no, /ul/67531/metadc330887/m2/1/high_re… · the conversion of phenylglyoxal to mandelic acid,...

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Gray, Bedii, Purification and Studies of Mammalian

Glyoxalase Enzymes. Doctor of Philosophy (Chemistry),

December, 1980, 110 pp., 5 tables, references, 96 titles.

The glyoxalase system, which has been known since 1913,

is widely distributed in nature. The system consists of two

enzymes, glyoxalase I and glyoxalase II. The reactions

catalyzed by the two enzymes are:

glyoxalase I

Methylglyoxal + glutathione

glyoxalase II

S-lactoylglutathione — ^

H 2 °

B-lactic acid + glutathione

Methylglyoxal is very unstable and undergoes oxidation

and polymerization reactions. One of the purposes of this

study was to find a simple, convenient and reproducible

method of methylglyoxal preparation. Another objective was

the purification of both glyoxalase enzymes employing

affinity chromatography as a major step. The purified

enzymes were to be characterized by chemical, physical and

kinetic properties as an approach to the understanding of

the biological function of the system.

The purification procedures were designed to minimize

any alteration of enzyme proteins and to give high recovery

of the activities. The purification of glyoxalase I was

accomplished by the use of both hydrophobic and affinity

chromatography. The homogeneous enzyme has a specific

activity of 944 units per mg protein. The enzyme has a

molecular weight of 43,000 daltons; it is a dimer and is

apparently composed of identical subunits of molecular

weight approximating 21,500 daltons.

The purification of glyoxalase II was accomplished by a

rapid, two-step affinity chromatographic scheme. The homo-

geneous enzyme exhibited a specific activity of 920 units

per mg protein, has a molecular weight of approximately

29,500 daltons and appears to be a monomer.

Inhibition studies revealed that nucleotides, nucleo-

sides, structurally related compounds, and also some

microtubule poisons are potent, cooperative inhibitors of

both glyoxalase enzymes. From the studies of nucleotides

and related compounds, it was concluded that aromatic

compounds which contain heterocyclic nitrogen and/or have

amino groups on the aromatic ring, are effective inhibitors.

Kinetic studies indicated that there is a common

binding site on glyoxalase I for glutathione, S-octyl-

glutathione, methylglyoxal, colchicine and the hemimercaptal

of methylglyoxal and glutathione. Colchicine also seems to

become a better inhibitor in the presence of increasing

concentrations of free glutathione or free methylglyoxal.

The substrate of glyoxalase I, the hemimercaptal of methyl-

glyoxal and glutathione, is a competitive inhibitor of

glyoxalase II.

Copyright by

Bedii Oray

1980

1 1 1

PREFACE

The author expresses his gratitude to Mr. Michael W.

Kellum for his help in the methylglyoxal synthesis pro-

ject and to Mrs. Vana L. Smith, Ms. Jana Jordan and

Mrs. Patricia Lambert for their technical assistance in

this research project.

The financial support of this investigation by the

Robert A. Welch Foundation of Texas, No. B-133, and North

Texas State University Faculty Research Fund is gratefully

acknowledged.

Bedii Oray

December, 1980

IV

TABLE OF CONTENTS

Page

LIST OF TABLES v i

LIST OF ILLUSTRATIONS i • • • • • • • • • • v n

Chapter

I. INTRODUCTION x

II. EXPERIMENTAL PROCEDURE n

General Methylglyoxal Synthesis Routine Glyoxalase I Assays Routine Glyoxalase II Assays Determination of Protein Concentration Column Material Preparation Purification of Glyoxalase I Purification of Glyoxalase II Concentration Procedures Molecular Weight Determination of Glyoxalase I

by Sedimentation Equilibrium Molecular Weight Determinations of Glyoxalase

Enzymes by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

Polyacrylamide Gel Electrophoresis Isoelectric Focusing Kinetic Studies

III. RESULTS AND DISCUSSION 22

Methylglyoxal Synthesis Purification of Glyoxalase I Purification of Glyoxalase II Molecular Weight Determinations Inhibition of Glyoxalase Enzymes by Nucleo-

tides, Nucleosides and a Series of Structurally Related Compounds

Inhibition of Glyoxalase Enzymes by Micro-tubule Poisons

Kinetics

IV. REFERENCES

LIST OF TABLES

T a b l e Page

I. Hydrophobic Chromatography of Glyoxalase I . . . 34

II. Purification of Glyoxalase I 37

III. Purification of Glyoxalase II 44

IV. I 5 0 Values (pM) for Some Glyoxalase Inhibitors . 68

V. Effect of Microtubule Poisons on Various Enzymes. 77

VI

LIST OF ILLUSTRATIONS

F i 9 u r e Page

1. (A) Effect of Temperature on Hydrolysis of Pyruvaldehyde Dimethyl Acetal

(B) Rate of Hydrolysis of Pyruvaldehyde Dimethyl Acetal 25

2. Effect of Acid Concentration on Hydrolysis of Pyruvaldehyde Dimethyl Acetal 27

3. Hydrolysis of Varying Levels of Pyruvaldehyde Dimethyl Acetal 29

4. Glyoxalase I Kinetic Study with Methylglyoxal Prepared by the Hydrolysis of Pyruvalde-hyde Dimethyl Acetal 32

5. Ethylamine—Sepharose Hydrophobic Chromatography of Swiss Mouse Liver Glyoxalase I 39

6. S-Octylglutathione-Sepharose Affinity Chromatog-raphy of Swiss Mouse Liver Glyoxalase I . . 41

7. First Oxidized Glutathione—Sepharose Affinity Chromatography of Swiss Mouse Liver Glyoxalase II 4 6

8• Second Oxidized Glutathione—Sepharose Affinity Chromatography of Swiss Mouse Liver Glyoxalase II 49

9. Sedimentation Equilibrium Ultracentrifugation of Glyoxalase I 5 2

10. Molecular Weight Determination of Glyoxalase I by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis 55

11. Molecular Weight Determination of Glyoxalase II by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis 57

12. Inhibition of Glyoxalase I by GTP, ATP, CTP and UTP 60

vii

F i c? u r e Page

25. Effect of Various Fixed Concentrations of Colchicine on the Glyoxalase I Activity at Increasing Levels of Free Glutathione with a Constant Level of Hemimercaptal . . 96

26. Effect of Various Fixed Concentrations of Colchicine on the Glyoxalase I Activity at Increasing Levels of Free Methylglyoxal with a Constant Level of Hemimercaptal . . 99

27. Inhibition of Glyoxalase II by the Hemimercaptal of Glutathione and Methylglyoxal 101

IX

INTRODUCTION

The conversion of phenylglyoxal to mandelic acid, when

rabbits were fed phenylglyoxal, led to the discovery of the

glyoxalase system in 1913 (1). Subsequently, the conversion

of phenylglyoxal and methylglyoxal (pyruvaldehyde) to

mandelic acid and lactic acid respectively was found in a

variety of animal and plant tissues, as well as in yeast

(2-4). At the time, the glyoxalase system was thought to be

involved in glycolysis, but Lohman demonstrated that the

glyoxalase system is not an essential part of glycolysis,

and that the system requires glutathione as a cofactor (5).

