bioinformatic and enzymatic characterization of the mapeg superfamily

Bioinformatic and enzymatic characterizationof the MAPEG superfamilyAnders Bresell1,*, Rolf Weinander2,*, Gerd Lundqvist3, Haider Raza3, Miyuki Shimoji3,Tie-Hua Sun3, Lennart Balk5, Ronney Wiklund6, Jan Eriksson6, Christer Jansson6, Bengt Persson1,4,Per-Johan Jakobsson2 and Ralf Morgenstern3

1 IFM Bioinformatics, Linkoping University, Sweden

2 Department of Medicine, Division of Rheumatology Unit, Karolinska Institutet, Stockholm, Sweden

3 Institute of Environmental Medicine Karolinska Institutet, Stockholm, Sweden

4 Centre for Genomics and Bioinformatics, Karolinska Institutet, Stockholm, Sweden

5 Stockholm Marine Research Centre, University of Stockholm, Sweden

6 Department of Plant Biology & Forestry Genetics, Swedish Agricultural University, Uppsala, Sweden

Keywords

MAPEG; microsomal glutathione

transferase; prostaglandin; leukotriene

Correspondence

R. Morgenstern, Institute of Environmental

Medicine, Karolinska Institutet, S-171 77

Stockholm, Sweden

Fax: +46 8 343849

Tel: +46 8 5248 7574

E-mail: [email protected]

*Both authors contributed equally to this

work

(Received 15 November 2004, revised 27

January 2005, accepted 3 February 2005)

doi:10.1111/j.1742-4658.2005.04596.x

The membrane associated proteins in eicosanoid and glutathione metabo-

lism (MAPEG) superfamily includes structurally related membrane proteins

with diverse functions of widespread origin. A total of 136 proteins belong-

ing to the MAPEG superfamily were found in database and genome

screenings. The members were found in prokaryotes and eukaryotes, but

not in any archaeal organism. Multiple sequence alignments and calcula-

tions of evolutionary trees revealed a clear subdivision of the eukaryotic

MAPEG members, corresponding to the six families of microsomal gluta-

thione transferases (MGST) 1, 2 and 3, leukotriene C4 synthase (LTC4),

5-lipoxygenase activating protein (FLAP), and prostaglandin E synthase.

Prokaryotes contain at least two distinct potential ancestral subfamilies, of

which one is unique, whereas the other most closely resembles enzymes that

belong to the MGST2 ⁄FLAP ⁄LTC4 synthase families. The insect members

are most similar to MGST1 ⁄prostaglandin E synthase. With the new data

available, we observe that fish enzymes are present in all six families, show-

ing an early origin for MAPEG family differentiation. Thus, the evolution-

ary origins and relationships of the MAPEG superfamily can be defined,

including distinct sequence patterns characteristic for each of the sub-

families. We have further investigated and functionally characterized repre-

sentative gene products from Escherichia coli, Synechocystis sp., Arabidopsis

thaliana and Drosophila melanogaster, and the fish liver enzyme, purified

from pike (Esox lucius). Protein overexpression and enzyme activity ana-

lysis demonstrated that all proteins catalyzed the conjugation of 1-chloro-

2,4-dinitrobenzene with reduced glutathione. The E. coli protein displayed

glutathione transferase activity of 0.11 lmolÆmin)1Æmg)1 in the membrane

fraction from bacteria overexpressing the protein. Partial purification of

the Synechocystis sp. protein yielded an enzyme of the expected molecular

mass and an N-terminal amino acid sequence that was at least 50%

pure, with a specific activity towards 1-chloro-2,4-dinitrobenzene of

11 lmolÆmin)1Æmg)1. Yeast microsomes expressing the Arabidopsis enzyme

Abbreviations

BSA, bovine serum albumin; CDNB, 1-chloro-2,4-dinitrobenzene; DEAE, diethylaminoethyl; FLAP, 5-lipoxygenase activating protein; LT,

leukotriene; MGST, microsomal glutathione transferase; PG, prostaglandin; PGES, prostaglandin E synthase; GST, glutathione S-transferase;

GPx, glutathione peroxidase; CuOOH, cumene hydroperoxide.

1688 FEBS Journal 272 (2005) 1688–1703 ª 2005 FEBS

Microsomal glutathione transferases (MGSTs) repre-

sent a recently recognized superfamily of enzymes

involved in detoxification, but also in specific biosyn-

thetic pathways of arachidonic acid metabolism. The

superfamily was termed the membrane associated

proteins in eicosanoid and glutathione metabolism

(MAPEG) and consists of proteins from mammals,

plants, fungi and bacteria [1]. The six members in

humans include 5-lipoxygenase activating protein

(FLAP) and leukotriene (LT) C4 synthase, which are

both involved in leukotriene biosynthesis [2,3];

MGST1, MGST2 and MGST3, which all are gluta-

thione transferases as well as glutathione dependent

peroxidases [4–7]; and finally, prostaglandin (PG) E

synthase (PGES), earlier referred to as MGST1-L1 [8].

PGES catalyzes the formation of PGE2 from PGH2,

which in turn is generated from arachidonic acid by

the prostaglandin endoperoxide synthase systems.

PGES has also been referred to as p53 induced gene

12 (PIG12) because the gene expression was found to

increase extensively following p53 expression [9]. The

relationships and other functional aspects of the

MAPEG enzymes have been reviewed [10].

Two groups of bacteria, purple bacteria and cyano-

bacteria, have been found to produce and maintain

significant levels of glutathione [11] and, interestingly,

also contain MAPEG members [1]. Glutathione was

observed in various species within the two groups,

among those in Escherichia coli, one of the most well

characterized species of purple bacteria [11]. The func-

tion of glutathione metabolism in bacteria may be pro-

tection against xenobiotics and ⁄or oxidative stress but

also as part of specific biosynthetic pathways [12].

Cyanobacteria produce oxygen by photosynthesis and

purple bacteria can use oxygen as a terminal electron

acceptor. Glutathione production in bacteria is thus

closely associated with those bacteria that generate or

utilize oxygen in specific biochemical pathways indica-

ting that glutathione metabolism originated in bacteria

at the time when an oxygen-containing atmosphere

developed on earth [11,12].

A low level of glutathione S-transferase (GST) activ-

ity has been demonstrated in E. coli but not in cyano-

bacteria [11]. Cytosolic GSTs have been identified in

various strains of bacteria [12] and in a few studies,

including those on Proteus mirabilis and E. coli, cyto-

solic GSTs have been purified and further character-

ized [13–15]. The three-dimensional structure of the

P. mirabilis cytosolic GST has also been determined

[16]. In Synechocystis sp. a gene homologous to cyto-

solic GST exists but has not been characterized further

[17]. In general, the enzymes involved in glutathione

metabolism in prokaryotes have not been so exten-

sively studied and therefore less is known about their

properties as compared to the corresponding proteins

in eukaryotes. Microsomal GST activity has not been

demonstrated in any prokaryotic organism.

