmol Oa consumed/min. Protein: 0.20 mg.
Final volume: 1 ml. MP, purified mitochondria.
5 mM CaCla. In order to prevent the formation of
linoleic acid hydroperoxide (10) the medium was bub-
bled with Argon. Aliquots (200 ~1) were taken at var-
ious times for lipid analysis. Polar lipids and
liberated fatty acids were extracted and were
chromatographed in two dimensions on silica plates
as previously described (11). For quantification and
free fatty acids analysis, lipid areas were scraped
from the plates and transferred into vials containing
4 ml of the transesterification mixture (CH,OH/
HaSOJbenzene, 100~5~5, v/v) and 100 pg behenic acid
as internal standard (11). The vials were bubbled
with argon, securely tightened with a Teflon-lined
cap, and placed in an oven at 68°C for 2 h. The sep-
aration of fatty acid methyl esters by gas chroma-
tography (Hewlett-Packard 5750, 10% DEGS on a
Varaport 30 column, temperature 175‘C) and quan-
tification of their parent lipids were done according
to Allen and Good (12).
Enzymes and other assays. Catalase (HsOa:HaOa

FIG. 4. Oxidation of succinate, NADH, and a-ketoglutarate by potato tuber mitochondria. Upper
traces: washed mitochondria (MW); lower traces: Percoll-purified mitochondria (MP). The concen-
trations given are the final concentrations in the reaction medium. The numbers on the traces refer
to nmol 02 consumed/min/mg of protein. In this particular experiment, the ratio of the total
protein added to the top of the Percoll gradient (input) to the total mitochondrial protein recovered
in fraction 2 (Fig. 1) was 1.45. Since most of the plant mitochondria isolated so far are depleted
of their endogenous thiamine pyrophosphate (TPP) it is necessary to add this cofactor in the
medium in order to trigger a-ketoglutarate oxidation. The rate of succinate oxidation by Percoll-
purified potato tuber mitochondria is within the range of 400 to 650 nmol Oa consumed/min/mg
of mitochondrial protein.
316 NEUBURGER ET AL.

oxidoreductase, EC 1.11.1.6) was assayed according
to the method of Van Ginkel and Brown (13). Lipox-
ygenase (an enzyme that catalyzes the addition of Oa
to linoleic acid, EC 1.13.11.12) was assayed polaro-
graphically in medium A. Linoleic acid was soluhi-
lized in Oa-free ethanol. The reaction was initiated
by addition of hydroperoxide-free linoleic acid (1 mM
final concentration) to a l-ml reaction cell containing
the appropriate fraction (14).
Protein was determined by the Folin-Ciocalteu
phenol reagent (15).
C@chrome determination Spectrophotometric
measurements were performed with a sensitive spec-
trophotometer (Aminco, DW2). The mitochondrial
concentrations of cytochromes were measured at low
temperature (77°K) from succinate anaerobic minus
oxidized difference spectra. The pairs of wavelengths
selected for measurement of individual cytochromes,
as well as the extinction coefficients, were those given
by Lance and Bonner (16).
Electron microscopy. Cell organelles were pelleted
and fixed 2 h at 4°C with 0.25% glutaraldehyde
the washing medium. The pellets were rinsed two
times (15 min each) with the washing medium and
were postfixed with 1% 0~0~ dissolved in the same
medium. After 2 h at 4°C the pellets were washed
four times (20 min each) with 0.1 M cacodylic acid
(pH 7.6) and incubated in 1% tannic acid (Mallinck-
rodt) in 0.1 M cacodylate buffer for 30 min at 22°C
according to the method of Simionescu and Simi-
onescu (17). We used tannic acid in order to enhance
contrast and to reveal the three-layered structure of
the membranes which is otherwise not readily visible
in thin sections. The samples were dehydrated
ethanol followed by propylene oxide, embedded in
Epon 812, and sectioned with a diamond knife. The
silver sections were stained with 7% uranyl acetate
in 50% ethanol for 30 min, counterstained with 3%
lead citrate for 10 min, and were examined with a
Philips 301 transmission electron microscope oper-
ated at an accelerating voltage of 80 kV.
RESULTS
Properties of Percoll-Puti$ed
Mitochondria
Figure 1 shows the position of mito-
chondria isolated from potato tubers after
centrifugation on a Percoll gradient con-
taining 0.3 M sucrose. Most of the mito-
chondria (fraction 2) appeared as a broad
band located beneath a yellow layer (frac-
tion 1) at the top of the gradient. In ad-
dition, heavy organelles (fraction 3) were
recovered as a sharp band sitting above
a clear colorless layer of Percoll at the
in
bottom of the tube. It is noteworthy that
the substitution of sucrose by 0.3 M man-
nitol led to a marked shift, downward, of
fraction 2 along the gradient and the mi-
tochondria aggregated near the bottom of
the tube just on top of fraction 3. In fact,
we have observed by using density mark-
ers (Density marker beds, Pharmacia)
that the total density range covered by
Percoll in sucrose is slightly different than
that of Percoll in mannitol. The apparent
in density of Percoll-purified mitochondria
is within the range 1.054 to 1.067. Conse-
quently, the apparent average density of
sucrose-purified mitochondria (see (4, 6))

