Vol. 268, No. 15, Issue of May 25, pp. 11217-11221,1993
Printed in U.S.A.

THE JOURNAL
OF BIOLOGICALCHEMISTRY
0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Human Glyoxalase I
cDNACLONING,EXPRESSION,ANDSEQUENCESIMILARITYTOFROM PSEUDOMONAS PUTIDA*GLYOXALASEI(Received for publication, September 22, 1992, and in revised form, January 19, 1993)

Nam-Soon Kim$, YuriUmezawaS, Satoshi Ohmuras, and Seishi Kato$$ll
From the SKanagawa Academy of Science and Technology (KAST), 3-2-1 Sakado, Kawasaki, Kanagawa 213 and the §Sagami
Chemical Research Center, 4-4-1 Nishi-Ohnuma, Sagamihara,Kanagawa 229, Japan

Glyoxalase I (EC 4.4.1.5) catalyzes the transformation of methylglyoxal and glutathione to S-lactoylglutathione. We have isolated human cDNA clones encodingglyoxalase I from a phorbol myristate acetatetreated U937 cDNA library. This cDNA encodes aprotein of 184 amino acids with a calculated M , of
20,719. The amino acid composition calculated fromthe deduced amino acid sequence agreed with thatreported for glyoxalase I purified from human erythrocytes. The Escherichia coli cells carryingthe expression vector of this cDNA acquired methylglyoxal resistance and the cell lysate showed the high activity ofglyoxalase I. The amino acid sequence of human glyoxalase I exhibited 57%identity with Pseudomonas putidu glyoxalase I at the C-terminal two-thirds, suggesting that the two enzymes may have originated froma common ancestor.

6) and reported to be present in most tumor cell lines (10).
The human glyoxalase I exists in three separable isoforms
which originate from two alleles in the genome (11).Thus
these isozymes have been used for population genetics as amarker enzyme (12).
We have taken a novel approach’ toobtaininghumanproteins. Itscore is the constructionof a human proteincDNAbank which contains thesequence determined andexpressible
cDNA derived from the entire mRNAspecies of human cells.
In this bank, we found a cDNA clone encoding a glyoxalase
I-like protein in which the amino acid sequence was similar
to glyoxalase I isolated from P. putidu. This paper shows thesequence analysis of this cDNA and its characterization by
expression in vivo and in vitro. This is the first reportof thecDNA cloningof mammalian glyoxalase I.
EXPERIMENTALPROCEDURES

Materials-Restriction and modified enzymeswere purchased fromTakaraShuzo,except for BstXIfrom New England Biolabs andGlyoxalase I (EC 4.4.1.5) catalyzes the transformation of reverse transcriptase from Seikagaku Kogyo. Methylglyoxal and glu(1) tathione were obtained from Sigma.
methylglyoxal andglutathionetoS-lactoylglutathione
which is converted to D-lactic acid by glyoxalase 11. This
Preparation of Poly(A)’ RNA-Human histiocytic lymphoma cellglyoxal pathway is found in cells of all organisms, suggesting line U937 cells were cultured in RPMI 1640 medium supplemented
a fundamental role of this system for maintaining their lives. with 10% fetal bovine serum. Total mRNAwas isolated from PMAZThe function of glyoxalase I is not yet clear, but it has been treated or untreated U937 cells by the guanidinium/cesium chloride
method (13). PMA treatmentwas carried out a t a concentration of 5
proposed that it may be concerned with the detoxificationof X
M for 12 h afteraddingPMA.Poly(A)+RNA was preparedmethylglyoxal which has been known as a potent cytotoxic using oligo(dT)-cellulose column chromatography (13).
metabolite (2).
Preparation of cDNA Library-A cDNA librarywas prepared using
Glyoxalase I has beenpurified from various sources such as a (dT)-tailed vector primer according to Pruitt’s method (14). The
mammalian tissues (3, 4), yeast (5, 6), and bacteria (7). A plasmid pTZ18RP1,’ which was constructed by inserting oligonuclecomparison between yeast glyoxalase I and the mammalian otides possessing four restriction enzyme sites (BstXI,EcoRV, KpnI,and NotI) between EcoRI and BamHI of pTZ18R (Pharmacia LKB
type showed that their molecular structures differ in many
Biotechnology Inc.), was used as a vector primer after tailing about
respects in spite of the similar mechanism of their catalytic 60 (dT)s to the KpnI sitefollowed by removing one (dT)-tailed end
reactions (6, 8). Yeast glyoxalase I consists of a single poly- by EcoRV digestion. The procedure and reaction conditions
for cDNA
peptide chain of M, = 32,000, while the mammalian enzymes synthesis were the same as those described in Pruitt’s report. The
consist of two identical subunits of M , = 20,000 26,000. ligation mixture was used to transform E. coli NM522 according toHanahan’s method (15).
Bacterial enzymes have not been well characterized except
Screening-A cDNAfragment
waslabeledwith [ C Y - ~ ~ P ] ~ C T P
for Pseudomonas putidu glyoxalase I ( 7 ) . P. putida glyoxalase (Amersham)using a random primer labeling kit (Takara Shuzo) and
I consists of a single polypeptide chain like yeast glyoxalase I used as a probe for screening by the colony hybridization method
but has different properties from yeast
glyoxalase 1. The gene (13).
for glyoxalase I has been cloned only from P. putidu (9).
Northern Blot Hybridization-Two pg of poly(A)+ RNA samples
Human glyoxalase I has beenpurified from erythrocytes (3, were electrophoresed through agarose gel containing formaldehyde-* The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked “aduertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate thisfact.
The nucleotide sequence(s) reported in thispaperhas been submitted
to theGenBankTM/EMBLDataBankwith accession number(s)013315.
ll To whom correspondence shouldbe addressed SagamiChemical
Research Center, 4-4-1 Nishi-Ohnuma, Sagamihara, Kanagawa 229,
Japan. Tel.: 81-427-42-4791; Fax: 81-427-49-7631.