Expressed sequence tag (EST) clones with open

reading frames (ORFs) similar to MAPEG proteins

have been found in E. coli, Synechocystis sp. and

Vibrio cholerae [1]. The Synechocystis sp. ORF dis-

played sequence similarity to the MAPEG subfamily

consisting of FLAP, LTC4 synthase and MGST2, and

also to the MGST3 subfamily but it could not be sig-

nificantly grouped to any of those two subfamilies,

whereas the E. coli and V. cholerae sequences form a

separate group [1]. Nothing is known, however, about

the enzymatic properties of any prokaryotic MAPEG

protein.

As the number of sequenced bacterial genomes has

increased considerably during recent years, we de-

signed this study to search further for MAPEG pro-

teins and functionally characterize representative gene

products. Database searches revealed various new gene

products, in some cases coexisting, with homologies to

the two MAPEG subfamilies (described above and in

[1]). We investigated representative gene products from

the E. coli and Synechocystis sp. bacteria further, to

gain insight into the function of these proteins and the

evolution of the MAPEG superfamily. Cloning and

overexpression demonstrated that both are membrane-

bound glutathione transferases.

showed an activity of 0.02 lmolÆmin)1Æmg)1, whereas the Drosophila

enzyme expressed in E. coli was highly active at 3.6 lmolÆmin)1Æmg)1. The

purified pike enzyme is the most active MGST described so far with a spe-

cific activity of 285 lmolÆmin)1Æmg)1. Drosophila and pike enzymes also

displayed glutathione peroxidase activity towards cumene hydroperoxide

(0.4 and 2.2 lmolÆmin)1Æmg)1, respectively). Glutathione transferase activity

can thus be regarded as a common denominator for a majority of MAPEG

members throughout the kingdoms of life whereas glutathione peroxidase

activity occurs in representatives from the MGST1, 2 and 3 and PGES sub-

families.

A. Bresell et al. Characterization of MAPEG members

FEBS Journal 272 (2005) 1688–1703 ª 2005 FEBS 1689

To understand the evolutionary relationships better

on a more global scale we also cloned and expressed

(or purified) MAPEG representatives from plant,

insect and fish. Together with earlier data on the frog

enzyme [18] these data define glutathione transferase

activity as a central property of MAPEG members

from a wide range of organisms and suggest ancestral

MAPEG members.

Results

MAPEG members from complete genomes

Over 130 MAPEG members were retrieved from

sequence databases and completed genomes, of which

less than half (56) were previously known members

according to the PF01124 entry in Pfam release 11 [19].

Multiple sequence alignments and hydrophobicity plots

were calculated (for a full alignment see supplementary

Fig. 1). Even though several members are distantly

related, all exhibit the typical MAPEG properties of

150 residue subunits with four hydrophobic regions,

compatible with four transmembrane regions [20,21].

Using information from completed genomes, we

have traced the evolutionary relationships of the

MAPEG members. The general relationships are

depicted in Fig. 1. MGST1, PGES and insect forms

have a common branch, compatible with their overlap-

ping substrate-specificities [22]. Likewise, MGST2,

FLAP and LTC4 synthase also show somewhat closer

relationships, indicating properties in common.

MGST3 forms a separate branch. The bacterial E. coli

and Synechocystis variants are found on separate bran-

ches. A detailed dendrogram is shown in Fig. 2.

The bacterial forms show distant relationships and

their exact grouping is not significant at all sites, as

indicated from their low bootstrap values (no asterisks

in Fig. 2). Furthermore, the bacterial forms are present

at three sites in the dendrogram. However, the group-

ing of the families MGST1, MGST2, MGST3, PGES,

FLAP and LTC4 synthase is significant. In a dendro-

gram without the bacterial forms, the grouping of these

families becomes even more evident (not shown).

Among the MAPEG sequences from fish, we find

members from all six branches (MGST1, MGST2,

MGST3, PGES, FLAP and LTC4 synthase), suggest-

ing that the origin of these forms dates back to before

the occurrence of vertebrates, i.e. more than 500 mya.

This dates the differentiation of the MAPEG forms

back to the late Cambrian multiplicity of eukaryotic

species. Notably, in the screenings we have not found

any members from the archaea kingdom, indicating

that the enzymatic activities of the MAPEG family are

not present in these species or that these activities are

catalysed by other enzymes. The absence of MAPEG

members in archaea is certainly consistent with the

lack of GSH in these organisms.

Cloning, expression and characterization

of selected MAPEG members

MGST homologues from Synechocystis and E. coli

After identifying MAPEG members in several bacterial

strains, the E. coli and Synechocystis sp. proteins were

Fig. 1. Schematic evolutionary tree of the MAPEG superfamily. The

evolutionary tree shows the relationships between the six MAPEG

families and three further groups (Insect, E.coliMGST cluster and

SynMGST cluster). A major subgrouping is visible with MGST1,

PGES and Insect in the upper part of the tree and the remaining

families ⁄ groups in the lower part. In the lower part, MGST2, FLAP

and LTC4 synthase have a close relationship, as judged by the

short branches between these enzymes.

Fig. 2. Detailed dendrogram of the MAPEG superfamily. The tree shows all presently known MAPEG forms, excluding species variants

which differ at only a single position. In the tree, the six families are clearly distinguished. The prokaryotic forms are found at three sites –

the E. coli cluster, the Synechocystis cluster, and the group of remaining forms, denoted Bacteria. Two further groups are marked, denoted

Insects and Waterliving. The branch lengths are proportional to the number of residue differences, with the scale bar indicating a 5% amino

acid difference. The fish forms, having representatives for all six MAPEG families, are marked with a fish symbol. Accession numbers refer

to the databases Uniprot, NCBI or ENSEMBL.

Characterization of MAPEG members A. Bresell et al.


selected for functional characterization of bacterial

MGST homologues. These homologues represent two

different groups of prokaryotic MAPEG members found.

The E. coli ORF, which we refer to as E.coliMGST,

encodes a 141 amino acid residue polypeptide with a cal-

culated molecular mass of 16.2 kDa. The Synechocystis

sp. ORF (from strain PCC6803 [23]) encodes a 137 resi-

due polypeptide with a predicted molecular mass of

15.4 kDa, which we refer to as SynMGST.