TABLE I

REPRESENTATIVE EXPERIMENT SHOWING THE RECOVERY OF PROTEIN, CYT OXIDASE, AND SUCCINATE
OXIDATION IN VARIOUS FRACTIONS OBTAINED AFTER CENTRIFUGATION OF WASHED MITOCHONDRIA
ON CONTINUOUS PERCOLL GRADIENT

Succinate oxidation Cyt c-dependent

Fraction Protein (state III) 0s uptake
(see Fig. 1) (md (pm01 Oz/min) (pm01 Or/min)

Input
(washed mitochondria) 64 18.5 28
Fraction 1 17 1.5 2.2
Fraction 2
(mitochondria) 40 16.4 24.6
Fraction 3
(peroxisomes) 4 nd” 0.2

Note. For measurement of succinate oxidation and cyt c-dependent Or uptake, see Materials and Methods.
o Not detected.
 PURIFICATION OF PLANT
AODcOOl
FIG. 5. Succinate reduced minus oxidized difference
spectra of potato tuber mitochondria at liquid nitro-
gen temperature (77°K). Mitochondrial protein con-
centrations: washed mitochondria (MW) = 3.33 mg/
ml; purified mitochondria (MP) = 3.33 mg/ml. Optical
path: 2 mm (Plexiglas cuvettes). Concentrations of
cyt o,oa in MW and MP are, respectively, 0.27 and 0.46
nmol/mg protein. In this particular experiment, ex-
pressed on a relative basis taking the protein con-
centration as unity, the succinate oxidation rates in
MW and MP were, respectively, 273 and 460 nmol Oa
consumed/min.
is much higher than that of Percoll-pu-
rified mitochondria. The mitochondria are
highly pleomorphic, sometimes filamen-
tous, and they differ in the relative pro-
portion of internal membranes (cristae)
and remaining matrix. It is possible that,
in vivo, mitochondria with large matrix
volume have a much higher content of sol-
uble enzymes and a higher density than
mitochondria with small matrix volume.
The isolated mitochondria (fraction 2)
appeared mainly in the condensed config-
uration and showed no contamination
with intact plastids or microbodies (Fig.
2). In addition, mitochondria exhibited in-
tact membranes and a dense matrix. The
intactness of purified mitochondrial prep-
arations was strengthened by the mea-
surement of KCN-sensitive ascorbate cyt
c-dependent O2 uptake (see Materials and
MITOCHONDRIA 317
Methods). For example, in Fig. 3, the rate
of O2 uptake by the intact mitochondria
was 5% of that recorded for osmotically
shocked mitochondria, giving an apparent
percentage intactness of 95%. In 120 ex-
periments, the percentage intactness thus
measured ranged from 80 to 98% with
mean value of 94%. We have shown that
there is a good agreement between direct
observations (Fig. 2) and the ascorbate-cyt
c assay (Fig. 3) when we were able to
make simultaneous measurements. Fur-
thermore, although there was some
variation in the result for individual
preparations, we have shown that there
was a broad measure of agreement be-
tween percentage of intact mitochondria
measured by the cyt c-dependent O2 up-
take and percentage of intact mitochon-
dria measured by the other assays (suc-
cinate:cyt c oxidoreductase; NADH:cyt c
oxidoreductase, see (6)).
Mitochondria purified in continuous
Percoll density gradients showed a mark-
edly higher rate of O2 uptake in state 3
than did unpurified mitochondria (Fig. 4,
Table I). In addition, ADP/O and respi-
ratory control ratios with succinate,
NADH, and a-ketoglutarate as substrates
were maintained or slightly improved fol-
I
0.1 A
L
( “o’““e,e*
1
FIG. 6. Absorption spectra of chloroform extracts
of (A) chloroplast envelope from spinach leaves and
(B) fraction 1 (Fig. 1). The protein contents of (A)
and (B) were 0.3 and 7.6 mg, respectively.
318 NEUBURGER ET AL.
lowing separation from contaminating
subcellular structures (Fig. 4; see also ref.
(18)). It is interesting to note that, ex-
pressed in terms of nanomoles per milli-
gram of mitochondrial protein, the con-
centration of the different cytochromes
was greater in the purified mitochondria
than in unpurified mitochondria (Fig. 5).
Furthermore, just as the state 3 oxidation
rates were more rapid in the purified mi-
tochondria, compared with unpurified mi-
tochondria, the cyt oxidase concentration
was similarly increased (see legend to Fig.