It was determined that glutathione, y-glutamyl-L-

cysteinylglycine, can be replaced as a coenzyme by

a-L~glutamyl-^-cysteinylglycine, B-^-aspartyl-L-cysteinyl-

glycine or Y-D-glutamyl-L-cysteinylglycine, though all are

less effective than glutathione (6,7). Other compounds

tested as coenzymes but found ineffective include oxidized

glutathione, cysteine, thioglycolic acid (5), Y-glutamyl-

cysteine and cysteinylglycine (8). Wieland et al. concluded

that glutathione derivatives which serve as the cofactor for

the glyoxalase system must possess a terminal free amino

group (9). This was based on the observations that

N-acetylglutathione is not effective as a cofactor, but that

N-DL-alanylglutathione and N-DL-valylglutathione could serve,

though less effectively than glutathione.

Methylglyoxal can be replaced by other aliphatic and

aromatic a-ketoaldehydes such as glyoxal, kethoxal, hydroxy—

pyruvaldehyde, phenylglyoxal and some substituted phenyl-

glyoxals (10,11). The ability of the a-ketoaldehydes to

serve as substrates for the glyoxalase system is impaired

when the side-group of the a-ketoaldehyde is sterically

crowded. 2,4,6-Trimethylphenylglyoxal is not a substrate

even though the related compound 2,4-dimethylphenylglyoxal

does function as a substrate (11).

In 1951 Racker showed that the glyoxalase system

consists of two enzymes (12), glyoxalase I (EC 4.4.1.5) and

glyoxalase II (EC 3.1.2.6). These two enzymes catalyze the

following reactions with methylglyoxal as the substrate:

0 O glyoxalase I

CH3-C-CH + GSH

(methylglyoxal) (glutathione)

0 OH glyoxalase II

CH3-C-CH-SG

(S-lactoylglutathione) B^O

OH I

CH -CH-COOH + GSH

(lactic acid)

It has been demonstrated that the glyoxalase system

forms stereospecifically S-g-lactoylglutathione and D-lactic

acid (13-16). The crucial step in the mechanism of glyox-

alase I action was found to be the transfer of a hydride

equivalent from C—1 to C—2 of the Oi—ketoaldehyde with the

resulting thioester formation (17,18). Hal et al. intro-

duced NMR evidence to support an enediol-proton transfer

mechanism for this step (19).

Davis and Williams showed that Mg is required by

glyoxalase I from calf liver, but may not be necessary for

the enzyme from yeast (20). Arronson et al. concluded using

atomic absorption spectrometry, that glyoxalase I from human

and porcine erythrocytes, as well as rat liver and yeast are

zinc-metalloenzymes (21).

Steady-state kinetics of glyoxalase I have been a con-

troversial subject, and two independent studies have given

rise to two different mechanisms for the action of

glyoxalase I. Kermack and Matheson proposed a two substrate

mechanism where free methylglyoxal and free glutathione are

the two substrates (22). On the other hand, Cliffe and

Waley supported a one substrate mechanism in which the true

substrate is a hemimercaptal formed nonenzymatically from

methylglyoxal and glutathione (23), as indicated below:

Methylglyoxal + glutathione k hemimercaptal

glyoxalase I

hemimercaptal > S-g-lactoylglutathione

Subsequently, a third mechanism has been proposed which

is a combination of the first two (24). This random-pathway

mechanism involved one- and two-substrate branches. The

two-substrate path involved glutathione and methylglyoxal as

the first and second substrates, respectively. The

one-substrate path utilized the hemimercaptal as the sole

substrate. Except at very low hemimercaptal and free

glutathione concentrations, the one—substrate pathway was

proposed to predominate. At higher hemimercaptal and free

glutathione concentrations the one-substrate pathway model

predicts a hemimercaptal-free glutathione competition.

Although the glyoxalase system has been known for many

years and has received considerable attention from various

investigators, the basic biological function of this system

remains to be elucidated. The possibility that the

glyoxalase system could be a step in carbohydrate metabolism

was eliminated by Lohman (5) by showing that the system is

not an essential step in glycolysis. It has been suggested

that the glyoxalase system may function metabolically by

providing a mechanism for the entry of hydroxypyruvic

aldehyde into the tricarboxylic acid cycle or the glycolytic

pathway (25). Support for this was the finding that

phosphohydroxypyruvic aldehyde is a substrate for glyoxalase

I, and the corresponding glutathione thioester could be

hydrolyzed by glyoxalase II to form D-3-phosphoglyceric

acid. However, there is no evidence for the natural occur-

rence of phosphohydroxypyruvic aldehyde (26).

It has also been postulated that the glyoxalase system

may participate in a cycle for the degradation of glycine

and threonine (27-29) via aminoacetone in view of the fact

that methylglyoxal is the deamination product of aminoace-

tone. Enzymatic conversion of aminoacetone to methylglyoxal

was first found in ox plasma, but subsequent studies found

this conversion in only a few other biological systems (30).

Glutathione and the glyoxalase system may possibly play

some role in the regulation of porphyrin synthesis by con-

version of some part of Y,6—dioxovalerate into tricarboxylic

acid cycle intermediates, y,6-Dioxovalerate has been found

to be a substrate for glyoxalase I and the resulting gluta-

thione-thioester is then hydrolyzed by glyoxalase II to form

D-a-hydroxyglutarate (31).