The ORFs encoding E.coliMGST and SynMGST

were amplified by PCR, the products cloned into an

expression vector and the DNA sequences were verified

against the EMBL database entries. Following hetero-

logous expression in E. coli, the membrane fractions

were assayed for enzyme activities. The membrane

fraction from cells overexpressing E.coliMGST cata-

lyzed the conjugation of 1-chloro-2,4-dinitrobenzene

(CDNB) with reduced glutathione with a specific

activity of 0.11 lmolÆmin)1Æmg)1. When a shorter con-

struct beginning from the alternative translation start

site of the E.coliMGST was expressed no activity was

detected. Incubation with N-ethylmaleimide (which

activates mammalian MGST1) did not affect the

activity of E.coliMGST. Membranes from cells over-

expressing the SynMGST also displayed glutathione

transferase activity. The glutathione conjugating activ-

ity towards CDNB was 1.7 lmolÆmin)1Æmg)1 for the

SynMGST membrane fraction. Neither LTC4 synthase

activity, nor any glutathione-dependent peroxidase

activity (towards cumene hydroperoxide or 5-hydrope-

roxy-eicosatetraenoic acid) could be observed in any of

the fractions. No activity could be detected with these

enzymes towards 1,2-epoxy-3-para-nitrophenoxypro-

pane or trans-phenylbut-3-en-2-one as substrates (sum-

marised in Table 1).

Partial purification of SynMGST

To characterize bacterial MGSTs further we concen-

trated on SynMGST. RT-PCR was used to confirm

that SynMGST is indeed expressed in the cyanobac-

teria (Fig. 3).

Having established gene expression of SynMGST in

the cyanobacteria and a functional overexpression of

recombinant protein in E. coli we made an attempt to

purify the protein for further characterization. Bacterial

membranes isolated from cells overexpressing recom-

binant SynMGST were solubilized in Triton X-100.

The recombinant SynMGST was also enzymatically

active upon detergent solubilization and the CDNB

conjugating activity was used to monitor subsequent

purification steps. The SynMGST is basic (the cal-

culated isoelectric point being 9.9) and could therefore

be expected to yield a purified product using meth-

ods developed for MGST1 [24]. However, although

the enzyme behaved in a predictable manner upon

hydroxyapatite batch chromatography, in cation

exchange chromatography the enzyme was recovered in

the flow-through fractions. Diethylaminoethyl (DEAE)

columns, likewise, did not retain the enzyme. Because

cation and anion exchange chromatography, in concert,

did retain most of the contaminating proteins, a parti-

ally purified protein was nevertheless recovered. In fact,

SDS ⁄PAGE (Fig. 4) shows that the protein is nearly

homogeneous. Furthermore, N-terminal amino acid

Table 1. Comparison of glutathione transferase and peroxidase

activity of MAPEG members expressed ⁄ purified from prokaryotes,

plant, nonmammalian and mammalian species. ND, not detectable.

Species

CDNB activity

(lmolÆmin)1Æmg)1)

CuOOH GPx

activity

(lmolÆmin)1Æmg)1)

Activity of purified enzyme

Human MGST1 [71,72] 1.9 0.04

Rat MGST1 [43] 2 0.08

Xenopus laevis, frog [18] 210 2.1

Esox lucius, pike 290 2.2

Synechocystis sp. 11 ND

(partially purified)

Activity in membrane fraction

after heterologous expression

Drosophila melanogaster 3.6 0.4

Arabidopsis thaliana 0.02 n.d.

Synechocystis sp. 1.7 n.d.

Escherichia coli 0.11 n.d.

800

1 2 3 4

400

200100

Fig. 3. RT-PCR. To demonstrate that the SynMGST gene was

expressed in Synechocystis 6803, total RNA was isolated and

amplified by PCR with SynMGST-specific primers, in the presence

(lane 4) or absence (lane 3) of reverse transcriptase. PCR amplifica-

tion from isolated total DNA, using the same primers (lane 2)

served as a positive control. Sizes in bp, deduced from a 100 bp

ladder (lane 1) are indicated.



sequence analysis of the predominant band displaying

the correct molecular mass, purified from the gel,

yielded the expected sequence. The partially purified

protein constitutes a major part of the preparation and

therefore the specific activities measured will be close to

those of the pure enzyme.

The enzyme is more active than its mammalian

counterparts and expressed extremely well. Assuming

that the protein was at least 50% pure, the purification

factor (12-fold) indicates that expressed SynMGST

constituted about 8% of the E. coli membrane asso-

ciated proteins. The specific activity of the partially

purified enzyme with 1-chloro-2,4-dinitrobenzene was

11 ± 0.4 lmolÆmin)1Æmg)1 (mean ± SD, n ¼ 3). The

activity was not affected by incubation with the sulf-

hydryl reagent N-ethylmaleimide in contrast to mam-

malian MGST1, which is activated several-fold by this

reagent.

MGST3 from Arabidopsis

When plant MGST3 was cloned and overexpressed

in a yeast expression system, the yeast microsomes

displayed a low glutathione transferase activity with

CDNB (0.02 lmolÆmin)1Æmg)1) that was not activa-

ted ⁄ inhibited by N-ethylmaleimide. Glutathione peroxi-

dase activity was not altered compared to that in

microsomes from yeast expressing the pYeDP60 vector

only (the negative control).

MGST1/PGES-like enzyme from Drosophila

The MGST from Drosophila was cloned and over-

expressed in E. coli where the isolated membrane

fraction displayed a high glutathione transferase acti-

vity (3.6 lmolÆmin)1Æmg)1) and glutathione peroxidase

activity (0.4 lmolÆmin)1Æmg)1). Addition of 1% (v ⁄ v)Triton X-100 to the membrane fraction resulted in a

slight increase in activity, whereas N-ethylmaleimide

had no effect on enzyme activity. The enzyme did not

display PGES activity.

MGST1/PGES-like enzyme from pike

MGST was successfully purified to apparent homogen-

eity (Fig. 4) from pike liver using protocols developed

for the rat enzyme. The N-terminal sequence of the

purified pike enzyme was determined using Edman de-

gradation. Sequence comparisons reveal that the pike

form purified is closely related to the MGST1 ⁄PGES

branch (Fig. 5). Of the N-terminal 47 residues, 22–28

residues are identical to fish MGST1 sequences, while

only 2–12 residues are identical to the fish sequences of

other MAPEG families.

The enzymatic properties of the pike MGST1-like

enzyme were extensively characterised (Table 1) dem-

onstrating that the protein has the highest glutathione

transferase activity of any MAPEG member detected

so far. As the enzyme displays similar substrate speci-

ficity to MGST1, including glutathione peroxidase

activity, the assignment to the MGST1 ⁄PGES sub-

family appears well founded.