5). Consequently, when expressed on a rel-
ative basis taking the cyt oxidase concen-
tration as unity, there was virtually no
difference between the purified and washed
mitochondria (Fig. 5).
Contaminants of Unputified
Mitochondrial Fractim
It is clearly seen from the ratio of total
protein added to the top of the gradient
to total protein collected in fraction 2
(Table I), that the contamination was very
important in unpurified potato tuber mi-
tochondria. Experiments were therefore
performed to characterize these contami-
nations.
Fraction 1 contained numerous small
smooth - surfaced vesicles (result not
shown). The spectrum of fraction 1 showed
a strong carotenoid absorption with the
typical three-banded spectrum in the blue
region (Fig. 6). On the other hand, Percoll-
purified mitochondria were almost devoid
of carotenoids (Fig. 7). In fact, we have
verified that almost all of the carotenoids
present in unpurified mitochondria prep-
arations were recovered in fraction 1. In-
terestingly, the full absorption spectrum
of a chloroform extract of the envelope
from spinach leaf chloroplasts was almost
identical to that of fraction 1 (Fig. 6). This
result strongly suggests that unpurified
potato mitochondria were appreciably
contaminated by amyloplast envelope
membranes (19,20). The presence of galac-
tolipids in fraction 1 (monogalactosyl-
diacylglycerol and digalactosyldiacylglyc-
erol were present in a ratio of 0.8:1) and
the absence of galactolipids in Percoll-pu-
I
wavelength (nm)
FIG. ‘7. Absorption spectra of ethanol extracts of
potato tuber mitochondria. MW, washed mitochon-
dria (3.2 mg protein; 0.32 pg carotenoid); MPS, mi-
tochondria purified on a sucrose gradient (3.2 mg
protein; 0.14 pg carotenoid); MPP, mitochondria pu-
rified on a Percoll gradient (3.2 mg protein; 0.03 pg
carotenoid).
rified mitochondria confirm this view. Un-
der these circumstances it is obvious that,
during the course of tissue grinding, con-
siderable fragmentation of the fragile
amyloplasts necessarily occurs and leads
to extensive contamination of the unpu-
rified mitochondria fraction by a variable
proportion of amyloplast envelope mem-
branes rich in galactolipids (19, 20) and
carotenoids (19, 20). To determine if su-
crose was as effective as Percoll in remov-
ing carotenoids from the mitochondria,
discontinuous sucrose gradients (6) were
tested. Figure ‘7 indicates that mitochon-
dria purified on sucrose gradients were
more contaminated with carotenoids than
mitochondria purified in Percoll.
Fraction 3 appeared as a homogeneous
population of organelles. Each have a sin-
gle bounding membrane and have a gran-
ular matrix (Fig. 8). A large crystalline
inclusion occurred within the matrix but
no ribosome and no internal membrane
system were ever present (Fig. 8). It is
obvious that fraction 3 contained almost
 PURIFICATION OF PLANT MITOCHONDRIA 319

FIG. 8. Electron micrographs of Percoll-purified potato tuber microbodies (fraction 3, Fig. 1).
Fixation methods described in the text. Note that the microbodies have a single bounding membrane
and a large crystalline inclusion within the granular matrix.

exclusively fully intact microbodies. As a 2 (see Materials and Methods). In addition,

matter of fact, estimation of the marker
enzyme catalase showed that sedimenta-
tion through a continuous Percoll gradient
eliminated microbody contamination in
mitochondrial fractions by eightfold com-
pared with that found in unpurified mi-
tochondrial preparations (Table II). Most
of this catalase was in soluble form and
could be eliminated by pelleting fraction
if the purification procedure is repeated
once (i.e., a second time through Percoll)
mitochondrial catalase activity can be re-
duced to less than 0.2% of that found in
unpurified mitochondria (Table II). These
results demonstrate that unpurified po-
tato mitochondria were also massively
contaminated by microbodies.
We have shown that all crude prepa-
320 NEUBURGER ET AL.