It has also been postulated that a-ketoaldehydes might

have a special function as regulatory molecules in cell

division (32-35). Szent-Gyorgyi et al. defined "retine" as

the agent that prevents cell division and it was proposed to

be a derivative of methylglyoxal. "Promine" was defined as

the agent which controls the action of "retine", and the

glyoxalase system can serve as "promine". This view, along

with the known cancerostatic and cytotoxic properties of

methylglyoxal (36-39), gave rise to the hypothesis that

selective inhibition of glyoxalase I should provide a means

of retarding cell division. This approach has been taken by

Vince and his co-workers (40-42), who have synthesized a

large number of S-substituted glutathione derivatives as

potential inhibitors of glyoxalase I and tested them with

the yeast enzyme. The most effective compounds were those

containing long S-alkyl chains or nonpolar S-aryl groups.

Methylglyoxal was once believed to be involved in

glucose catabolism as a component of the so-called non-

phosphorylating glycolysis (43). When phosphorylated

compounds were identified as the intermediates in glucose

breakdown, the formation of methylglyoxal was considered to

be due to nonenzymatic side-reactions and thus of little or

no importance (44). Methylglyoxal gained more importance

after the reports on an enzyme from Escherichia coli,

Pseudomonas saccharophila and Proteus vulgaris which

catalyzes the conversion of dihydroxyacetone phosphate to

methylglyoxal and inorganic phosphate (15,45,46). This

enzyme, methylglyoxal synthase, appears to constitute the

first step of a glycolytic bypass from triosephosphates to

pyruvate via methylglyoxal and g-lactate. Methylglyoxal

synthase is not detected in liver, skeletal muscle, and

brain of the rat, nor in erythrocytes, lymphocytes, skeletal

muscle, kidney, liver, brain and cardiac muscle of the

human, or in other species, including crab, crayfish,

shrimp, ascarid, snail, lizard, frog, clam, starfish,

jellyfish, sponge, yeast (Saccharomyces cerevisiae and

Candida albicans), algae (Chlorella sp.), fungi (Aspergillus

sp., Trichophytan sp. and Penicillum sp.) and Euglena

gracilis (47). The absence of methylglyoxal synthase in

these organisms and tissues raises a question as to the role

of the glyoxalase system, since it is present in the tissues

or organisms apparently lacking methylglyoxal synthase.

Two recent reports provide evidence that methylglyoxal

is most probably the true substrate for the glyoxalase

system (48,49). Methylglyoxal was isolated from beef liver

as its 2,4-dinitrophenylhydrazone and identified by com-

parison to synthetic methylglyoxal (48). Sato et al. (49)

concluded that methylglyoxal formation in rat liver cells is

a metabolic process, and is largely derived from dihydroxy-

acetone phosphate.

Over the years, glyoxalase I has received considerable

attention from numerous investigators. The first attempt to

purify glyoxalase I was by ammonium sulfate precipitation of

crude tissue homogenates (2). Glyoxalase I has been

partially purified from calf liver (50), from livers of

8

normal DBA/lJ mice and from the same mice bearing a lympho-

sarcoma (51), from the livers of rats of various ages (52),

and from porcine erythrocytes (53). Nearly homogeneous

preparations of glyoxalase I have been obtained from rat

liver, erythrocytes, brain and kidney (11); homogeneous

preparations from mouse liver (54,55) and from rat liver

(56) have been reported in the literature.

A nearly homogeneous preparation of glyoxalase II from

human liver with a very low yield has been reported by

Uotila (57). Recently, glyoxalase II has been purified to

homogeneity from mouse liver and rat erythrocytes (58,59).

Some recent reports in the literature have suggested

that the glyoxalase enzymes are involved in the polymer-

ization of tubulin to form microtubules (60,61,62). The

microtubules are proteinaceous organelles that are present

in virtually all eucaryotic cells. The microtubules are

composed of subunits assembled into elongated tubular

structures having an indefinite length? they are capable of

rapid changes in length by assembly or disassembly of the

subunit proteins, the tubulins, by such conditions as tem-

perature changes, hydrostatic pressure changes, and by

certain drugs such as colchicine and vinblastine. The

microtubules are associated along with other proteins with

the formation of complex assemblies such as the mitotic

spindle, centrioles, cilia and flagella, axonemes, and

neurotubules, and are a determinant of cell shape and

motility (63).

Colchicine, the best known of all microtubule poisons is

believed to combine at a specific site with the tubulin

dimer (M.W. 110,000) in a 1:1 ratio (64). It has been found

that colchicine binds to tubulin and prevents the in vitro

assembly of microtubules (65); whereas lumicolchicine, the

irradiation product of colchicine, neither binds to tubulin

nor prevents microtubule assembly. Vinblastine, one of the

Vinca alkaloids, which in very low concentrations causes

precipitation of tubulin granules (thereby inhibiting

assembly), has a binding site on tubulin which is different

from that of colchicine (64). Additionally, the two binding

sites on tubulin for the guanosine nucleotides are distinct

(64). The colchicine binding site on tubulin is also the

binding site for the microtubule poison, podophyllotoxin

(66). Another drug, griseofulvin, does not apparently block

the in vitro polymerization of tubulin as measured by

electron microscopy (66); this drug may not therefore

directly affect microtubules, but may act on other processes

essential for normal completion of cell division.

There were several objectives of the studies reported

herein. The first objective was to devise a simple,

convenient, and reproducible method of methylglyoxal prepa-

ration, since methylglyoxal is very unstable even stored

10

under nitrogen in the cold. Consequently, the tedious and

cumbersome preparation and standardization of methylglyoxal

solutions have been periodically required when continuing

glyoxalase I assays were being conducted.

A second objective was to develop new schemes for the

purifications of both glyoxalase enzymes employing affinity

chromatography as a major step. In this manner, it was

hoped that higher overall yields could be obtained, and any

alteration of enzyme protein resulting from the harsh

methods previously employed could be avoided.

A third objective involved a comparative study of the

structure and composition of the purified glyoxalase

enzymes, employing chemical, physical and kinetic charac-

terizations. It was anticipated that questions relating to

a possible structural relationship between the two

glyoxalases might be answered by the application of these

methods.

Finally, experiments were to be conducted which would

bring additional insights as to the biological function of

the glyoxalase system. One approach to be taken involved a

search for cellular metabolites which might affect the

activity of glyoxalases, either activators or inhibitors.