Sequence patterns of the MAPEG members

For the MGST1–3, FLAP, LTC4 synthase, PGES and

Insect family clusters we generated sequence patterns,

shown in Table 2. These patterns are all 100%

unambiguous when scanned against Swiss-Prot and

TrEMBL, i.e. no nonmembers are ranked higher than

SynMGST

MGST MGST1RatPike

1mg/lane 1mg/lane

MGST1Rat

75 ng 25 ng kDa

Mrmarkers

kDa

Mrmarkers

45

31

21.5

14.4

10

2015

150 ng

Fig. 4. SDS ⁄ PAGE analysis of purified

SynMGST and pike MGST. The protein was

fractionated on SDS ⁄ PAGE (15%) and

visualized by silver staining. Major proteins

were detected that comigrated with purified

RatMGST1 (17 kDa).



the lowest ranked true member. These patterns are

more specific then the existing PROSITE pattern

PS01297 (FLAP ⁄GST2 ⁄LTC4S: G-x(3)-F-E-R-V-[FY]-

x-A-[NQ]-x-N-C) [25]. The patterns are selected based

on conserved regions in the sequence. Notably, the

PGES pattern is located at the beginning of loop one

and for FLAP it is located in the third hydrophobic seg-

ment. All of the remaining patterns are located at the

end of first loop (Fig. 6). Both the first and third loop

are located on the cytosolic side of the membrane and

are regions earlier postulated to host the active site

[21,26]. Furthermore, the patterns of the two very sim-

ilar families of PGES (earlier denoted MGST1-like) and

MGST1 do not overlap, even though they both are

located in the first loop.

For the classical FERV pattern, which is a part of

PS01297, we note that it is still included in the two

new and more specific patterns of MGST2 and LTC4

synthase. The last member of PS01297 is FLAP for

which the novel pattern is located in the third loop.

The reason for the similarity and location of these pat-

terns could be a result of short evolutionary time

rather than gain of new features as FLAP, MGST2

and LTC4 synthase have been detected only in higher

eukaryotes to date. However, all patterns in Table 2

will be useful in genome characterizations and func-

tional annotations.

Discussion

The MAPEG family

We have characterized the MAPEG family and found

the eukaryotic forms to consist of six families, while

the prokaryotic forms are clustered at two sites or

more, depending upon whether the E. coli cluster (top)

and the bacterial cluster (bottom) are separated or not

(Fig. 2). The SynMGST branches with the cluster of

Fig. 5. Alignment of pike MGST1 with homologous forms. The N-terminal fragment of pike MGST1 is multiply aligned with other MAPEG

fish members. Positions identical between the pike form and any other fish form are shown in bold. It can be seen that most of the bold

amino acid residues are found within the MGST1 family, supporting evidence for the pike form to belong here. A dendrogram is shown to

the left of the alignment, calculated from the aligned sequences.

Table 2. Sequence patterns for the different MAPEG families.

Family Pattern Position

FLAP P-A-A-F-A-G-x(0,1)-L-x(0,1)-Y-L-x(2)-R-Q-K-Y-F-V-G-Y 123

LTC4 synthase G-P-P-E-F-[DE]-R-[IV]-[FY]-R-A-Q-[AV]-N-[CS]-[ST]-E-Y-F-P 66

MGST1 E-R-V-R-R-[ACG]-H-x-N-D-[IL]-E-N-[IV]-[IV]-P-F-[FLV]-[AGV]-I 92

MGST2 V-[ST]-G-[APS]-[LP]-[DE]-F-[DE]-R-x-F-R-A-x(0,1)-Q-x(0,1)-N-[CNS]-[ALV]-E 63

MGST3 F-N-C-[AIV]-Q-R-[AGS]-H-[AQ]-[NQ]-x(2)-E-x(2,3)-P 90

PGES M-Y-[AIV]-[IV]-A-[IV]-I-T-G-Q-[IMV]-R-L-R-[KR]-K-A-x-A-N 47

Insect D-P-x-V-E-R-V-R-R-A-H-x-N-D-x-E-N-I-L-P 87



FLAP, MGST2 and LTC4 synthase, while E.coli-

MGST branches earlier, before the divergence of

MGST3 from the previous cluster. However, it should

be kept in mind that the early branches have low boot-

strap values and that the order might change when

more data become available. Interestingly, several bac-

terial species contain multiple MAPEG forms. For

example, the Caulobacter crescentus has three different

forms – one in the SynMGST cluster, one in the Ecoli-

MGST cluster and one in the large bacterial cluster

(Fig. 2). We checked whether any of the MAPEG

members were encoded by plasmids, but we did not

find any MAPEG members among known plasmid

sequences.

Mutiplicity of MAPEG members is also seen in

insects. Both Drosophila and Anopheles show multiple

forms, but these forms are more closely related than

the multiple forms of bacterial species. As judged from

sequence comparisons, the insect multiple forms have

appeared independently in each species, probably

reflecting adoption to the environment. Interestingly,

Drosophila also has multiple gene families of cytosolic

GSTs [27].

Extensive searches in archaea only revealed possible

homologues related to transport proteins. If these rela-

tionships are real they might give a link to ancestors

with different functions, which were later recruited as

detoxification enzymes.

Upon examination of the eukaryotic MAPEG

forms, we found that the subdivision into six different

families is present already in fish, dating this diver-

gence to 500 mya. These findings agree in general with

the known well developed capacity of fish xenobiotic

metabolism [28] and raises the possibility of arachi-

donic acid based signalling. Zebrafish express both

cyclooxygenase (cox)-1 and -2 and the primary

prostaglandin end product is PGE2 [29]. Furthermore,

the bleeding time as a measurement of platelet activa-

tion was sensitive to inhibition of cox-1 but not of

cox-2, i.e. similar to the situation in humans. Incuba-

tion of whole blood from rainbow trout with calcium

ionophore resulted in the biosynthesis of leuko-

triene B4 suggesting an intact leukotriene pathway

including phospholipase, 5-lipoxygenase and LTA4

hydrolase in this species [30]. Thus, the fish kingdom

seems to contain a similar biosynthetic capacity to

humans to oxidize arachidonic acid. In plants, leuko-

triene B4 has been demonstrated in nettles [31] prob-

ably as part of its defence mechanism. In various

species of corals, large amounts of prostaglandin-rela-

ted compounds are found [32]. Here the prostaglandin-

like compounds may constitute structural elements of

the organism or be part of their chemical defence.