(13). RNA was transferred from the gel to a nylon filter which was
as in the screening.
used for hybridization under the same conditions
Sequencing-Single-stranded DNAs were isolated from the culture
medium of the transformant carrying a cDNA vector after infection
by helper phage M13K07 andused as a template for sequencing. The
sequencing reaction was performed by the dideoxy method using a

S. Kato, S. Sekine, N.-S. Kim, Y. Umezawa, N. Abe, T. Aoki, and
S. Oh, manuscript in preparation.
The abbreviations used are: PMA, phorbol myristate acetate;bp,
base pairs; PAGE, polyacrylamide gel electrophoresis.

11217

This is an Open Access article under the CC BY license.

11218Human Glyoxalase I cDNA Cloning and Expression

(a)

(b)
0

8s85 2 sI-

FIG.1. Isolated cDNA clones andNorthern blot analysis. a, restriction
map of cDNA, the sequencing strategy,
andtheschematic representation of isolated clones. The solid box represents theopen reading frame. PHGLOI-2 is the&st clone orbtainedby the random sequencing. The deleted clones were prepared to sequence the entire region. 6, 2
pg of poly(A)+RNA isolated from PMAtreated or untreated U937 cells weretransferred onto a nylon membrane after
electrophoresis and hybridized with the
EcoRI fragment of cDNA. kb, kilobase.

.

.

.

E"=

wnzm

.

K

x

w

2 WbP)

1
f

i

Q)

.

.

*

.

1

PMA

,

+

-

"

