Planta (1995)197:369-375
9 Springer-Verlag1995
Glyoxysomal malate dehydrogenase and malate synthase from
soybean cotyledons (Glycine m a x L.): enzyme association,
antibody production and cDNA cloning
Nicolas Guex, Hugues Henry*, Jean Flach**, Hannes Richter, Francois Widmer
Institute of Plant Biology and Physiology of the University, CH-1015 Lausanne, Switzerland
Received: 26 August 1994/Accepted: 26 January 1995
Abstract. In order to investigate a possible association
between soybean malate synthase (MS; L-malate
glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and
glyoxysomal malate dehydrogenase (gMDH; (S)-malate:
N A D § oxidoreductase, EC 1.1.1.37), two consecutive en-
zymes in the glyoxylate cycle, their elution profiles were
analyzed on Superdex 200 H R fast protein liquid chroma-
tography columns equilibrated in low- and high-ionic-
strength buffers. Starting with soluble proteins extracted
from the cotyledons of 5-d-old soybean seedlings and
a 45% ammonium sulfate precipitation, MS and g M D H
coeluted on Superdex 200 HR (low-ionic-strength buffer)
as a complex with an approximate relative molecular
mass (Mr) of 670000. Dissociation was achieved in the
presence of 50 m M KC1 and 5 m M MgC12, with the
elution of MS as an octamer of M r 510 000 and of g M D H
as a dimer of Mr 73 000. Polyclonal antibodies raised to
the native copurified enzymes recognized both denatured
MS and g M D H on immunoblots, and their native forms
after gel filtration. When these antibodies were used to
screen a k ZAP II expression library containing cDNA
from 3-d-old soybean cotyledons, they identified seven
clones encoding gMDH, whereas ten clones encoding MS
were identified using an antibody to SDS-PAGE-purified
The nucleotide sequence data reported in this paper have been
submitted to Genbank and assigned the accession numbers L01628
for gMDH and L01629 for MS.
Present addresses:
*Centre Hospitalier Universitaire Vaudois, LCC, CH-1011
Lausanne, Switzerland
** Laboratory of Plant Biochemistry and Physiology of the Univer-
sity, CH-1211 Geneva 4, Switzerland
Abbreviations: mCS = mitochondrial citrate synthase (citrate
oxaloacetate-lyase, EC 4.1.3.7); FPLC = fast protein liquid
chromatography; (c, cp, g, m)MDH = (cytosolic, chloroplastic,
glyoxysomal, mitochondrial) malate dehydrogenase ((S)-malate:
NAD § oxidoreductase, EC 1.1.1.37);Mr = relative molecular mass;
MS = malate synthase (L-malate glyoxylate-lyase,CoA-acetylating,
EC 4.1.3.2)
Correspondence to: F. Widmer; FAX: 41 (21)692 41 95; E-mail:
nguex@ulys.unil.ch
P l a n t ~
MS. Of these cDNA clones a 1.8 kb clone for MS and
a 1.3-kb clone for g M D H were fully sequenced. While
88% identity was found between mature soybean g M D H
and watermelon g M D H , the N-terminal transit peptides
showed only 37% identity. Despite this low identity, the
soybean g M D H transit peptide conserves the consensus
R(X6)HL motif also found in plant and mammalian
thiolases.
Key words: Enzyme complex - Glycine - Glyoxysome -
Malate dehydrogenase Malate synthase
Introduction
The glyoxylate cycle (glyoxysomal citrate synthase and
aconitase, isocitrate lyase, malate synthase and glyoxy-
somal malate dehydrogenase) bypasses the two decar-
boxylative steps of the citric-acid cycle and redirects the
carbon flow toward gluconeogenesis. The two enzymes
specific to this metabolic pathway (isocitrate lyase and
malate synthase) were first found in bacteria and plants
(for a review, see Beevers 1979). Recent biochemical stud-
ies have shown that these two enzymes are also present in
some mollusks (Benevides et al. 1989) as well as in mam-
malian liver (Davis et al. 1989), and that their activity
increases in the brown adipose tissue of black bears during
hibernation (Davis et al. 1990).