Recently, two coral (Gersemia fruticosa) cyclooxygen-

ases were cloned and functionally characterized, and

found to catalyze the formation of PGF2a, PGE2 and

PGD2 (presumably through nonenzymatic conversion

of PGH2) as well as unspecified hydroxyeicosatetra-

enoic acids [33]. It is also suggested that an ancestral

gene coding for cyclooxygenase was duplicated before

A

B

Fig. 6. Hydrophobicity plot. (A) The plot

shows the mean value of hydrophobicity

(solid lines) and standard deviation (dashed

lines). Values are calculated according to

Kyte and Doolittle [53] using an 11-residue

window. The positional numbers follow a

multiple sequence alignment of all MAPEG

members. (B) The plot shows the number

of sequences present at each position. The

four hydrophobic segments, corresponding

to the transmembrane regions are visible as

peaks in (A).



the divergence of the modern cyclooxygenase-1 and -2.

It would be interesting to know at what time during

development the MAPEG proteins (specifically PGE

synthase and FLAP ⁄LTC4 synthase) were associated

with the cyclooxygenase and lipoxygenase protein

families, respectively. At the introduction of these

MAPEG proteins a more specialized level of product

control must have occurred, allowing for the specific

metabolism of the products derived from cyclooxygen-

ases and lipoxygenases into the end products known

today.

Structural implications

Now that over 100 different MAPEG forms are avail-

able, a limited number of conserved residues have

appeared. Two of these, Glu81 and Arg114 (human

MGST1 positional numbering), are found in the puta-

tive transmembrane segments 2 and 3, respectively.

According to electron crystallographic structure deter-

mination of MGST1 [34] and LTC4 synthase [35] and

hydrophobicity properties, the MAPEG forms all

appear to contain four transmembrane regions. MGST1,

LTC4 synthase and PGES [22] are all trimeric

proteins. At the tight border between transmembrane

region 2 and 3, some of the sequences have a Gly-Pro

sequence, typical of a sharp bend. Interestingly, the

almost strictly conserved charged residues mentioned

above are both spaced by exactly 15 residues from the

Gly-Pro bend, strengthening a role for structural

charge interactions. In addition, Asn78 is conserved in

almost all MAPEG members. This residue faces the

cytosol, positioned just before the second transmem-

brane segment, and is probably involved at the active

site. In fact, mutation of these residues in MGST1

seriously affects activity (unpublished observations).

Mutation of the residue corresponding to Arg114

(Arg110) in human mPGES-1 also abolishes activity

[36]. Similarly Arg130, facing the cytosol and adjacent

to the fourth transmembrane segment, is conserved in

nearly all members. The sequence patterns diagnostic

for the PGES and FLAP families are both found

in regions facing the cytosol, thus implying that they

represent family specific regions of the active site

and ⁄or substrate-binding areas.

Observations on the proteins

E.coliMGST and SynMGST represent the first charac-

terized prokaryotic members of the MAPEG super-

family. It was therefore of strong interest to determine

their catalytic properties. Both enzymes efficiently cata-

lyze a glutathione transferase reaction and conse-

quently may be involved in detoxification. In contrast

to human MGSTs 1, 2 and 3, no glutathione peroxi-

dase activity could be detected. Our results thus

demonstrate that both of these highly divergent pro-

karyotic MAPEG members indeed are microsomal

glutathione transferases.

SynMGST, MGST2, and LTC4 synthase to some

extent, align with a postulated lipid binding site of

FLAP (amino acids 48–61) [37–39]. In addition, Syn-

MGST contains conserved arginine and tyrosine resi-

dues implicated in LTC4 synthase activity [40].

However no such activity could be detected, logically

coinciding with the fact that 5-lipoxygenase (forming

the substrate) as well as other lipoxygenases are found

later in evolution [41]. However, recently a 15-lipoxy-

genase was characterized as a secretable enzyme

in Pseudomonas aeruginosa [42] and is, to the best of

our knowledge, the first example of a lipoxygenase in

bacteria.

The cyanobacteria, Synechocystis spp., represent an

interesting model system for further studies of the bio-

logical functions of SynMGST. Knock out experi-

ments, as well as studies of the effects caused by

environmental factors such as light and oxygen on

SynMGST gene expression, will provide important

information about the biological function. Moreover,

if the MGSTs represent common bacterial components

involved in glutathione metabolism mediating cell sur-

vival, they may constitute possible targets for the

development of novel antibiotics.

N-ethylmaleimide, activity and activation

Mammalian MGST1 is activated by sulfhydryl rea-

gents and its relatively modest activity towards CDNB

is increased by 20-fold (from 3 lmolÆmin)1Æmg)1 to

60 lmolÆmin)1Æmg)1) [43]. An MGST has been purified

from Xenopus laevis that was extremely active

(200 lmol min)1Æmg)1) but on the other hand very sen-

sitive to sulfhydryl reagents [44]. The pike enzyme is

also inactivated by N-ethylmaleimide (not shown).

Synechocystis, Arabidopsis and Drosophila MGSTs

appear to represent a third category, namely enzymes

that are insensitive to sulfhydryl reagents. In the case

of Synechocystis and Drosophila enzymes, this is

accounted for by the fact that no cysteine residues are

present and probably explains why SynMGST is an

exceptionally stable protein (in our experience). The

catalytically active form of E.coliMGST contains

three cysteine residues but was not activated by

N-ethylmaleimide. Instead a slight inhibition of the

activity towards CDNB was observed. Apparently,

none of the cysteines is situated at an accessible posi-



tion that is critical for enzyme activity of the

E.coliMGST. In conclusion, sulfhydryl reagent activa-

tion ⁄ inactivation cannot be used as a criterion to iden-

tify MAPEG MGST1 members as the activation has

been detected so far only with mammalian MGST1.

Also, the closest relative of MGST1, PGES, is inacti-

vated by N-ethylmaleimide [22] as well as LTC4 syn-

thase [45]. It is evident that cysteine is not involved in

the catalytic mechanism of several MAPEG members,

but could well be relevant for PGES and LTC4 syn-

thase, which harbour cysteines at unique positions.

Conclusion

We have identified several new MAPEG proteins by

sequence homologies with proteins in various databases.

The mammalian members can be traced back 500 mya

as all six families can be found in fish, consistent with a

role in eicosanoid signalling. The gene products

from two representative bacterial strains, E. coli and

Synechocystis sp. were cloned and overexpressed in

E. coli. In addition, plant, insect and fish MAPEG mem-

bers were characterized. As a common denominator,

most MAPEG members catalyze glutathione conjuga-

ting activity towards CDNB or a specific substrate such

as LTC4, some with remarkable efficiency. The enzymes

represent early MAPEG members in their respective

phylogenetic classes and thus create a defined basis for

understanding this superfamily.