EXPERIMENTAL PROCEDURE

General. Swiss mice (24-27 g) were purchased from

Timco, Houston, Texas. Yeast glyoxalase I, hexokinase, glu-

coses-phosphate dehydrogenase, glucose phosphate isomerase,

a-glycerophosphate dehydrogenase, triosephosphate isomerase,

aldolase, phosphofructokinase, g-lactate dehydrogenase,

glutathione and oxidized glutathione were obtained from

Sigma Chemical Co., St. Louis, MO. 3-Hydroxy-3-methyl-

glutaryl-CoA reductase assays were conducted by Dr. R. E.

Thompson and co-workers, glycogen synthase and glycogen

phosphorylase assays by Dr. B. G. Harris and co-workers, and

S-adenosylmethionine synthetase and cyclopropane fatty acid

synthase assays by Dr. D. D. Smith. Pyruvaldehyde dimethyl

acetal and alkylamines were purchased from Aldrich Chemical

Co., Milwaukee, Wis.? semicarbazide•HC1 and imidazole were

obtained from Eastman Organic Chemicals, Rochester, N.Y.

Nucleotides, nucleosides and structurally related compounds

were purchased either from Sigma Chemical Co., St. Louis,

MO. or from Calbiochem, Los Angeles, CA.

S-g-Lactoylglutathione was prepared and purified by the

procedure of Uotila (67). S-Octylglutathione was prepared

by reacting 1-bromooctane with glutathione employing Method

A of Vince ejt a_l. (41). Lumicolchicine was prepared by

11

12

irradiation of colchicine with ultraviolet light utilizing

the method of Wilson and Friedkin (68).

All other chemicals were the highest purity obtainable

from commercial sources. All enzymes were assayed with

the usual assay procedures obtainable from the literature.

All chromatography columns were run at 4°, and enzyme

preparations were stored at -30°.

Methylglyoxal synthesis. The synthesis of methyl-

glyoxal (pyruvaldehyde) was accomplished by the hydrolysis

of pyruvaldehyde dimethyl acetal, as indicated below:

0 H + 0 0 II II II

CH.-C-CH(OCH0)0 > CH -C-CH

Pyruvaldehyde dimethyl acetal (10.0-605 pi, 0.083-5.02

mmol), 2.5 ml of 10% H2S°4' an

13

Alexander and Boyer (70) were employed to quantify

methylglyoxal.

Routine glyoxalase I assay. Glyoxalase I activity was

determined by a modification of the procedure of Racker

(12). The reaction mixture used was: 7.9 mM methylglyoxal,

1.0 mM glutathione, 200 mM imidazole•HC1 (pH 7.0), and 16 mM

MgSO^. The reaction mixture was allowed to stand for at

least 10 minutes at room temperature to ensure equilibration.

The hemimercaptal concentration was calculated to be 0.7 mM

using K = 3.1 mM (41). The enzymatic production of

S-g-lactoylglutathione ( E ^ = 3.37 mM - 1™" 1) was

followed at 240 nm for 2 minutes at 25° on a Beckman DBG

recording spectrophotometer. The reaction was initiated by

the addition of the enzyme preparation (1-20 jal) to 3.0 ml

of reaction mixture, and initial rates were determined by

the slope of the linear portion of the plot. The reference

cell contained all reaction mixture components with the

exception of the enzyme preparation. A unit (I.U.) of

glyoxalase I activity is defined as the amount of enzyme

catalyzing the formation of 1 jumol of S-g-lactoylglutathione

per minute in the routine enzyme assay system. Specific

activity is expressed as units per mg protein.

Routine glyoxalase II assay. The reaction mixture used

was: 0.5 mM S-D-lactoylglutathione in 100 mM potassium

phosphate (pK 7.0). The enzymatic disappearance of

14

S-D-lactoylglutathione ^240 ~ ^*37 ^ c m w a s

followed at 240 nm for 5 minutes at 25° on a Beckman DBG

recording spectrophotometer. The reaction was initiated by

the addition of the enzyme preparation (5-75 jal) to 3.0 ml

of reaction mixture, and the initial rates were determined

by the slope of the linear portion of the plot. A unit

(I.U.) of glyoxalase II is defined as the amount of enzyme

catalyzing the conversion of 1 jumol S-D-lactoylglutathione

per minute in the routine enzyme assay system.

Determination of protein concentration. Protein con-

centrations were determined by the colorimetric Coomassie

Blue procedure of Bradford (71). Crystalline bovine serum

albumin was used as the protein standard. The protein

levels in the effluent of the chromatography columns were

monitored by their absorbance at 280 nm.

Column material preparation. The preparations of

hydrophobic and affinity chromatography column materials

were based on the method of Cuatrecasas (72). To 100 ml of

washed Sepharose 4B, 25 g of finely divided cyanogen bromide

was added with stirring. The pH was maintained at 10.5 by

the addition of 5 N NaOH. The temperature was maintained

between 18 to 20° by the addition of ice to the reaction

mixture. When all cyanogen bromide was dissolved (12-15

minutes), the activated Sepharose was quickly washed with a

minimum of 10 volumes of cold 0.1 M sodium carbonate (pH

15

10.2). The step of washing the activated Sepharose was

accomplished in less than 90 seconds. For hydrophobic

chromatography columns, the appropriate n-alkylamine (3.6

mmoles) which was dissolved in 100 ml of wash buffer was

then added with gentle stirring to the activated Sepharose.

The mixture was stirred overnight at 5°. It was then

washed extensively with distilled water and placed in the

buffer used for chromatography. For the preparations of

affinity chromatographic column materials, oxidized

glutathione and S-octylglutathione were coupled to Sepharose

under the same conditions. The degree of substitution of

the ligands was not determined.

Purification of glyoxalase I. Swiss mice were

sacrificed either by asphyxiation in CO^ or by cervical

dislocation. The livers were immediately removed and

homogenized (0 for 45-60 seconds at medium speed with a

Virtis homogenizer) in three volumes of a solution of 1 mM

potassium phosphate (pH 7.0), 1 mM MgS04 and 20% glycerol.

The homogenate was centrifuged at 100,000 x g for 1 hour.

The supernatant fraction thus obtained (approximately 225 ml

per 100 g liver) was designated as the crude preparation of

glyoxalase I and contained 25-30 mg protein/ml. When the

livers were not used immediately they were frozen in dry ice

and then stored at -30°.