While isocitrate lyase (ICL; t h r e o - D s - i s o c i t r a t e
glyoxylate-lyase, EC 4.1.3.1) and malate synthase (MS) are
restricted to the glyoxysomes, malate dehydrogenase
(MDH) is found in cytoplasm (cMDH; malate-aspartate
shuttle), mitochondria (mMDH; citric acid cycle), gly-
oxysomes (gMDH; glyoxylate cycle) and chloroplasts
(cpMDH; Gietl 1992). Peroxisomal/glyoxysomal target-
ing has been intensively studied by Gould et al. (1988,
1989), who were the first to report that the signal peptide
necessary for import of peroxisomal firefly luciferase was
a C-terminal SKL. These authors tested between six and
eight mutations for each position of the SKL tripeptide
370
(no double m u t a t i o n however) and identified tripeptides
that retained peroxisomal targeting capacity (tripeptides
xyz with x = S, A or C; y = K, R or H; z = L). Usually
located at the carboxy-terminal end, signal peptides have
been occasionally found at an internal location of the
protein (catalase, a c y l - C o A oxidase), or in a cleavable
amino-terminal transit peptide ( g M D H , acetoacyl-CoA
thiolase; for a review, see de H o o p and Ab 1992). Al-
t h o u g h the carboxy-terminal S K L tripeptide has been
extensively studied, at least two other mechanisms for
i m p o r t into peroxisomes/glyoxysomes have been pro-
posed. Osumi et al. (1992) have studied the N-terminal
transit peptide of the rat peroxisomal thiolase and identi-
fied a G H L tripeptide whose histidine residue appeared to
possess a targeting function. Recent data on yeast thiolase
(Glover et al. 1994), and plant g M D H (Gietl et al. 1994)
have further d e m o n s t r a t e d that an R(X6)HL motif is
essential for the import of these enzymes into glyoxy-
somes/peroxisomes. A third mechanism has been en-
visaged for the i m p o r t of a c y l - C o A oxidase and ICL,
which do not have any SKL, G H L or one of their conser-
vative equivalents (Behari and Baker 1993).
N u m e r o u s studies on m M D H using methods such as
gel filtration, electrophoresis and kinetic techniques have
suggested that this enzyme can be a c o m p o n e n t of multiple
enzyme clusters (for details see Discussion). While the
published results are from in-vitro experiments, they sug-
gest that similar clusters could exist in vivo. An in-vivo
organization of the citric acid cycle has indeed been re-
ported (a sequential complex of enzymes, or (metabolon),
on the basis of the relative kinetic advantages of slightly
disrupted m i t o c h o n d r i a over completely solubilized
m i t o c h o n d r i a (Robinson et al. 1987). In addition, the
efficiency of enzyme clusters with regard to substrate
transfer has recently been demonstrated by the study of
a fusion m M D H - m i t o c h o n d r i a l citrate synthase (mCS)
protein in Saccharomyces cerevisiae (Lindbladh et al.
1994a, b).
T o further study the possibility of complex formation
a m o n g enzymes involved in a specific biochemical path-
way, we investigated MS and g M D H , two consecutive
enzymes of the glyoxylate cycle. It has been shown that
MS from s o y b e a n cotyledons can undergo structural in-
terconversions depending on ionic strength (Henry et al.
1992), and that MS obtained from c u c u m b e r seeds can be
found as m o n o m e r i c (5S), octameric (19S; p r e d o m i n a n t
form) or multimeric (100S) species. This high-relative-
molecular-mass (Mr) species was often recovered in the
microsomal fraction of sucrose gradients, and was there-
fore long believed to be an MS precursor synthesized in
the endoplasmic reticulum (for a review, see Beevers 1979).
K611er and Kindl (1980), however, suggested that the 100S
form was not a m e m b r a n e - b o u n d species but rather an
aggregated form. The cytoplasmic 5S m o n o m e r i c form is
n o w believed to be the precursor of the larger MS forms
(Kindl et al. 1980) and it has been shown that this 5S form
c a n n o t initiate oligomerization (19 S) until it is seques-
trated into glyoxysomes (Kruse and Kindl 1983a). The
19S form, in turn, reversibly aggregates into the 100S
form (Kruse and Kindl 1983b). In contrast, no aggregated
form was observed for cottonseed MS (Turley and
Trelease 1987), which had been purified either as
N. Guex et al.: Glyoxysomal malate dehydrogenase and malate synthase
a dodecamer of 750 k D a ( > 20S) or as a m o n o m e r
of 63 k D a (5S) (Trelease et al. 1987). The in-vitro evidence
based on gel filtration presented here shows that MS and
g M D H from germinating cotyledons can associate into an
active complex. In addition, the p r o d u c t i o n of antibodies,
the construction of a c D N A expression library and the
isolation of clones encoding MS and g M D H are reported.