- -

n
28s

-

- .
fkb\

pHGLO,-,4
POlYA

pHGLOI-2
pHGLOI-3

polyA

I
polYA

pHGLOI-4 -polyA

fluorescent dye-labeled primer kit(Applied Biosystems). The reaction
mixture was analyzed on a 373A automated DNA sequencer (Applied
Biosystems). The deleted cDNA clones were prepared using a deletionkit (Takara Shuzo) and used to sequence the entire region of the
cDNA.
Vector Construction for Expression in Escherichia coli-The EcoRI
fragment of the cDNA, containing the entirecoding region for glyoxalase I, was subcloned into pUC19. Then the coding sequence wasamplified by the polymerase chain reaction method using two synthetic oligonucleotide primers: 5'-CCGACGTCATGGCAGAACCGCAGCCCCCG-3' including an AatII site followed by the sequence of 21 nucleotides beginning with the initiation codon of theglyoxalase I cDNA and a M13 reverse primer. The amplified productwas digested with AatII and HindIII, and then the fragment was
purified by agarose gel electrophoresis. This fragment was inserted
between the AatII and Hind111 sites of expression vector pMPRA3(16) which hasa tac promoter,a ribosome binding sequence of
metapyrocatechase gene, and a rrnBTIT2 terminator. The resultant
plasmid was denoted as pMGLOI.
Expression of Glyoxalase I in E. coli-To test methylglyoxal resistance, the E. coli cells carrying the pMGLOI were cultured at 37 "C inthe minimal medium (0.7% K,HPO,,0.3%KH,PO,,0.5%glucose,
0.1% (NH,),SO,, 0.01% MgSO.) containing 50 pg/ml ampicillin, 1
mM isopropyl-l-thio-8-D-galactopyranoside,and various concentrations of methylglyoxal. The growth of E. coli cells was traced spectrophotometrically at 600 nm for 52 h.
To prepare the cell lysate, the transformantwas incubated at 37 "Cin the minimal medium containing 50 pg/ml ampicillin and 1 mM
isopropyl-1-thio-8-D-galactopyranoside.
The cells were harvested,sonicated, and centrifuged at 16,000 rpm in a 1.5-ml tube. The
supernatant was used for glyoxalase I assay and gel electrophoresis.
Assay for Glyoxalase I Actiuity-The activity of glyoxalase I was
assayed spectrophotometrically at 240 nm in a 1-ml reaction mixture
containing 100 mM potassium phosphate buffer, pH 6.6,l mM reduced
glutathione, and 2.7 mM methylglyoxal(1). The protein concentration
was determined by the Bradford method using a kit (Bio-Rad). The
specific activity was defined as micromoles of methylglyoxal transformed per min/mg of protein at 28 'C.
The activity staining was performed according to Kompfsmethod(12). The position of glyoxalase I was seen as a colorless band against
a blue background. Yeast glyoxalase I (Bethesda Research Laboratories) was used as a control.
In Vitro Translation-The plasmid containing the glyoxalase I
cDNA was digested with Not1 and thenused for in oitro transcription
to prepare acapped RNA. In vitro transcription was carried out using
a kit (Boehringer Mannheim) according to the protocol described by
the supplier. The capped RNA was translated in vitro in a reaction
mixture containing [:"S)methionine (Amersham) using rabbit reticulocyte lysate (Promega). The product was analyzed on an 8-16%
SDS-PAGE gel.

cDNA clones encodeda protein similarto P. putida glyoxalase
I. Thisclone,pHGLOI-2,contained
a cDNAinsert of2la). Northernblothybridizationusing
kilobasepairs(Fig.
this cDNA fragmentas a probe showed three bands (Fig. Ib):a main band of 2.2 kilobases and two faint bands of 1.4 and1.0 kilobases. The pattern and strength of the bands wereindependent of PMA stimulation. Since thesize of the mainband was longer than that of the cDNA insert, this clone
seemed to contain a truncated cDNA. After screening thelibrary using the cDNA fragmentas a probe, several positive
clones were obtained in which four independent clones areshown with the restriction enzyme mapFig.
in la. T h e longest
clone pHGLOI-1 contained the cDNA insert of 2.1 kilobase
pairs.
Sequence Analysis-The determined nucleotide sequenceand the deduced amino acid sequence are shown in
Fig. 2.
The cDNA contains
a total 2,127 bp includinga 5"noncoding
region of 87 bp,anopenreadingframeof 555 bp, a 3'noncoding region of 1,351 bp, and a poly(A) tail of 134 bp.
The open reading frame encodesa protein of 184 amino acidresidues with a calculated M, of 20,719. The sequence surrounding the initiation codon is in good agreement with theconsensus sequence for initiation of translation in vertebrates(17). The putative poly(A) addition
signal is found at position
1,971. The short clone pHGLOI-4 has the same sequence as
pHGLOI-1 up to position 902, followed by a poly(A) t a i l of
55 bp.
The amino acid composition was calculated from the deduced amino acid sequence and compared with that reported
for human erythrocyte glyoxalase I (6). When the first methionine was omitted, the values agreed with each other within
experimental error as shown in TableI. This result supports
theassumptionthattheobtainedcDNAencodeshumanglyoxalase I.
The deduced amino acid sequence was compared with thatfrom theP. putida gene (9) which is the only example reported
for the glyoxalase I gene. Fig. 3 shows that human glyoxalase
I is longerthan P. putida glyoxalase I by20aminoacid
residues, 9 N-terminal residues, and 11 C-terminal residues.
T h e first third of the sequence from the N-terminal showedno similarity, but the remaining two-thirds showeda sequence
to position
similarity of 57% in 106 residues from position 62
167 (from 53 to 158 for P. putida).
Expression in E. coli-To examine the glyoxalase I activity
RESULTSof the encoded protein, the expression vector pMCLOI wasconstructed. TheE. coli cells transformed with pMGLOI were
Cloning of Glyoxalase I cDNA-cDNA clones were picked
at random from the PMA-stimulated
U937 cDNA library and cultured in a medium containing various concentrations of
their terminal sequencesof about 400bp were determined. As methylglyoxal. At a concentration of 5 mM, the cells carryinga result of the similarity search, we found that one of these pMGLOI could grow at 37 "C for 48 h, but the control host