Experimental procedures

Materials

Oligonucleotides were synthesized by KEBO, (Stockholm,

Sweden). Pfu DNA polymerase was purchased from Strata-

gene (La Jolla, CA, USA). pGEM T-vector was from

Promega (Madison, WI, USA). Gel extraction and plasmid

isolation kits were from Qiagen (Hilden, Germany). DNA

sequencing kit (ABI PRISM Dye Terminator Cycle Sequen-

cing Ready Reaction Kit) was obtained from Perkin-Elmer

(Boston, MA, USA). Hydroxyapatite (Bio-Gel HTP) was

from Bio-Rad (Hercules, CA, USA).

Sequence comparisons

In the search for new members of the MAPEG superfamily

a set of representative members were selected as seeds. The

seeds were the human member proteins of MGST1-3 (Uni-

prot-Swissprot identifiers P10620, Q99735 and O14880);

FLAP (P20292); LTC4 synthase (Q16873) and PGES

(O14684). Two bacterial members, SynMGST (P73795) and

E.coliMGST (P64515), were additionally selected to com-

plement the six human forms. The eight seeds were used as

query sequences in the search for homologues using fasta

[46] against Swissprot release 41.24 [47], TrEMBL release

24.13 [47] and 138 completely sequenced genomes. Further

screenings were performed against the NCBI non-redund-

ant protein database using psi-blast [48]. Finally, to fetch

unverified translations of MAPEG members the NCBI EST

database (excluding human and mouse) [49] was searched

using tblastn [48]. The resulting nucleotide sequences from

the EST search were translated using getorf from the

emboss package [50]. The open reading frames were filtered

by a minimum size of 100 amino acid residues and flanked

by start and stop codons. These homology searches resulted

in nearly 1000 redundant amino acid sequences which were

followed by an extensive work of manual filtering to obtain

a non-redundant set of sequences by removing duplicates

and non-EST supported alternative splicings.

Multiple sequence alignments and dendrograms

To study the relationships between the new members of the

superfamily we calculated multiple alignments using clu-

stalw [51] on the resulting sequences from the homology

searches. Dendrograms were obtained using neighbor-join-

ing method in the clustalw package and protpars from

the phylip package [52]. An unrooted tree was generated

based on the complete set of sequences of all superfamily

members. To also visualise the more general relationships

of the families included in MAPEG an unrooted consensus

tree was produced. The consensus sequences of the families

of MGST1–3, FLAP, LTC4 synthase, PGES, SynMGST

cluster, E.coliMGST cluster and Insect cluster were gener-

ated by the cons program from the emboss package. A

hydrophobicity plot was generated to verify the structural

similarities of the proteins. It was based on the multiple

sequence alignment of the complete superfamily and calcu-

lated according to Kyte and Doolittle [53] using a window

of 11 residues.

Pattern detection

To characterize the MAPEG families further we extracted

patterns compatible to the PROSITE database [25,54].

These patterns are helpful in annotation of new sequences

and model the unique motifs of a family. The patterns were

generated by the program pratt version 2.1 [55,56]. pratt

was run on sequences from each of the MGST1-3, FLAP,

LTC4 synthase, PGES and Insect families by setting the

maximal pattern length parameter to 20. The best ranked

patterns of each family, shown in Table 2, were selected

and tested for unambiguousness by performing a scan

against Swiss-Prot and TrEMBL with the program fuzz-

pro from the emboss package. The degree of unambiguous-

ness was defined as the fraction of member ranked higher

than the first occurring non-member.



Determination of SynMGST mRNA expression

RT-PCR analysis was performed in a PCR buffer contain-

ing 1 mm dNTPs, 1.5 mm MgCl2, 1 lm of the appropriate

primer and 50 ng template (final volume 50 lL). Two

20-mer synthetic oligonucleotides, 5¢-GGGCGGGCTCGG

GCTAAATA-3¢ (sense primer) and 5¢-GGTTGGATCTC

GGTAAATGG-3¢ (antisense primer) (DNA technology,

ApS, Aarhus, Denmark), were designed to anneal within

the ORF of SynMGST. The antisense primer, complement-

ary to the coding strand, was annealed to the SynMGST

mRNA by incubating the mixture at 70 �C for 2 min, fol-

lowed by a temperature shift to 42 �C at a speed of

2 �CÆmin)1. After an additional 30 min at 42 �C, 2.5 units

of AMV reverse transcriptase (Pharmacia Biotech, Uppsala,

Sweden) was added, and primer extension reactions were

carried out for 30 min. Finally, the AMV reverse transcrip-

tase was heat inactivated at 95 �C and the extension prod-

uct was PCR amplified by the addition of sense primer and

2.5 units of Taq polymerase (Pharmacia Biotech). The tem-

perature cycles were 95 �C, 1 min, 55 �C, 1 min and 72 �C1 min, repeated 25 times. The PCR product was run on a

1% agarose gel with a 100 bp ladder and visualized with

ethidium bromide. As positive and negative controls, DNA

and RNA were used as templates, respectively, without the

addition of reverse transcriptase. Preparations of Synecho-

cystis 6803 DNA and RNA were performed as described

previously [57].

Isolation and cloning of the SynMGST

and E.coliMGST

The coding sequence for the SynMGST, corresponding to

the complementary strand of the nucleotide sequence from

89 254 to 89 667 in the Synechocystis genome, accession

number D90909, was amplified from genomic DNA by

PCR. Oligonucleotide primers were constructed to incor-

porate suitable restriction sites (NdeI-HindIII) into the

5¢- and 3¢ ends of the product. Sense primer, 5¢-GAGA

GAGGATCCATATGACAAAAACCGAGTTAC-3¢, NdeI

site; antisense primer, 5¢-GAGAGAAAGCTTCAAAACT

GGGACAGTTG-3¢, HindIII site.

The same method was used to amplify the coding

sequence for the E.coliMGST, corresponding to the nucleo-

tide sequence 10 655–11 080 in the E. coli genome with a

GTG start. Sense primer, 5¢-GAGAGACATATGCCA

TCGGCCATTTTAAAG-3¢; antisense primer, 5¢-GAGA

GAAAGCTTCTAACGCAGGGAGAAAAC-3¢. An alter-

native start site would be the in-frame ATG, 30 nucleotides

downstream of the GTG. This coding region was amplified

using the sense primer 5¢-GAGAGACATATGGTAAGC

GCGCTGTACGCC-3¢.PCR was performed with 0.2 mm dNTPs, 2 mm MgCl2,

0.25 lm of the respective primer, about 0.1 pmol of tem-

plate and 0.5 U of Pfu polymerase. The temperature cycles

were 30 s at 94 �C, 1 min at 40 �C and 2 min at 72 �C,repeated 10 times, followed by 30 s at 94 �C, 1 min at

50 �C and 2 min at 72 �C, repeated 20 times. Finally, the

reaction was terminated by a 7 min extension at 72 �C. ThePCR product was isolated by agarose gel electrophoresis,

purified from the gel and digested with NdeI and HindIII.