Materials and methods
Plant material. Soybean (Glycine max L., cv. Maple arrow) seeds
were obtained from Schweizer Samen AG, Thun, Switzerland, and
germinated for 3 or 5 d in darkness as previously described (Ruchti
and Widmer 1986). Cotyledons were harvested after removal of testa
and primary leaves.
Clarified crude extract preparation. Fresh cotyledons (5-d old seed-
lings) were frozen in liquid nitrogen, pulverized in a coffee grinder
and homogenized at 4c'C in 2 volumes (e.g. 100 ml for 50 g) of
100 mM Tris-HCl (pH 8.0) containing 5 mM EDTA, 5 mM
dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride. The
slurry was then filtered through four layers of cheesecloth. After two
centrifugations at 24000"9 for 30 min at 4~ (model J2-21, JA-20
rotor; Beckman, Mfinchen, Germany), the supernatant was precipi-
tated at 45% saturation of ammonium sulfate. The pellet obtained
after centrifugation at 20 000-9 for 30 min at 4~ was resuspended in
0.1 volume of 25 mM Tris-HCl (pH 8.0). Additional centrifugations
(20000"9 at 4'~C) were performed until a clear supernatant was
obtained for gel filtration.
Sepharose CL-6B 9elfiltration. The first column (diameter = 5 cm;
flow rate = 1.5 mlmin-1) containing 982 ml of Sepharose CL-6B
gel (Pharmacia, Diibendorf, Switzerland) was used to recover the
aggregated MS/gMDH complex. The second column (diameter =
1.6 cm; flow rate = 0.2 mlmin -1) containing 66 ml of Sepharose
CL-6B gel (Pharmacia) was used to purify MS and gMDH after
dissociation due to equilibration in high-ionic-strength buffer
(Henry et al. 1992; "Mg 2+ shift"; for other details, see legend to
Fig. 1).
Diethylaminoethyl (DEAE)-Sepharose CL-6B anion exchange. A
DEAE-Sepharose CL-6B column (diameter = 1.6 cm; flow
rate = 0.5 mlmin- 1) containing 60 ml of anion-exchange gel (Phar-
macia) was used to recover nonadsorbed MS and gMDH after
equilibration at pH 9.0 in high-ionic-strength buffer (25 mM Tris-
HC1 buffer containing 5 mM MgCIz and 50 mM KC1; Henry et al.
1992).
Superdex 200 HR gel filtration and analysis. A Pharmacia Superdex
200 HR fast protein liquid chromatography (FPLC) column (dia-
meter = 1 cm; flow rate = 0.4 mlmin 1) containing 24 ml of gel
was used to analyze the association/dissociation properties of
MS/gMDH. Proteins from the FPLC fractions were diluted in
SDS-PAGE sample buffer (50 mM Tris-HCl, pH 6.8; 4% SDS; 12%
glycerol), run on N-[2-hydroxyl-l,l-bis(hydroxymethyl)ethyl]-
glycine (Tricine)-SDS PAGE gels (10% T, 3% C) (Schaggen and
Jagov 1987) and blotted on a polyvinylidene difluoride (PVDF)
membrane (Millipore, Volketswil, Switzerland). The blots were
treated with antibodies raised against native MS/gMDH (see below)
and immunoreactive proteins were revealed by chemiluminescence
with the ECL reagent (Amersham, Ziirich, Switzerland). The
autoradiographs were scanned on a laser densitometer (Molecular
Dynamics, Sunny Valley, Calif., USA) at a resolution of one pixel for
50 ~tm and the quantitation of the bands was done with the help of
ImageQuant software (Molecular Dynamics, Sunnyvale, Calif,
USA).