16

At the outset of glyoxalase I purification, sufficient

crude preparation to give 6.2 grams of protein was placed on

a 2.5 x 45 cm ethylamine-Sepharose (hydrophobic) column. The

column material had been previously equilibrated with a

solution of 1 mM potassium phosphate (pH 7.0), ImM MqSO 4

and 20-6 glycerol. After loading, the column was washed with

the equilibration solution until the concentration of

protein being eluted was quite low (monitored at 280 nm). A

linear phosphate gradient (1-50 mM, pH 7.0, a total of 7

liters) in 5 mM MgSO^ and 20% glycerol was used to elute

glyoxalase I. The phosphate concentrations were determined

by the method of Chen et al. (73).

Pooled active fractions from the ethylamine-Sepharose

column were then added to a 1.5 x 20 cm S-octylglutathione-

Sepharose affinity chromatographic column which had been

equilibrated with a solution of 10 mM potassium phosphate

(pH 7.0), 5 mM MgSC>4 and 20% glycerol. After sample

application, the column was washed with the equilibration

solution until no protein could be detected in the effluent.

The column was then treated with 400 ml of 50 mM imidaz-

ole *HC1 (pH 7.2) solution containing 5 mM MgSO^ and 20%

glycerol. Homogeneous glyoxalase I was then eluted by the

addition of the above imidazole buffer medium containing 20

mM glutathione.

17

Purification of glyoxalase II. Swiss mice were sacri-

ficed by cervical dislocation. The livers were immediately

removed and homogenized (0° for 2 minutes at medium speed

with a Virtis homogenizer) in two volumes of a solution of

10 mM potassium phosphate (pH 7.0) and 20% glycerol. The

homogenate was centrifuged at 100,000 x g for 1 hour. The

supernatant fraction thus obtained was designated as the

crude preparation of glyoxalase II. When the livers were

not used immediately they were frozen in dry ice and stored

at -30°.

Sufficient crude preparation to give 7.0 grams of

protein was placed on a 2.6 x 70 cm oxidized glutathione-

Sepharose affinity chromatographic column. The column

material had been previously equilibrated with a solution of

10 mM potassium phosphate (pH 7.0) and 20% glycerol. After

loading, the column was washed with the equilibration

solution until the bulk of the protein was eluted. The

column was then washed with 350 ml of a solution of 50 mM

potassium phosphate (pH 7.0) and 20% glycerol, and again

with the equilibration solution (600 ml). A gradient of a

competitive inhibitor of glyoxalase II, S-octylglutathione,

(1 mM to 5 mM, a total of 2 liters) prepared in the equil-

ibration solution, was used to elute glyoxalase II.

Pooled active fractions from the first column were

diluted two-fold with the equilibration solution. This

18

diluted enzyme preparation was then added to a 2.6 x 40 cm

oxidized glutathione-Sepharose affinity chromatographic col-

umn, which had been previously equilibrated with a solution

of 10 mM potassium phosphate (pH 7.0) and 20% glycerol.

After sample application the column was washed with the

equilibration solution/ then with 150 ml of a solution of 75

mM potassium phosphate (pH 7.0) containing 20% glycerol, and

again with the equilibration solution (250 ml). Homogeneous

glyoxalase II was then eluted by addition of a solution of

2.0 mM S-octylglutathione in the equilibration solution.

Concentration procedures. The pooled active fractions

from the affinity columns employed for the purifications of

both enzymes were placed in a Millipore 142 mm Hi-Flux Cell

ultrafiltrator. The enzymes were concentrated to a volume

of approximately 50 ml. Further concentrations of the

enzyme preparations were carried out with Millipore

immersible CX ultrafiltration units which have a molecular

weight cut-off of 10,000 daltons.

Molecular weight determination of glyoxalase I by

sedimentation equilibrium. Sedimentation equilibrium exper-

iments were conducted in a Beckman-Spinco Model E analytical

ultracentrifuge equipped with RTIC temperature control and

electronic speed control. The meniscus depletion method

(74,75) was used in the An-D rotor in a 12 mm double sector

cell with sapphire windows. Glyoxalase I (0.420 mg/ml) was

19

extensively dialyzed in K H2 P°4' 9/1* Na^HPO^, 4.3

g/l (pH 7.413); P=1.0020. Centrifugation was carried out

for 30 hours at speeds of 28,000 and 32,000 rpm.

Molecular weight determinations of glyoxalase enzymes

by sodium dodecyl sulfate polyacrylamide gel electro-

phoresis. For the molecular weight determinations of

glyoxalase I the method reported by Fairbanks et al. (76)

was employed. Gels were prepared with a 5.6% polyacrylamide

concentration in a solution of 40 mM Tris-HCl (pH 7.4), 20

mM sodium acetate, 2 mM EDTA and 1% sodium dodecyl sulfate,

and stained with 0.2% Coomassie Blue-R in an acetic acid/

methanol/water (8:46:46, v/v) solution. After staining for

at least 2 hours, the gels were destained in an acetic

acid/methanol/water (10:45:45, v/v) solution. Bovine serum

albumin (67,000), ovalbumin (45,000) and ribonuclease A

(13,700) were used as standards for molecular weight deter-

minations . Standards and samples were dialyzed against a

solution composed of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA and

0.1% 2-mercaptoethanol, they were made 2% in sodium dodecyl

sulfate and incubated at 100° for two minutes, before the

electrophoresis run.

For the molecular weight determinations of glyoxalase

II the method of Maizel (77) was employed. Gels were

prepared with a 7.5% polyacrylamide concentration in a

solution of 200 mM sodium phosphate (pH 7.0) and 1% sodium

20

dodecyl sulfate, and stained with 0.2% Coomassie Blue-R in

an acetic acid/methanol/water (8:46:46, v/v) solution.

After staining for at least 2 hours, the gels were destained

in an acetic acid / methanol / water (10:45:45, v/v)

solution. Ovalbumin (45,000), glyceraldehyde-3-phosphate

dehydrogenase (37,000), triose phosphate isomerase (26,500)

and hemoglobin (16,500) were used as standards for molecular

weight determinations. Before the electrophoresis runs,

samples and standards were dialyzed against 200 mM sodium

phosphate (pH 7.0) and 0.1% 2-mercaptoethanol, they were

made 2% in sodium dodecyl sulfate and incubated at 100°

for two minutes.

Polyacrylamide gel electrophoresis. Polyacrylamide gel

electrophoresis was performed according to the method of

Maizel (77). Gels were prepared with a 7.5% polyacrylamide

concentration in a solution of 2.5 mM Tris • HC1, 20 mM

glycine (pH 9.0). The staining and destaining of the gels

were carried out by the same procedures as described above

for the sodium dodecyl sulfate polyacrylamide gel electro-

phoresis experiments.