The resulting product was gel purified and ligated into the

bacterial expression vector pSP19T7LT as described previ-

ously [58]. Ligated plasmids were transformed into E. coli

XL-1 Blue as described previously [59]. Plasmids were isola-

ted from a number of clones and cleaved with NdeI and

HindIII followed by agarose gel electrophoresis to verify

the size of the inserts. Selected inserts were sequenced on

an Applied Biosystems (Foster City, CA, USA) 373A auto-

mated DNA sequencer using a dye terminator cycle sequen-

cing kit.

The expression construct containing the correct coding

sequence for both the SynMGST and E.coliMGST was

transformed into E. coli BL21 (DE3) (that harboured the

plasmid pLys SL [60]) using the same protocol. Glycerol

stocks were prepared and stored frozen at )70 �C for sub-

sequent use as starting material for the expression experi-

ments.

Overexpression of SynMGST and E.coliMGST

and preparation of membrane fraction

Small aliquots (1–2 lL) of bacterial glycerol stock were

grown in 2· YT medium overnight at 37 �C. The cultures

were diluted 1 : 100 into 2 L of Terrific Broth medium

containing ampicillin (75 lgÆmL)1) and chloramphenicol

(10 lgÆmL)1) in a 5 L flask placed in a thermostated water

bath. The culture was oxygenated by air bubbling and

grown until the D600 was 0.4–1.2. Expression was then

induced by the addition of 0.4 mm isopropyl thio-b-d-gal-actoside, the temperature was switched to 30 �C and the

culture allowed to grow for another 4 h. Thereafter, cells

were pelleted and resuspended in 200 mL 15 mm Tris ⁄HCl,

pH 8.0, 0.25 m sucrose, 0.1 mm EDTA, 1 mm glutathione

(TSEG buffer). Lysozyme was added to a final concentra-

tion of 0.2 mgÆmL)1, and the mixture was gently stirred for

30 min at 4 �C. The resulting spheroplasts were pelleted

(8000 g, 10 min), resuspended in 200 mL TSEG and lysed

by four 30 s sonication pulses from an MSE Soniprep

150 sonifier (Beckenham, Kent, UK) at 40–60% of maxi-

mum power. Magnesium chloride was added to a final con-

centration of 6 mm and DNA and RNA were hydrolyzed

by incubation with DNaseI (4 lgÆmL)1) and RNase A

(4 lgÆmL)1) for 30 min at 4 �C with gentle stirring. Cell

debris was removed by centrifugation at 5000 g for 10 min.

The supernatant was then centrifuged at 180 000 g for 2 h

and the membrane pellets were suspended in 10 mm

potassium phosphate, pH 7.0, 20% (v ⁄ v) glycerol, 0.1 mm

EDTA, 1 mm glutathione.



Purification of SynMGST

Solubilisation and partial purification of SynMGST was

performed at 4 �C as follows. Membranes were solubilised

by the addition of an equal volume of 10 mm potassium

phosphate, pH 7.0, 20% (v ⁄ v) glycerol, 0.1 mm EDTA,

1 mm glutathione, 10% (v ⁄ v) Triton X-100 and 15 min

incubation on ice. Insoluble particles were removed by

centrifugation (100 000 g, 20 min). Solubilized membranes

were adsorbed to 10 g of hydroxyapatite equilibrated with

10 mm potassium phosphate, pH 7.0, 20% (v ⁄ v) glycerol,

0.1 mm EDTA, 1 mm glutathione, 1% (v ⁄ v) Triton X-100

(hereafter referred to as buffer A), for 15 min. Hydroxyapa-

tite elution was performed by a batch procedure where the

hydroxyapatite was pelleted by a low speed centrifugation

pulse and washed with two volumes of buffer A, followed

by one volume of 50 mm potassium phosphate in buffer A.

SynMGST was eluted with 0.4 m potassium phosphate in

buffer A and desalted by dialysis for 20 h against two chan-

ges of 60 volumes of buffer A.

Cation-exchange chromatography was performed on a

HiTrap SP, 5 mL (Pharmacia Biotech) column equilibrated

with buffer A. The eluate from the hydroxyapatite chroma-

tography step was loaded on the column and elution of

SynMGST was monitored by enzyme activity measure-

ments (see below). As no activity was retained on the col-

umn, the flow-through was applied onto a DEAE-Sephadex

A-25 (Pharmacia Biotech) anion exchanger equilibrated

with buffer A. As no enzyme activity was retained on either

ion exchanger, the protein content of the flow-through from

the DEAE-Sephadex was examined by SDS ⁄PAGE. A pre-

dominant protein band, comigrating with rat MGST1, was

observed. This band was also observed in fractions from

the other purification steps. The band was cut out from the

gel followed by elution of the protein and the N-terminal

amino acid sequence was determined using an Applied Bio-

systems 477A instrument with on-line analysis of the phe-

nylthiohydantoin amino acids.

Cloning and overexpression of Drosophila

and Arabidopsis MAPEG members

D. melanogaster

The (EST) clone (GenBank AF111426, Mgst1 gene) was

purchased from Invitrogen (Stockholm, Sweden). This

clone encodes Drosophila MGST-like protein. Primers were

designed to introduce a NdeI restriction site immediately

upstream of initiation codon and a HindIII site immediately

downstream of the stop codon. Forward primer, 5¢-gagacatATGGCCAGCCCCGTGGAACT-3¢; reverse primer

5¢-cccaagcttTCAGAAGGCGGCCGAG-3¢ (lower case indi-

cates CLAMP and restriction site). Drosophila MGST was

amplified using PCR (94 �C for 2 min, followed by 35

cycles of 45 s at 94 �C, 30 s at 50 �C and 3 min at 72 �C,

with a final extension of 10 min at 72 �C) with Pfu DNA

polymerase. The PCR product was subcloned into pGEM-T

Easy vector for sequencing, using a PerkinElmer 373 auto-

mated sequencer (Applied Biosystems) using T7 and Sp6

primers. The EST clone showed an additional nucleotide

sequence GAAGA (position 29–33) and C (position 35) as

compared to GenBank AF111246 [61]. In the EST clone,

the asparagine at position 42 of GenBank AF111246was

replaced with a lysine. The cDNA was digested with NdeI

and HindIII and inserted between these sites in the

pSP19T7LT vector. The protein was expressed in E. coli

BL21(DE3) by a slightly modified method compared to

SynMGST. Protein induction with 0.8 mm isopropyl thio-

b-d-galactoside was carried out at 30 �C. After low speed

centrifugation (4000 g) the resulting supernatant was centri-

fuged at 105 000 g for 60 min. The membrane fraction was

resuspended in 0.05 m potassium phosphate buffer (pH 7.4)

containing 0.25 m sucrose and 0.3 mm EDTA. The freshly

prepared membrane fraction was assayed for GST and

glutathione peroxidase (GPx) activities. The protein concen-

tration was measured by the Lowry method using BSA as a

standard [62].