N. Guex et al.: Glyoxysomal malate dehydrogenase and malate synthase
Antibodies to native MS/gMDH. Fractions with activity for
M S / g M D H were collected after Mg 2+ shift (Sepharose CL-6B gel
filtration) and further purified using a DEAE-Sepharose CL-6B
column (Henry et al. 1992). The copurified proteins were adsorbed
onto nitrocellulose powder and used as immunogen in rabbits. The
serum was collected after two months and used directly on Western
blots, and to screen the expression library.
Antibodies to denatured MS. Purified soybean MS (Henry et al.
1992) was excised from SDS-PAGE gels stained with CuC12 (Lee et
al. 1987). The CuCI 2 and Tris-SDS buffer were successively washed
out using 0.1 M EDTA (pH 8.0) and phosphate-buffered saline
(PBS). The gel blocks were homogenized with Freund's adjuvant
and injected into rabbits. The serum was collected after two months
and used directly to screen the expression library.
Construction and immunoscreening of soybean cDNA expression
library. Total RNA was extracted from the cotyledons of 3-d-old
dark-grown soybean seedlings using guanidine thiocyanate accord-
ing to the method of Chomczynski et al. (1987) as modified by
Wadsworth et al. (1988). cDNA was synthesized from 5 gg of
poly(A)+RNA enriched by oligo (dT)-cellulose chromatography
(Aviv and Leder 1972). EcoRI adaptators containing a NotI restric-
tion site were added to both ends of the eDNA and cloned into the
phage vector k ZAP II (Stratagene, Ztirich, Switzerland) (Short et al.
1988). The recombinant phages of two different eDNA expression
libraries (1105 and 3105 independent clones, respectively) were
immunoscreened either with the antibodies to native M S / g M D H or
with those to denatured MS. pBluescript vectors containing eDNA
inserts were excised from ~, ZAP II, and the size of inserts were
determined on 1% agarose electrophoresis gels after NotI restric-
tion.
Sequencing of DNA. The eDNA clones were sequenced using
double-stranded templates and the dideoxy chain-terminating
method (Sanger et al. 1977) with [~t-35S]dATP as the radioactive
label. T3 and T7 primers were used to initiate sequencing at both
ends of the insert. The internal sequence of g M D H was obtained by
the Exometh method (Sorge and Blinderman 1989). The pBluescript
plasmid was opened with SacI (3' overhang) and BamHI (5' over-
hang). An Exometh TM II sequencing kit (Stratagene) was used to
perform exonuclease III digestion and sequencing reactions with
modified nucleotides (5-methyl-dCTP and 7-deaza-dGTP). DdeI or
RsaI were used to digest each timepoint before loading the se-
quencing gel. The internal part of the MS c D N A was sequenced
using MS-specific synthetic primers (Oligos Etc. Inc., Wilsonville,
Ore. USA).
Enzymatic assays. Malate dehydrogenase was assayed essentially
according to the method of Walk and Hock (1977) and MS accord-
ing to the method of Miernyk et al. (1979).
Protein contents were measured by the Coomassie assay (Bradford
1976) as modified by Peterson (1983), using bovine serum albumin as
the standard.
Results
Association~dissociation of a putative MS/MDH complex:
preparative purification. During attempts to purify
g M D H from crude extracts of soybean cotyledons, the
possible association of this enzyme with MS under condi-
tions of low ionic strength was suggested by the coelution
of both enzyme activities with a high-Mr value.
A 10-ml clarified crude extract was loaded onto
a Sepharose CL-6B column equilibrated with a low-
ionic-strength buffer (Henry et al. 1992). The results are
shown in Fig. 1A. Peak I contains a high'Mr complex
(approx. 670 kDa) of coeluting M D H and MS activities (it
371
1.5
0.6
0.5I ' (~'
0
1.0
.,2, 0.25 /',~- 2
.0.5 <
09
I I
400 600 800 1000
Elution volume (ml)
i0[o toit~
C 02 C
25
i,\ o,
20 30 40 50 60
Elution volume (ml)
Fig. IA, B. Sepharose CL-6B gel filtration of various forms of MS
and MDH. A Elution of MS and M D H with 25 mM Tris-HC1
(pH 8.0). The activities of MS and M D H coeluted in peak I (high-
Mr complex). B Elution of the pooled fractions of peak I (A) in the
same buffer containing 5 m M MgCI/ and 50 mM KC1. Both MS
and M D H were assayed enzymatically. Most of the high-Mr com-
plex (A) disaggregated into peak II (B). Vo. void volume
should be noted that only a minimal amount of the total
M D H activity coeluted with the aggregated MS). The
corresponding fractions were pooled and applied onto
a Sepharose CL-6B column equilibrated with a high-ionic-
strength buffer. Most of the activities of MS and M D H
found in peak I of the first column (low-ionic-strength;
Fig. 1A) were shifted into peak II of the second column
(high-ionic-strength buffer; Fig. 1B). In addition, the shift
resulted in a tenfold purification step (Henry et al. 1992).