Isoelectric focusing. Isoelectric focusing was per-

formed in a LKB Multiphor Electrophoresis Unit according to

the instructions of the manufacturer. A pH gradient of

3.5-10 was used with 1% (w/v) ampholyte concentration.

21

Kinetic studies. It was assumed that the hemimercaptal

of methylglyoxal and glutathione is the true substrate of

glyoxalase I, and that the dissociation constant for the

hemimercaptal is 3.1 mM (41). The appropriate amounts of

both methylglyoxal and glutathione are added together to

form, nonenzymatically, the required hemimercaptal

concentration before the addition of the enzyme. Since

equilibrium solutions of hemimercaptal would also contain

glutathione and methylglyoxal, the concentrations of

glutathione and methylglyoxal after equilibration are

designated as free glutathione and free methylglyoxal.

Therefore, experiments utilizing hemimercaptal will state

not only hemimercaptal concentration, but also either free

glutathione or free methylglyoxal levels. For the exper-

iments involving the inhibition of glyoxalase II by the

hemimercaptal, the free glutathione level was 0.3 mM. This

level of glutathione does not inhibit the human glyoxalase

II (57).

RESULTS AND DISCUSSION

Methylglyoxal Synthesis. Methylglyoxal, for use in en-

zymatic studies, is usually obtained by vacuum (54) or steam

distillation (10) of a commercial 40% solution in order to

eliminate polymerized methylglyoxal. The pale green distil-

late may then be passed through an anion exchange resin

(54,78) to obtain acid-free methylglyoxal. Methylglyoxal is

then standardized by Friedemann's titration method (69)

after oxidation with H2°2' o r enzYmatically (79), or by

the semicarbazide method of Alexander and Boyer (70). This

tedious method of distillation, removal of acidic contami-

nants, and quantitation is time consuming. Furthermore,

methylglyoxal is very unstable and readily undergoes

polymerization and oxidation reactions (80). The true

methylglyoxal content of solutions stored for even short

time periods is questionable at best.

For studies involving repetitive assays of glyoxalase

I, a simple, convenient, and reproducible method for the

preparation of methylglyoxal is needed. The rapid and

direct method for the conversion of pyruvaldehyde dimethyl

acetal to methylglyoxal (81), which does not require a

quantitation analysis with each preparation, serves as a

22

23

convenient procedure for the preparation of methylglyoxal

for use in glyoxalase I assays.

The effect of temperature and time on the hydrolysis of

the acetal are shown in figures 1A and IB respectively.

From figure 1A, it is apparent that the hydrolysis proceeds

at an appreciable rate only at temperatures approaching

100°. As can be seen (Figure IB), the concentration of the

methylglyoxal produced (disemicarbazone assay) is highest

after a 25 minute hydrolysis. This time period was used in

all subsequent hydrolysis procedures. As subsequently

indicated, the hydrolysis reaction is virtually quantitative

when the process is carried out at 100° for 25 minutes.

The effect of H 2S°4 c o n c e n t r a tion on the hydrolysis of

pyruvaldehyde dimethyl acetal is shown in Figure 2. Five

percent H 2S 0

4 w a s chosen as the acid concentration for the

hydrolysis, since this was the lowest concentration that

gave essentially quantitative hydrolysis under the experi-

mental conditions employed.

The effect of increasing pyruvaldehyde dimethyl acetal

concentrations on the extent of hydrolysis was studied. As

shown in Figure 3, the response plot is linear up to an

acetal concentration of at least 450 mM. Each point in the

plot represents essentially a quantitative conversion to

methylglyoxal. It is probable that even higher levels of

24

Figure 1

A. Effect of Temperature on Hydrolysis of Pyruvaldehyde

Dimethyl Acetal

Pyruvaldehyde dimethyl acetal (200 pi, 1.65 mmole) was

hydrolyzed in a final volume of 5.0 ml of 5% H SO for 2 4

30 minutes. Ten pi of a 1:50 dilution of the hydrolysate

was added to 3.0 ml of 0.067 M semicarbazide•HC1 in 0.1 M

sodium phosphate ( PH 7.4). After 15 minutes, the absorbancy

at 286 nm was used to determine the methylglyoxal concen-

tration.

B. Rate of Hydrolysis of Pyruvaldehyde Dimethyl Acetal


hydrolyzed in a final volume of 5.0 ml of 5% H SO at ° _ 2 4

00 c. The conditions used to determine methylglyoxal

concentrations are given in the legend of figure 1A.

25

t CD

i in *

a 00

i CM •

1 T~ •

o • o o* o o O 9 9 2 w

Z

2

o o

26

Figure 2

Effect of Acid Concentration on Hydrolysis of

Pyruvaldehyde Dimethyl Acetal


hydrolyzed in a final volume of 5.0 ml at 100°C for 25

minutes in the presence of varying levels of . '^ie

conditions used to determine methylglyoxal concentrations

are given in the legend of figure 1A.

27

286

6 * H2SO4

28

Figure 3

Hydrolysis of Varying Levels of Pyruvaldehyde

Dimethyl Acetal

Each point represents a separate hydrolysis process. The

abscissa represents the acetal concentration in the hydro-

lysis mixture. The ordinate represents the methylglyoxal

concentration formed after the hydrolysis. Hydrolysis

experiments were carried out at 100°C for 25 minutes in a

final volume of 5.0 ml of 5% H2SC>4. The conditions used

to determine methylglyoxal concentrations are explained in

the legend of figure 1A.

29

£

%

o LU

X cr o LL

O

100 200

[ACETAL] , mM

30

acetal can be employed with a similar stoichiometric

conversion resulting.

In a separate study, triplicate samples from each of

six individual hydrolysates were quantitated for methyl-

glyoxal concentration by the semicarbazide method. The

original concentration of pyruvaldehyde dimethyl acetal in

each hydrolysis flask was 330 mM. After hydrolysis, the

concentration of the resulting methylglyoxal was found to be

329 mM (S.D. = +7). From these data it is apparent that

hydrolysis goes to completion.

Methylglyoxal prepared by this procedure was used for

the glyoxalase I kinetic studies. Two separate hydrolyses

and enzyme assays were conducted on two consecutive days,

and the response plot shown in Figure 4 is a composite of

those two experiments. It is obvious that a single curve

fits the data points obtained from both experiments.