Human Glyoxalase I cDNA Cloning
Expression
andFIG. 2. Nucleotide sequence of human glyoxalase I cDNA and the deduced amino acid sequence. The osterisks indicate a termination codon. The
open reading frame is preceded by an inframetermination
codon. The clone
pHGLOI-4 is terminated at position 902( A ) and followed by poly(A) tail.The
putative poly(A) additionsignalsare
underlined.

11219

11220

Human Glyoxalase I cDNA Cloning and Expression

or transformants carrying the cDNA-removed vector could
used as a template for in vitro translation using rabbit reticnot. This acquisitionof methylglyoxal resistance suggests that ulocyte in a reaction medium containing [35S]methionine.The
the expressed glyoxalase I degraded methylglyoxal. This was translation product shows one band at the molecular weight
further confirmedby the direct measurementof the glyoxalase of 24,000 on a SDS-PAGE gel (Fig. 4b), which was larger thanI activity of the lysate. The lysateof host E. coli showed weak the expected value, 20,719.
activity (0.02 Fmol/min/mg protein) originating from itsown
DISCUSSION
glyoxalase I. Ontheotherhand,thelysate of pMGLOI
carrying cells showed 110-fold higheractivity (2.2 Fmol/min/During the construction of a human protein cDNA bank,
mg protein). Glyoxalase I is known to contain one atom of we found a cDNA clone encoding a glyoxalase I-like protein
zinc persubunit. However, theaddition of ZnS04 in thewhich showed great similarity to the aminoacid sequence of
culture medium had no effect on the glyoxalase I activity of glyoxalase I from P. putida. Using this as a probe, the fullthe lysate.
length cDNA of 2.1 kilobase pairs was cloned and sequenced.
To estimate themolecular weight of the expressed product, The amino acidcomposition calculated from the deduced
the lysate was analyzed by SDS-PAGE. Since theexpression amino acid sequence agreed with that of glyoxalase I purified
level was too low, we could not see a clear band corresponding from humanerythrocytes. The expression product of theto glyoxalase I on the gel. However,when the lysate was cDNA in E. coli showed glyoxalase I activity. From these two
separated by native gel electrophoresisandthenactivity
results, we concluded that the isolated cDNA encodes humanstaining was carried out, a colorless band showing glyoxalase glyoxalase I. However, there was some discrepancy between
I activity appeared on the gel with ablue background as the molecular weights of 20,719 calculated from the deduced
shown in Fig. 4a. The positionof the bandwas different from amino acid sequence and 24,000 determined by SDS-PAGE
that of yeast glyoxalase I.
analysis of i n vitro translation product, which is similar to
In Vitro Translation-To estimate the molecular weight of the reportedvalue for the purified protein (6, 11).This differthe encoded protein, i n vitro translation was performed. Since ence may be within theallowable range of deviation of molecour cDNA cloning vectorhas a T 7 RNA polymerase promoter ular weights determined by SDS-PAGE.
upstream of the cDNA insert, it is easy to prepare the capped The strength of the hybridization band on the Northern
RNA corresponding to thecDNA. The transcribed RNAwas blot and the existence of many positive clones suggest theabundant expression of glyoxalase I in U937 cells. The glyoxTABLEI
has beenalase I activity inpolymorphonuclearleukocytes
Amino acid composition of human glyoxalase I predicted from amino
reported to increase asa result of PMA stimulation (18).
acid hydrolysis and nucleic acid sequence
However, the PMA stimulation did not cause a remarkable
Amino acidBy hydrolysis"
From sequencechange in thelevel of glyoxalase I mRNA in U937. Two faintresidueslmol subunit
extra bands of short size were recognized by the Northern
24
24.4
blot hybridization. At least one of them resulted from the
Asx
10.0
10
Thr
alternative poly(A) addition, because the corresponding short
10.5
10
Ser
cDNA was cloned.
17.9
16
Glx
Human glyoxalase I shared 57% amino acid residues with
12 Pro11.8
P.
putida glyoxalase I at the C-terminal two-thirds. This
was
12
13.5
Gb
an unexpected resultbecause glyoxalase I has been classified
13
12.2
Ala
4.4
4
into twogroups, mammaliantype(rat,pig, human)and
CYs
6.1
6
Val
microbial type (yeast, E. coli, Rhodospirillum rubrum), which
4.8
6 (7)
Met
remarkably differ in the number of subunits (two and one),
8.7
9
Ile
the
isoelectric point (4.8 and 7.0), and the molecular weight
17.0
17
Leu
(23,000
per subunit and32,000) (cf. Table I1 in Ref.6). On the
6.1
6
TY
9
8.3
other hand,P. putida glyoxalase I has been shown to have the
Phe
3.0
3
His
characteristics of the mammalian typeexcept for the number
17.4
18
LYs
of subunits (7). This and our results suggest that P. putida
2.2
3
Trp
glyoxalase I belongs to the mammalian type, and thustwo
the
4.8
5
Ark!
enzymes originate from a common ancestor. However, thegreat differences in the amino acid composition, the peptide
Total
183
183
The original data were recalculated on 183 residues per subunit mapping, and the isoelectric point between mammalian andyeast enzyme imply a difference in their origins in evolution
omitting the first methionine.