The fractions of peak II (Fig. 1B) were pooled, concen-
trated (YM10 membrane; Amicon, Wallisellen, Switzer-
land) and loaded onto a DEAE-Sepharose CL-6B column
equilibrated with a high-ionic-strength buffer at pH 9.0.
Under these conditions neither MS nor M D H bound to
the matrix (data not shown). This purified M S / M D H
entity was either used as native immunogen to produce
antibodies to M S / M D H or run on SDS-PAGE gels to
recover MS and produce antibodies to this denatured
enzyme. While antibodies to native M S / M D H recognized
both denatured M D H and MS on Western blots, antibod-
ies to denatured MS recognized only MS (data not
shown). The antibodies were utilized in expression-clon-
ing MS and MDH.
Association~dissociation of a putative MS/MDH complex:
analysis. In order to rationalize the association~dissocia-
tion phenomena appearing to occur between MS and
M D H in low- and high-ionic-strength buffers, their elu-
tion profiles were further investigated using a calibrated
372
670 158 44 17 k D a
, r~ r~ ~1
50
+ 0.6
.,---
25
Ia
o ~0.0
12000 -
'
l B
8000 -
0 10000
I o
91-
a
4000 - v
z;
0-
i i i i i i
6 10 14 18
E l u t i o n v o l u m e (ml)
Fig. 2A, B. Superdex 200 HR F P L C gel filtration of various forms
of MS and MDH. Buffer: 25 m M Tris-HCl (pH 8.0). A Elution
profiles of M D H and MS assayed enzymatically. The activities of
MS and M D H coeluted in peak I. B. Elution profiles of M D H and
MS immunodetected on Western blots using the antibodies to
native coeluted M S / M D H (chemiluminescence with ECL reagent);
the picture of the scan used to quantify MS and M D H bands was
scaled and aligned to match the elution profiles. Coelution of MS
and M D H was observed for peak I and Ibis, while the 140 kDa
isoform of M D H (see peak II in A) did not react with the antibodies
F P L C gel filtration column. These profiles were analyzed
both enzymatically and immunochemically.
A 50-#1 clarified crude extract was loaded onto
a Superdex 200 HR F P L C column equilibrated with low-
ionic-strength buffer (Fig. 2A) or onto the same column
equilibrated with high-ionic-strength buffer (Fig. 3A). The
gel filtration profile in low-ionic-strength buffer has two
distinct activity peaks for M D H (Fig 2A, peaks I and II,
with approximate Mr values of 670 000 and 140000, re-
spectively). For both Figs. 1A and 2A, most of the total
M D H activity is concentrated in peak II, without associ-
ation proclivity with MS. A fraction of the M D H activity
coelutes with MS (Fig 2A, peak I), as already observed
with the Sepharose CL-6B column (Fig. 1A), and this
indicates a possible aggregation between MS and an
M D H isoform (coelution of a high-Mr M D H / M S com-
plex was also observed on other columns with different
separation ranges, including a Pharmacia Superose 6HR
F P L C column and a Sepharose CL-4B column; data not
shown). Plant M D H is usually resolved as a dimer (Mr
70000) with a subunit Mr of 35 000 (Habig and Racusen
1974). The higher Mr (140 000) estimated for M D H peak
N. Guex et al.: Glyoxysomal malate dehydrogenase and malate synthase
II (Fig. 2A) suggests either elution as a tetramer, or ag-
2 0.8 gregation of M D H with other proteins, as already con-
sidered by various authors (Srere et al. 1973; Datta et al.
1985; Robinson et al. 1987; Beeckmans et al. 1989; Fahien
et al. 1989; Queiroz-Claret and Queiroz 1992). This sec-
ond peak has a slight bulge on its left side, which corres-
0.4 E
O~ ponds to an intermediate peak with a Mr of 270000, as
E will be demonstrated below (Fig. 2B, peak Ibls).