In kinetic studies using glyoxalase I, substrate con-

centrations are usually calculated from the dissociation

constant of the hemimercaptal (41). The required substrate

concentrations are prepared by the addition of appropriate

amounts of both methylglyoxal and glutathione. It has been

found that methylglyoxal is very unstable, even when stored

under nitrogen in the cold. Consequently, the tedious and

cumbersome preparation, removal of acidic contaminants and

standardization of methylglyoxal solutions have been

31

Figure 4

Glyoxalase I Kinetic Study with Methylglyoxal Prepared

by the Hydrolysis of Pyruvaldehyde Dimethyl Acetal

The abscissa values represent the hemimercaptal concen-

tration derived from the equilibrium mixture of glutathione

and methylglyoxal. For each data point represented, the

methylglyoxal was obtained from separate hydrolyses of

pyruvaldehyde dimethyl acetal (varying concentrations).

Hydrolysate (0.25 ml) and an appropriate amount of glu-

tathione were diluted to a final volume of 10.0 ml. The

free glutathione level after the nonenzymatic equilibrium

reaction was 0.3 mM (see Experimental Procedure). Homo-

geneous glyoxalase I (4 I.U.) was used as enzyme source in

the assays. The plot is a composite of two separate

experiments conducted on consecutive days. • represents the

data obtained the first day; #the second day.

32

a 0 I# k 9 E 1 o z

(U|IU/VV)A

33

periodically required when continuing glyoxalase I assays

are being conducted. This method of stoichiometric

hydrolysis of pyruvaldehyde dimethyl acetal to methylglyoxal

is simple, reproducible, and permits the quick preparation

of fresh solutions of methylglyoxal of known concentrations

on a daily basis.

Purification of glyoxalase I. The purification of

Swiss mouse liver glyoxalase I was accomplished by use of

both hydrophobic and affinity chromatography. The choice of

a specific hydrophobic grouping for the first purification

step was made after a study of a variety of alkyl chains

covalently linked to Sepharose 4B. As the length of the

hydrophobic chain is increased, glyoxalase I binds more

tenaciously, and the concentrations of phosphate or imidaz-

ole buffers necessary to elute the enzyme increases greatly

(Table I). With hydrophobic columns prepared with alkyl

chains of six carbons or less, hemoglobin and other unde-

sired proteins were not bound, but passed through in the

washings. The ethylamine-containing hydrophobic column was

therefore chosen for use in the purification of glyoxalase

I, since the enzyme could be eluted at low ionic strengths

and much of the contaminating protein was not initially

bound, giving better purifications. The glyoxalase II

activity was eluted with the early fractions containing the

bulk of the protein.

34

TABLE I

HYDROPHOBIC CHROMATOGRAPHY OF GLYOXALASE I

b Eluting Agent

Liganda Liganda

Imidazole(M) Phosphate(M)

Ethylamine 0.02

n-Propylamine 0.1 0.08

n-Butylamine 0.4 0.2

n-Pentylamine 0.7 0.8

n-Hexylamine 1.0 1.5

n-Octylamine 2.0 -

n-Decylamine 4.0 —

Details of coupling of alkylamines to CNBr-activated

Sepharose are given in Experimental Procedure.

b The concentrations of imidazole and phosphate buffers

necessary to elute glyoxalase I from the corresponding

column materials.

35

A variety of glyoxalase I inhibitors which could

possibly serve as affinity chromatography ligands have been

prepared (20,41,82-84). S-(10-Aminodecyl)glutathione has

been successfully employed as a ligand for the affinity

chromatographic purification of glyoxalase I from DBA/lJ

mouse liver (55). Unfortunately, affinity columns prepared

with this ligand have low capacities for glyoxalase I. Fur-

thermore, these columns cannot be used repeatedly on crude

preparations because of the degradation of the glutathione

moiety by endogeneous glutathionase activity. In an effort

to minimize the latter problem, glutaryl-S-(10-aminodecyl)-

^-cysteinylglycine (83) was studied as a possible ligand for

affinity chromatography, but it was found to be totally

ineffective. It was therefore concluded that the covalent

linkage of the ligand to Sepharose should be with an -amino

group (not present in the glutaryl-containing compounds) for

effective affinity chromatography. S-Octylglutathione, a

potent inhibitor of glyoxalase I (41), was studied as an

affinity ligand and was found to be quite effective. The

capacity of the column material to bind glyoxalase I

activity is high, and the enzyme is specifically eluted in

good yield by additions of glutathione. A single pass of

the active fractions from the hydrophobic chromatography

through the S-octylglutathione-Sepharose column was

sufficient to purify glyoxalase I to homogeneity.

36

Table II summarizes the purification data of Swiss

mouse liver glyoxalase I. The 1162-fold purified enzyme

exhibited a specific activity of 944 units per mg protein.

The enzymatic activity has been shown to be associated with

a single protein species which has been purified to apparent

homogeneity. The above purification factor is based on a

100,000 x g supernatant fraction obtained from a crude cell

homogenate.

Figure 5 shows the protein concentration, the

glyoxalase I activity and the phosphate concentration

profiles obtained on an ethylamine-Sepharose hydrophobic

chromatographic column. The phosphate concentrations were

determined by the method of Chen et al̂ . (73). A phosphate

gradient was used to elute glyoxalase I activity. It should

be noted that better purifications were achieved by gradient

elution than by increasing the ionic strength in a stepwise

manner.

Affinity chromatography using S-octylglutathione as the

immobilized ligand proved to be invaluable as a purification

step. Figure 6 shows the protein concentration and

glyoxalase I activity profiles obtained on an S-octyl-

glutathione-Sepharose affinity chromatographic column. To

elute the glyoxalase I activity from the affinity column, 20

mM glutathione was required. This concentration of an

eluant may be considered quite high; however,

w h-3 CQ <

H w CO < 1

3 s o

fa o

£ o H Eh C u H fa H PS P fa

u 0 •p rH 0 rH 0 0 •H -P .p •H 03

o >i cn

ftUH

>1 -P

37

CN • O CN

V0

CN KO

o CN VQ • • •

1—1 o CN LO

CN cr>

i—i 00 v£> • • •

o V0 CO 1—1 as

•H • CO ĉ rH > D 00 00 r-*H • 00 r̂ ID •P H CN CN 0 —' <

d) E — o o LO 3 H CN 00 H £ CM CN in 0 — rH >

3 a) c rH c •H Cn

0 Q* e r-H *H (d >1 -P 0) rH •P 0 >1 0 fd rC O

-P 1 fa u W CO

38

Figure 5

Ethylamine-Sepharose Hydrophobic Chromatography of

Swiss Mouse Liver Glyoxalase I

Experimental details of chromatography and enzyme assays are

described in Experimental Procedure. Crude preparation (220

ml) was applied to a 2.5 x 45 cm preequilibrated ethylamine-

Sepharose column. Fraction volumes were 20 ml. The arrow

marks the application of potassium phosphate gradient (1-50

mM, pH 7.0). o — o , glyoxalase I activity, AA/min (20 pi of

each fraction was used in the assay); • •, protein concen-

tration, mg/ml; x — x , phosphate concentration, mM.