..

..

1' MAEPQPPSGGLTDEAALSCCSDADPSTRDFLLQQTMLRVKDPKKSLDFYTRVLGMTLIQK* * : :::..
MSLNDLNTLPALLPKRTLPLRNSFQPHHRARQGHREVADSIPACLVS~

1"

FIG. 3. Sequencealignment of human glyoxalase I (upper)and P.putida glyoxalase I (lower) (9). The
asterisks indicate exact matches and thecolons conservative substitutions.

61' CDFPIMKFSLYFLAYEDKNDIPKEKDEKIAWALSRKATLELTBNWGTEDDATQSYHNGNS** ******** * ** : :* : ****** *** ** **:**:
52" SDFVEAKFSLYFLJUWDPATIPADDDARHQWMKSIPGVLELTHNHGTERDADFAYHDGNT
121 1 D P R G F G H I G I A W D W S A C K R F E E L G D D G I M C G L A F I Q D P D G Y W I E I L N P ~******** : ******

*** *

* *:

**:* **** **112" DPRGFGHICV~PDWAACERFEALQVPFQ~SDGRMNBLAFIRDPMVTGSK
181' ATLM

Human Glyoxalase I cDNAExpression
Cloningand11221

may be due to the difference in molecular structure and/or
theparticipation of otheramino acidresidues. P. putida
glyoxalase I contains a further 6 His residues, which may heMrconcerned with the stabilizationof zinc by chelation.
97,40(3Three isozymes have been reported for glyoxalase I purified
from human erythrocytes (11). These correspond totwo hom69,00(D " wodimers and one heterodimer
composed of two subunits show46,OOl3
ing different electrophoretic properties. This polymorphism
was used for population genetics as a marker. Since no difference in the aminoacid sequences was found in all the cDNAs
30,001cloned from the U9.77 cDNA library, the genotype of U9.77
for glyoxalaseI may be a homozygote. If the cDNA originating
21,501from another allele could be cloned, these cDNAs could be
used
as new probes to examine thepolymorphism in a glyox14,30l3.
alase I gene.
The obtainedcDNA can be used to measure the expression
level of glyoxalase I in various tissuesat various stages, which
will provide useful informationtoinvestigatetherole of
FIG.4. Expression products of human glyoxalase I in vivo glyoxalase I in regulation of cell proliferation. ThecDNA canand in vitro.a, activity staining after electrophoresing on a native be also used to produce a large amount of recombinant glyoxpolyacrylamide gel. 1 , the lysate of host E. coli; 2 and 3, the lysate of alase I which will help in investigating the catalytic reaction
recombinant E. coli carrying pMGLOI; 4 , yeast glyoxalase I. b, SDS- mechanism of glyoxalase I.
(b)

-

1

PAGE analysis of the in vitro translation product. The size of theprotein marker is given on the left.