0.2 Immunoblot analysis of the elution profile under low-
ionic-strength-conditions revealed three peaks of im-
munoreactive M D H (Fig. 2B, peaks I, Ibi s and II, with
approximate Mr values of 670000, 270000 and 30000
20000
respectively). Coelution of MS and M D H was again ob-
served in peaks I and Ibis. Peak Ibls (Fig. 2B) corresponds
15000 to the bulge on the left side of M D H activity peak II
+ of Fig. 2A, and peak II of Fig. 2B might correspond
to a small amount of enzymatically inactive monomer
of MDH. Interestingly, the antibodies to the native
HA M S / M D H entity do not recognize the M D H isoform(s)
co
5000 observed in peak II of Fig. 2A. The enzyme form(s) corres-
ponding to both peaks II (Figs. 1A, 2A) is (are) therefore
definitely distinguishable from the coeluting M S / M D H
molecular species with respect to its (their) antigenic prop-
erties. Since it is reported below that the antibodies to the
native MS/MDH entity identified seven cDNA clones all
encoding gMDH, this leads to the conclusion that the
M S / M D H entity is more precisely an MS/gMDH associ-
ation. Consequently, the M D H activity characterizing
both peaks II (Figs. 1A,2A) presumably originated from
m M D H and/or cMDH (cpMDH activity being excluded
since it is NADP specific).
When a 50-#1 clarified crude extract was loaded onto
the same Superdex 200 HR column equilibrated with
high-ionic-strength buffer (Fig. 3), the complex disag-
gregated and MS was eluted as a 510-kDa octamer (peak
I on Fig. 3A, B), whereas gMDH was eluted as a 73-kDa
dimer (Fig. 3B). Immunodetection of M D H and MS also
showed that a fraction of the presumed high-M, complex
remained unshifted at approx. 670 kDa and that a frac-
tion of peak Ibi~ (Fig. 2B) similarily remained practically
unshifted. The m M D H and/or cMDH 140-kDa peak
found in low-ionic-strength buffer (Fig. 2A; peak II) disag-
gregated into a 45-kDa activity peak that still did not
cross-react with the antibodies.
Clonin9 and analysis of MS and gMDH cDNAs. While
seven cDNA clones, all encoding gMDH, were identified
by immunoscreening the soybean cotyledon cDNA
expression library with antibodies to native coeluted
MS/MDH, ten clones encoding MS were purified using
antibodies raised against denatured MS.
Eight of these MS clones have approximately the same
insert size (1.8 kb), as determined on 1% agarose gels, and
are nearly full length. Comparison with the other plant
MS sequences found in the databases indeed shows that
approximately 10-16 nucleotides (depending on the
clone) are missing at the 5' end to reach the translation
start codon. One clone is shorter (1.65 kb) and one is
longer (about 2.4 kb). This longer clone has an unusually
long 3' untranslated sequence of about 800 nucleotides
compared to about 160 for all the other clones. The
farthermost 3'-end putative polyadenylation signal sequence
N. Guex et al.: Glyoxysomal malate dehydrogenase and malate synthase 373

670 158 44 17 kDa

m, ,m m m
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10 14 18 W 351 VSFIRS 356
Elution volume (ml)
Fig. 4. Comparison of soybean (S; this work) and watermelon (W;
Gietl 1990) gMDHs. The first amino acid of soybean gMDH may be
serine, threonine, alanine or proline. The transit peptides are shown
in bold. The DNA sequence analysis and comparison were achieved
using the GCG programs (Devereux et al. 1984)

Fig. 3A, B. Superdex 200 HR FPLC gel filtration of various forms -3O -20 -10 -1
of MS and MDH. Buffer: 25 mM Tris-HC1 (pH 8.0) containing
5 mM MgC12 and 50 mM KC1. A Elution profiles of M D H and MS i. ?NSGASDRISRIAGHLRP. . .Q R E D D V C L K R S D C R ~ . .
assayed enzymatically. B Elution profiles of M D H and MS im- 2. MQ P I PDVNQR IARI SAHLH PPKSQMEE S SALRRANCP~..
munodetected on Western blots using the antibodies to native co-
eluted MS/MDH; the picture of the scan used to quantify MS and 3. MEKAINRQSILLHHLRPSSSAYTNESSLSASVCk..