rations of intact mitochondria isolated in isolating mitochondria free from en-

from potato tubers were more or less con-
taminated by harmful hydrolases such as
nonspecific lipolytic acyl-hydrolase(s) (31).
When intact Percoll-purified mitochon-
dria were incubated at 25°C in suspending
medium containing 5 mM CaC&, practi-
cally no degradation of phospholipids oc-
curred within an hour (Fig. 9). In contrast,
in the case of unpurified mitochondria,
addition of 5 mM CaC& to the medium in-
duced a rapid hydrolysis of total mito-
chondrial phospholipids within an hour.
During the course of the phospholipid
breakdown, the fatty acid moieties of
phospholipids were mostly recovered in
the form of free fatty acids. These results
demonstrate that unpurified potato mito-
chondria were also contaminated by an
unknown cell fraction containing a cal-
cium-dependent lipolytic acyl-hydrolase
activity. It is possible that this contami-
nating enzyme (membrane bound? solu-
ble?) could derive from the vacuolar sys-
tem. In support of this suggestion, we have
observed that the unpurified mitochondria
retained a variable percentage of vacuolar
protease( DNAse(s), and RNAse(s) ac-
tivities (results not shown). In marked
contrast, extramitochondrial acidic hy-
drolases were practically absent in Per-
toll-purified suspension. Sucrose gradient
centrifugation did not appear to be as ef-
fective as Percoll gradient centrifugation
dogenous RNAse, DNAse, and lypolitic
acyl-hydrolase (Fig. 9).
Finally, with potato tuber mitochondria
preparations, the lipoxygenase activity re-
covered in the purified mitochondria frac-
tion represented less than 5% of the total
activity present in the unpurified mito-
chondrial fraction (Table II). In fact, from
95 to 100% of the lipoxygenase activity
initially layered onto the gradient was re-
covered in fraction 1. Again, we have
found that isolation of mitochondria on
Percoll gradients appeared to be more ef-
fective in removing lipoxygenase activity
than centrifugation on comparable su-
crose gradients. These results, in contrast
with a recent report (21), clearly indicated
that Percoll purified mitochondria are de-
void of lipoxygenase activity (see also Sie-
dow and Girvin (14)).
DISCUSSION
The results presented in this article
demonstrate that potato tuber mitochon-
dria purified by centrifugation in density
gradients of modified silica sol (Percoll)
are highly intact and retain a high rate
of substrate-dependent O2 consumption.
They approach ultimate purity as judged
by electron microscopy and analysis using
marker enzymes. Since on a cyt oxidase
basis the rate of succinate oxidation by

TABLE II

REPRESENTATIVEEXPERIMENTSHOWINGTHE RECOVERYOF PROTEIN,CATALASE,AND LIPOXYGENASEIN

VARIOUSFRACTIONSOBTAINEDAFTERCENTRIFUGATIONOFWASHED MITOCHONDRIA
ON CONTINUOUS PERCOLL GRADIENT

Fraction Protein Catalase Lipoxygenase

(see Fig. 1) (mid (pm01 Oa/min) (pm01 Oa/min)

Input
(washed mitochondria) 52 1000 187
Fraction 1 14 90 162
Fraction 2
(mitochondria) 33 120 19
Fraction 3
(peroxisomes) 3 768 2

Note. If the purification procedure is repeated once (i.e., after pelleting fraction 2 and layering the pellet
on top of a new Percoll gradient) mitochondrial catalase and lipoxygenase activities can be reduced to less
than 2 rmol O.Jmin and 0.1 rmol OJmin, respectively.
 PURIFICATION OF PLANT MITOCHONDRIA 321

TIME(hours)

FIG. 9. Rate of endogenous phospholipid breakdown during ageing of potato tuber mitochondria
induced by 5 mM Caa+. The reaction medium contained 0.3 M mannitol; 10 mM MOPS buffer, pH
6.5; 5 mM CaCl,; and a suitable amount of mitochondria (10 mg protein/ml). The mixture, bubbled
with Argon was shaken at 25°C. With time, 200-4 aliquots were taken for analysis (see Materials
and Methods). (A) Washed mitochondria; (B) mitochondria purified on sucrose gradient (see also
(31)); (C) mitochondria purified on Percoll gradient.