REFERENCES
1 . Racker. E. (1951) d . Riol. Chem. 1 9 0 , 6 8 5 - 6 9 6
2. Aronnson, A:C.. and ,Mannervik. H. (197i) Htnrhrm. J. 165. M3-501)
J. Mannervik. R.. Aronsson. A:(:., and Tilhelin. G. (19R'L) Mrlhodr Enzymol.
90. .s:i5-541
4. Ora?. H. and Norton. S. J. (1982) M r f h d n Enzvmol. 9 0 . 5 4 2 - 5 4 6(6). Oneexplanationforthis
issueinvolves assumingthe
existence of two evolutionary distinct enzymes which havebeen called by the same name,glyoxalase I, because of similar
5. Marmstil. E..and Mannervik, H. (197Rl PEHS M f . 8 5 . 2 7 5 - 2 7 8catalytic propertiesin spite of the different structural features 6. Marmstil, E.,Aroncicion. A,-('., and Mannervik. H. (1979l Htruhrm. d . 183.
2:lL:iIland possibly different functions in each cell. The direct com5. Rhee.H.-I..Murata.
K.. andKimura.A.
(19%) Rinrhrm. Riophv.7 RP.~.
l.i m ~ r n r r n 1- d.l 99.1-WlQ...... ....
parison in amino acid sequences of both enzymes must wait
A. Larsen. K.. Aronsann, A:(:., Marmstll. E.. and Mannervik.R. (19R-S) Comp.
for the gene cloningof yeast glyoxalase I.
Himhrm. f ' h w o / . 82R.625-638
9. Rhee. H.-I.. Saio. N.. \hrrata. K.. and Kimura. A. (191uI)Agrir. Rid. Chrm.
Human glyoxalase I forms a dimer, butP. putida glyoxalase5. _9_ ')*)A.1-')')AG.I-.., -I...
I acts as a monomer. On comparing the aminoacid sequence 10. Castro. 1'. M.. Soderntrom. M.. Carlherg. I.. Widernten. M.. Platt. A,. and\lannervik.
(1990) (hrrrnogencw 11, 156:J-l576
of the two enzymes, no significant similarity could be found 1 1 . Aronsson, A . TH.
. . Tihhelin. G., and .Mannervik. H. 11979) Anal. Rinrhrm.
92.
:i90-:193
in the N-terminal region; thus this region may be concerned
f. .I.. Hisshort. S., Cuanmann. S.. and Ritter. H.(1975) Humangrwfik
with the dimerization. Thetwo enzymes have been shown to 12. Knrn
2i! 141-1.1:1
13.
Mnnlatis.
T..Frit.sch. E. F.. and Samhrnnk. J. (1982) Molecular C h icontain one zinc atom per subunit. Our sequencing results
f h n r o l o p M o n u d . Cold SprinR Harhnr hhoralory. ('old Spring Ha%:,show that the two enzymes contain a conserved sequence,
SY
14. Pruitt. S. C. (1988) Cenr ( A m s t . ) 66. 121-134
His-Xl2-His-Xln-His-XIl-Cys,
which is a candidate for a zinc 15.
Hanahan, D.(1983) J . Mol. Hiol. 166,557-5x0
binding site referring to the other metallo(Zn)enzymes (19). 16. Kohatake. E.. Ikariyama. Y.. Ailawn. M.,Miwa. K.. and Kato. S. ( 1 9 9 0 1 J .
f h f r r h n f d .16. 87-96
The activity of P. putida glyoxalase I has been reported to be 17. Kozak. M.11987) Nurbic Arid.7 Res. 15. 812.5-8148
18. (;illespie. E. (19x1) Hiorhrm. Hiophys. RPS.('ommun. 9 8 , 4 t 3 - 4 7 0
unaffected by EDTA which completely inhibits the activity
19. Rrown. K.S.,Huguet. .I.. and ('urtis. N..I. (1983) in Mrrol lorn* I n Hioh irol
Swfvms (SigeI. H.. ed) Vnl. 15, pp. 55--W.hlnrrel 1)ekker. New Yorfof mammalian glyoxalase I including the human type. This_.

~