M D H bands was scaled and aligned to match the elution profiles.
4. MEKAINRQQVLL?HLRPS??????????SASVC~..
Under high-ionic-strength, coelution affecting MS and M D H was
less pronounced 5. MQRLQVVLGHLRG...PADSGWMPQAAPC...
6. MHRLQVVLGHLAG...RSESSSALQAAPC...
7. MSESVGRTSAMI-IRLQVVLGHLAG...RSESSSALQAAPC...
8. MSQRLQSIKDHLVLSAMGLGESKRKNSLLEK...
( A A U A A A ) at + 156 is not conserved in the 2.4-kb clone. 9. M.DRLNQLSGQLK.PNAK..QSILQKNPDDV...
This long extension is n o w under study in order to under-
i0. M.DRLNNLATQLEQNPAKGLDAITSKNPDDV...
stand its function. The sequence of the longest 1.8-kb
clone has been submitted to G e n b a n k and assigned the Fig. 5. Comparison of the transit peptides of various gMDHs and
accession n u m b e r L01629. As for other MSs, a C-terminal thiolases. Conserved amino acids are shown in bold. 1. Soybean
S K L tripeptide signal is present. gMDH (this work). 2. Watermelon gMDH (Gietl 1990). 3. Cucum-
The seven purified g M D H clones were analyzed by ber thiolase (Preisig-Miiller and Kindl 1993). 4. Mango thiolase
(unpublished, accession number X75329); only those amino acids
1% agarose gel electrophoresis, which yielded six clones that could be deduced with precision from the out-of-flame sequence
with a 1.3-kb insert and one clone with a 1.1-kb insert. deposited in the database have been shown; unpredictable amino
A c o m p a r i s o n at the amino-acid level with watermelon acids are indicated by question marks. 5. Human thiolase (Bout
M D H s (Gietl 1990; Gietl et al. 1990) demonstrates 88% et al. 1988). 6. Rat thiolase A (Hijikata et al. 1987). 7. Rat thiolase
and 66% identity with mature g M D H and m M D H re- B (Bodner and Rachubinski 1990). 8. Saccharomyces cerevisiae
spectively (Fig. 4). Alignment at the transcript level shows thiolase (Einerhand et al. 1991). 9. Candida tropicalis thiolases
A and B (unpublished, accession numbers D17320 and D17321,
that six clones start at G + 17 from the watermelon respectively). 10. Yarrowia lipolytica thiolase (Berninger et al. 1993)
g M D H translation start codon, and one at C + 8. T w o
putative polyadenylation signal sequences ( A A U A A and
A A U A U A A ) were identified by sequence analysis in the 3' Discussion
untranslated sequence. C o m p a r i s o n a m o n g g M D H and
thiolase presequences is dealt with below (see Fig. 5, The purification of g M D H from intact glyoxysomes is
Discussion). The soybean g M D H sequence has been sub- a r d u o u s because of the fragility of o n e - m e m b r a n e or-
mitted to G e n b a n k and assigned the accession n u m b e r ganelles. The peculiar gel-filtration behavior of M D H
L01628. isoforms in low- and high-ionic-strength buffer thus
374 N. Guex et al.: Glyoxysomal malate dehydrogenase and malate synthase
provides the basis for an unconventional isolation proced-
ure for g M D H (see Figs. 1-3). It now appears that pre-
vious studies on the aggregation behavior of MS under
low-ionic-strength conditions overlooked its possible as-
sociation with g M D H and that the decameric MS struc-
ture postulated by Henry et al. (1992) is likely to corres-
pond to an association between octameric MS and tet-
rameric g M D H .
Our finding of M D H activity eluting as complexes of
high Mr (670 k D a and 140 kDa) is not unusual; indeed,
elution profiles of plant M D H s with apparent high-Mr
values of 5 0 0 k D a and 2 8 0 k D a have been reported
(O'Sullivan and Wedding 1972; Habig and Racusen 1974).
Although the high-Mr M D H isoforms are very similar to
the most commonly found low-Mr isoform with respect to
optimal pH, K m for malate, and inhibition by various
unreactive substrate analogs, they differ in their elec-
trophoretic properties and ability to reduce 3-acetyl-
pyridine-deamino-NAD + (Habig and Racusen 1974).