A. thaliana

A plant cDNA displaying sequence homology to human

MGST3 was cloned from an Arabidopsis thaliana cDNA

library from immature green siliques [63]. Primers for the

complete cDNA were designed as follows: forward primer

1, 5¢-ATGGCGGCGATTACAGAATT-3¢, reverse primer1,

5¢-TCAAGCAAGGATCAGAGTGA-3¢. The sequence was

obtained from the TIGR database (At1g65820 68408.m06848

putative microsomal glutathione S-transferase). The same

PCR method was used as described for Drosophila and then

Taq DNA polymerase was added to the PCR mix. Five

additional cycles were performed to add A overhangs to

the linear fragment. The PCR product was subcloned into

the pGEM-T Easy vector for sequence analysis. No muta-

tion was detected. To confirm whether the putative MGST3

is expressed in A. thaliana, northern blot analysis using the

cDNA was carried out in seedlings (data not shown). To

examine the catalytic activity the A. thaliana MGST3 was

expressed in W(R) Saccharomyces cerevisiae strain WAT11

[64]. Reformatting and cloning the MGST3 into the expres-

sion vector pYeDP60 was performed by PCR amplification

using Pfu DNA polymerase. The specific primers to intro-

duce a BamHI site (indicated in bold) immediately

upstream of the initiation codon (start of uppercase) were

designed as follows: forward primer 2, 5¢-cgggatccATGG

CGGCGATTACAGAATTTC-3¢. To obtain the cDNA with

suitable restriction sites, forward primer 2 and T7 primer

were used for PCR with Pfu DNA polymerase as the pGEM-

T Easy vector has an EcoRI site downstream of the stop

codon of MGST3. The PCR product was digested with



BamHI and EcoRI and then the reformatted BamHI-EcoRI

fragment was subcloned into BamHI and EcoRI sites of the

pYeDP60 vector. The nucleotide sequence of the cDNA was

determined with a PerkinElmer model 373 automated

sequencer using forward primer 1 and reverse primer 1. No

mutation was detected. The transformed yeast cells were

cultured and the microsomes were prepared by differential

centrifugation. The microsomes were washed with 0.15 m

Tris ⁄HCl buffer (pH 8.0) twice by ultracentrifugation. The

microsomal membranes were resuspended in 0.05 m potas-

sium phosphate buffer containing 0.25 m sucrose and

0.3 mm EDTA. The freshly prepared suspension was assayed

for GST and GPx activities within 4 h. Microsomal proteins

were quantified by the method of Lowry et al. [63].

Protein analysis of pike MGST

Pike liver MGST was purified essentially as was the rat

enzyme [43]. The enzyme displayed a single band upon

SDS ⁄PAGE analysis. The following procedure was used to

prepare samples for amino acid sequence determination.

For removing Triton X-100 from the enzyme solution, the

microsomal GST solution (0.9 mgÆmL)1) was dialysed

against 0.4 m Tris ⁄HCl, pH 8.0, 2 mm EDTA, 8 m urea.

Triton X-100 in the solution was determined by measuring

the absorbance at 275 nm. When Triton X-100 was

removed, the protein was dialysed against H2O for 48 h.

The amino acid sequence of the intact protein was deter-

mined by automated Edman degradation using an Applied

Biosystems 477A instrument with on-line detection of

amino acid phenylthiohydantion derivatives.

Enzyme assays

Enzyme activity with 1-chloro-2,4-dinitrobenzene (CDNB)

and 1,2-epoxy-3-para-nitrophenoxypropane (0.5 mm) was

assayed in 0.1 m potassium phosphate, pH 6.5, containing

1%, 0.1% or 0% (v ⁄ v) Triton X-100 at 30 �C as indicated

[65]. Activity with trans-phenylbut-3-en-2-one was measured

according to [66]. When activity was determined in bacterial

membranes and crude fractions, the concentration of Triton

X-100 was increased to 1% in order to avoid an increase in

turbidity that has been observed with bacterial membrane

fractions. GPx activity towards cumene hydroperoxide or

phospholipid hydroperoxide was determined in a coupled

assay system as described previously [67]. LTC4 synthase

activity as well as glutathione-dependent peroxidase activity

towards 5-hydroperoxy-eicosatetraenoic acid was measured

as described previously [5,7].

Gel electrophoresis and protein determination

SDS ⁄PAGE was performed according to Laemmli [68] in

15% polyacrylamide gels. Purified rat MGST1 and Rain-

bow molecular weight markers (Amersham, Little Chalfont,

Buckinghamshire, UK) were used as standards. Protein

bands were visualized by staining with Coomassie Brilliant

Blue R-250 or silver staining as described [69]. Protein was

determined by the method of Peterson [70] with BSA as

standard.

Acknowledgements

We thank Jan–Ove Jarrhed and the National Super-

computer Centre in Linkoping, Sweden, for valuable

support on the computer side. Per L. Petterson is

gratefully acknowledged for performing PGE synthase

measurements. Technical assistance by Gudrun Tibbe-

lin is gratefully acknowledged. Financial support from

the Swedish Research Council (13x-12564, 13x-12573),

the Swedish Cancer Society, the Swedish National

Board for Laboratory Animals, the Swedish Society of

Medicine, the Magnus Bergvall, Harald Jeansson, the

Research Committee, FMHS, UAE University (HR

was on a supported leave from UAE University, Al

Ain, UAE) and Carl Tryggers foundations, Karolinska

Institutet and Linkoping University is gratefully

acknowledged.

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Supplementary material

The following material is available from http://www.blackwellpublishing.com/products/journals/suppmat/EJB/EJB4596/EJB4596sm.htm

Fig. S1. Full alignment of all MAPEG members.



bioinformatic and enzymatic characterization of the mapeg superfamily

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