unpurified mitochondria is equal to that
recorded for Percoll-purified mitochon-
dria, it is clear that the physiological in-
tegrity of the mitochondria is maintained
during the purification process. The ability
to support ascorbate cyt c-dependent O2
evolution seems to be a suitable ground on
which to base estimates of the proportion
of mitochondria with an intact outer mem-
brane. This quick assay which does not
overestimate or underestimate the pro-
portion of intact mitochondria should be
applicable to purified or unpurified mito-
chondria.
The passage of potato tuber mitochon-
dria through a Percoll density gradient is
an advantageous step because it not only
removes extramitochondrial membranes
such as amyloplast membranes, contain-
ing carotenoids and galactolipids, and mi-
crobodies, but also removes contaminating
hydrolases such as lipolytic acyl-hydro-
lase(s). In fact, although the mitochondria
are rapidly separated from the broken
cells during isolation, there is a brief as-
sociation of the mitochondria with the
content of the vacuole before they are dis-
persed in the suspending medium. There-
fore, these harmful hydrolases might have
become associated with the outer mito-
chondrial membrane. It is interesting to
note that the soluble lipolytic acyl-hydro-
lase from potato tuber is normally found
in cell-free supernatant and does not re-
quire Ca2+(22,23). Consequently, it is pos-
sible that once it is bound to the mem-
brane system the lypolitic acyl-hydrolase
becomes Ca2+ dependent. Percoll consists
of colloidal silica particles of X-30 nm
diameter which have been coated with
polyvinylpyrrolidone (PVP). It seems likely
therefore that PVP may act beneficially
by binding these hydrolases. In support of
this suggestion the purification of potato
mitochondria by centrifugation through
a discontinuous sucrose density gradient
does not remove the totality of these con-
taminating hydrolases.
The method outlined above (see Mate-
rials and Methods), when followed care-
fully, will produce mitochondria with a
high degree of intactness. The crucial
point in the whole procedure is the grind-
ing of the tissue. In order to obtain intact
mitochondria it is absolutely necessary to
restrict the grinding procedure to a min-
imum. Longer blending improves the yield
of recovered mitochondria but increases
the yield of envelope-free mitochondria
which have resealed following rupture and
loss of matrix content (results not shown).
These results also raise the problem of
322 NEUBURGER
subcellular distribution of the lipoxygen-
ase in plant tissues (25). It is clear that,
in potato, lipoxygenase is not associated
with mitochondria (see also 14) and per-
oxisomes. Since the fragile potato tuber
amyloplasts are completely destroyed dur-
ing the course of tissue grinding, owing to
the presence of a big starch grain in their
matrix space, no firm conclusions can be
drawn on the association of lipoxygenase
with plastids (envelope membranes and
stroma). Preliminary experiments carried
out in our laboratory indicate that most
of the plant mitochondria isolated so far
are more or less contaminated with im-
mature plastids of variable size and con-
sequently of variable density. This is par-
ticularly conspicuous in mitochondria
obtained from cauliflower florets. In this
case, Percoll-density gradient fraction-
ation strongly suggests that the lipoxy-
genase is mainly associated with heavy
organelles having a density similar to
plastids. In addition, the low lipoxygenase
activity found in Percoll-purified cauli-
flower floret mitochondria could be en-
tirely attributable to the presence of small
contaminating plastids having the same
density as that of mitochondria. All these
results together strongly suggest that li-
poxygenase could be associated with im-
mature plastids (see also (26, 27)).
Finally, these results demonstrate that
a clear preparation of fully intact micro-
bodies can be obtained from a non-green
tissue using equilibrium Percoll density
gradients (see also (28)). Equilibrium su-
crose-density gradients of homogenates
also show nearly complete separation of
microbodies and mitochondria (29, 30).
However in this case, experiments with
microbodies are difficult, especially for
transport studies, since they may be bro-
ken upon dilution for assaying and since
assays in dense sucrose are diffusion lim-
ited (30). In contrast, Percoll appears to
possesscharacteristics which make it more
suitable in studies on the physiological
properties of microbodies.
ACKNOWLEDGMENT
The authors wish to thank Dr. Jacques Joyard for
samples of spinach leaf chloroplast envelope.
ET AL.
REFERENCES
1. BONNER, W. D. (1967) in Methods in Enzymology
(Estahrook, R. W., and Pullman, M. E., eds.),
Vol. 10, pp. 126-133, Academic Press, New
York.
2. LATIES, G. (1974) in Methods in Enzymology
(Fleischer, S., and Packer, L., eds.), Vol. 31, pp.
589-600, Academic Press, New York.
3. BEEVERS, H. (1961) Respiratory Metabolism in
Plants, Row, Peterson, Evanston, Ill./White
Plains, N. Y.
4. BAKER, J. E., ELFVIN, L.-G., BIALE, J. B., AND
HONDA, S. I. (1968) Plant Physid 43, 2001-
2011.
5. MOREAU, F., AND LANCE, C. (1972) Biochimie 54,
1335-1348.
6. DOUCE, R., CHRISTENSEN, E. L., AND BONNER,
W. D. (1972) Biochim Biophys. Acta 275,148-
160.
7. PERTOFT, H., AND LAURENT, T. C. (1977) in
Methods of Cell Separation (Catsimpoolas, N.,
ed.), Vol. 1, pp. 25-65, Plenum, New York.
8. DELIEU, T., AND WALKER, D. A. (1972) New Phy-
toL 71, 201-225.
9. JEFFREY, S. W., DOUCE, R., AND BENSON, A. A.
(1974) Proc Nat. Acad. Sci USA 71. 807-810.
10. HAUROWITZ, F., SCHWERIN, P., AND YENSON,
M. M. (1941) J. BioL Chem 140,353-359.
11. DOUCE, R., AND JOYARD, J. (1980) in Methods in
Enzymology (San Pietro, ed.), Vol. 69, pp. 290-
301, Academic Press, New York.
12. ALLEN, C. F., AND GOOD, P. (1971) in Methods in
Enzymology (San Pietro, A., ed.), Vol. 23, pp.
523-547, Academic Press, New York.
13. VAN GINKEL, G., AND BROWN, J. S. (1978) FEBS
L&t. 94, 284-286.
14. SIEDOW, J. N., AND GIRVIN, M. E. (1980) Plant
PhysioL 65, 669-674.
15. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L.,
AND RANDALL, R. J. (1951) J. BioL Chem 193,
265-275.
16. LANCE, C., AND BONNER, W. D. (1968) Plant Phy-
sioL 43, 756-766.
17. SIMIONESCU, N., AND SIMIONESCU, M. (1976) J.
Ceu. BioL 70, 608-621.
18. JACKSON, C., DENCH, J. E., HALL, D. O., AND
MOORE, A. L. (1979) Plant PhysioL 64, X0-153.
19. FISHWICK, M. J., AND WRIGHT, A. J. (1980) Phy-
tochemistry 19,55-59.
20. DOUCE, R., AND JOYARD, J. (1980) in The Bio-
chemistry of Plants (Stumpf, P. K., ed.), Vol.
4, Lipids: Structure and Function, pp. 321-362,
Academic Press, New York.
21. DUPONT, J. (1981) PhysioL Plant. 52, 225,232.
22. GALLIARD, T. (1980) in The Biochemistry of
Plants (Stumpf, P. K., ed.), Vol. 4, Lipids:
Structure and Function, pp. 85-116, Academic
Press, New York.
 PURIFICATION OF PLANT MITOCHONDRIA 323