It cannot be ruled out that the postulated association
between g M D H and MS observed under low-ionic-
strength conditions (coelution at 670 kDa) might have
in-vivo significance. Indeed, complex formation between
consecutive enzymes in a metabolic pathway has been
suggested for enzymes of glycolysis (Brooks and Storey
1991), the Krebs cycle (Srere 1987, Sumegi et al. 1990), the
Calvin cycle (Gontero et al. 1988), the malate-aspartate
shuttle (Fahien et al. 1989) and the multienzyme complex
involved in glycine decarboxylation (Oliver et al. 1992).
Special interest has been given to m M D H , which was
shown to associate with various enzymes, such as
fumarase (Beeckmans et al. 1989) or citrate synthase (Srere
et al. 1973; D a t t a et al. 1985). Furthermore, the kinetic
advantage of an immobilized three-enzyme system con-
sisting of m M D H / m C S and lactate dehydrogenase has
been reported (Srere et al. 1973). Association of m M D H
was also postulated with either glutamate dehydrogenase,
or with a preformed complex of mitochondrial aspartate
aminotransferase and a-ketoglutarate dehydrogenase
(Fahien et al. 1989). While the first association is believed
to participate in amino-acid degradation, the second one
is involved in the malate/aspartate shuttle feeding into
gluconeogenesis and ureogenesis. Another association
was identified in crassulacean-acid-metabolism plants
where c p M D H was found complexed with phospho-
enolpyruvate carboxylase (PEPCase, Queiroz-Claret and
Queiroz 1992). While P E P C a s e activity rapidly disap-
peared upon disruption of the P E P C a s e - M D H complex,
reactivation of P E P C a s e activity was achieved in vitro
in the presence of M D H . Taken together, regulation of
M D H activity in the cases listed above might conse-
quently result from the effects of metabolites such as
glutamate, citrate and a-ketoglutarate, as well as from its
association proclivity for multienzyme complexes belong-
ing to various biochemical pathways.
Similarities among soybean and watermelon gMDHs, plant,
mammalian and yeast thiolases. A high amino-acid se-
quence similarity is observed between mature soybean
and watermelon g M D H s (88% identity; Fig. 4), and
amino acids participating in substrate binding and cataly-
sis are conserved (Guex et al. 1993). In contrast, only 37%
of the amino acids from the transit peptide are strictly
conserved. Nevertheless, as recently elucidated by various
authors, amino acids essential for targeting are conserved
for plant gMDHs, as well as for plant, mammalian and
yeast thiolases (Fig. 5; Gietl et al. 1994; Glover et al. 1994;
Lehnerer et al. 1994). In addition to the conserved R(X6)HL
motif, a cysteine (Cys) residue located at, or one amino acid
before, the cleavage site of the transit peptide also charac-
terizes all transit peptides except those of yeast thiolases.
Since it has been reported that yeast thiolases presequences
are not cleaved upon import into peroxisomes (Gietl et al.
1994; Glover et al. 1994), the conserved Cys of cleavable
transit peptides could be of importance for their processing.
However, this hypothesis can now be questioned by the
recent evidence that the Yarrowia lipolytica presequence is
cleaved upon import (Nuttley et al. 1994). It is interesting to
note that yeast and plant peroxisomal/glyoxysomal M D H s
seem to be characterized by distinct organellar targeting
systems. The Saccharomyces cerevisiae peroxisomal M D H
possesses an SKL C-terminal tripeptide (Steffan and
McAlister-Henn 1992), whereas the N-terminal transit
peptide distinctive of plant g M D H s (Gietl 1990; this work)
might have been acquired at a later stage of evolution.
Conclusion
The identification of MS and g M D H in a 670-kDa com-
plex and the isolation of c D N A clones for both enzymes
provide the basis for further in-vitro and in-vivo studies of
the M S / g M D H complex. The cDNAs will be particularly
useful to study gene expression in response to the chang-
ing metabolic needs of the cotyledon and other parts of
the plant.
We are most greatful to Dr. A. Chanson (Institute of Plant Biology
and Physiology, University of Lausanne, Lausanne, Switzerland) for
critical reading of the manuscript.
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