23. HASSON, E. P., AND LATIES, G. (1976) Plant Phy-
siol 57. 142-147.
24. GUNNING, B. E. S., AND STEER, M. W. (1975) Ul-
trastructure and the Biology of Plant Cell,
Arnold, London.
25. GALLIARD, T., AND CHAN, H. W. S. (1980) in The
Biochemistry of Plants (Stumpf, P. K., ed.),
Vol. 4, Lipids: Structure and Function, pp. 131-
161, Academic Press, New York.
26. GROSSMAN, S., BEN AZIZ, A., ASCARELLI, I., AND
BUDOWSKI, P. (1972) Phytohmtitry 11, 509-
514.
27. DOUILLARD, R., AND BERGERON, E. (1979) Physid
V&g. 17,749-768.
28. METPLER, I. J., AND BEEVERS, H. (1980) Plant
PhysioL 66, 555-560.
29. BEEVERS, H. (1980) in The Biochemistry of Plants
(Stumpf, P. K., ed.), Vol. 4, Lipids: Structure
and Function, pp. 117-130, Academic Press,
New York.
30. TOLBERT, N. E. (1981) Annu. Rev. Biochem 50,
133-157.
31. BLIGNY, R., AND DOUCE, R. (1978) Biochim Bie
phys. Acta 529,419-428.