39

P R O T E I N ( m g / m l )

( u | u i / w )

A l l A i l D V I 3 S V 1 V X O A 1 9

40

Figure 6

S-Octylglutathione-Sepharose Affinity Chromatography

of Swiss Mouse Liver Glyoxalase I


described in Experimental Procedure. Pooled fractions (1280

ml) from the hydrophobic chromatography column were applied

to a 1.5 x 20 cm preequilibrated S-octylglutathione-

Sepharose column. Fraction volumes were 6.9 ml. The arrow

marks the application of glutathione (20 mM). o — o , glyox-

alase I activity, AA/min (10 pi of each fraction was used in

the assay); • — • , protein concentration, jag/ml.

41

PROTEIN ( p g / m l ) O 0 O

N

* CJ O O

(UJU3/VV) A1IAI1DV I 3SV1VX0A10

42

S-octylglutathione is a very potent inhibitor of glyoxalase

I (41), and it would be expected that relatively high

concentrations of glutathione would be needed to elute the

enzyme.

Polyacrylamide gel electrophoresis and sodium dodecyl

sulfate polyacrylamide gel electrophoresis were conducted on

the purified glyoxalase I. The amount of the enzyme applied

to the gels varied from 8 to 20 jig protein. The gels ex-

hibited only one band with both electrophoretic procedures;

no minor bands could be detected either visually or by

densitometric tracings of the gels.

In other studies, attempts were made to use affinity

chromatography as a one-step purification procedure starting

with the crude preparation; purifications up to 240 fold

have been achieved. Modifications of absorption and elution

conditions could be further investigated to improve this

purification factor.

Purification of glyoxalase II. The purification of

Swiss mouse liver glyoxalase II was accomplished by use of

affinity chromatography. Some glyoxalase I inhibitors, such

as various S-alkyl glutathiones, were studied as possible

affinity ligands. The column materials prepared with these

compounds do bind the glyoxalase II activity, but it has

been found that effective elution of the enzyme was not

possible. Oxidized glutathione was studied as an affinity

43

ligand and was found to be quite effective. Affinity

columns with this ligand readily bound glyoxalase II and had

a high capacity. The enzyme was selectively eluted with the

addition of 2 mM S-octylglutathione, a linear competitive

inhibitor of glyoxalase II (K^ =1.5 mM). The passing of

the enzyme preparation through the same kind of affinity

chromatographic column two times was sufficient to purify

glyoxalase II to homogeneity.

Table III summarizes the purification data of Swiss

mouse liver glyoxalase II. The overall yield obtained was

80% with a 2479-fold purification, and the enzyme exhibited

a specific activity of 920 units per mg protein. The

enzymatic activity has been shown to be associated with a

single protein species which has been purified to apparent

homogeneity. The above purification factor is based on a

100,000 x g supernatant fraction obtained from a crude cell

homogenate.

Figure 7 shows the protein concentration and the glyox-

alase II activity profiles obtained on the first oxidized

glutathione-Sepharose affinity chromatographic column. All

the glyoxalase I activity was eluted with the early

fractions containing the bulk of the protein. Elution of

glyoxalase II from the first column was accomplished by a

gradient of the linear competitive inhibitor, S-octylgluta-

thione. Before the addition of the pooled active fractions

H

W A CQ < Eh

H H

W CO <

J

3

o

fa o

25 O H Eh <

U H fa H &

D ft

u 0 -P a H rd rH fa rd

G CD O >

•H O -P rd a

•H MH •H ft U CD 3 •P ft CQ

' f t

1—i 0) 0) cN> -P

•rH '—' CO

rd O > i 1 ^

•H -p O) MH -H g •H > O *H • 1

rd -P 0 -P

-Prd •H ^ > •

H ID

a h

G o

•H •P a rd u fa

O o • •

rH CO ON r^

CN CN

CO

CN

If) G\

CN

o

LO CD

00

CN CO • • •

o o rH VD

ro VO i n o VD

rH rH rH

o o o CO vo LO 00 VO

G G G E 0 E

•H rH 4-> H 0 rd 0 O

a 1 rd i o ft a CO Q) CO CO U CO o ft o

o

a) -P G H3 CO 0

o JH •H Q) a fa CO

44 a 0

•H -P rd

-P G a) o G 0 u

rd

a> G 0

•H £

-P rd •P 3

CO a) rd

£ * > i i N >10 G -P g

•H 0 > £

•H •P i g o 0 -P v rd

i—i l

I 0 CO

LO

*0 (1) G

•H rd •P G O a

H -p

X •H e

•H -P a rd

u •rH MH •H O i ft rd ft

rd

0) TJ •P (D MH

> i O rd CO CO rd

CO 0) rd

H 0) •P rd e

•H X 0 u ft ft rd

CO •H

rd

45

Figure 7

First Oxidized Glutathione-Sepharose Affinity Chromatography

of Swiss Mouse Liver Glyoxalase II


described in Experimental Procedure. Crude prparation (380

ml) was applied to a 2.6 x 70 cm preequilibrated oxidized

glutathione-Sepharose column. Fraction volumes were 21 ml.

The arrow marks the application of the S-octylglutathione

gradient (1 to 5 mM) . o—o, glyoxalase II activity, -AA/min

(75 pi of each fraction was used in the assay); • • ,

protein concentration, mg/ml.

46

PROTEIN (mg/ml)

- o

e (uiui/VV)

AJLIMJOV II aSVlVXOAlO

47

to the second affinity chromatographic column, the fractions

were diluted two-fold to decrease the S-octylglutathione

concentration. The reason for the dilution of the pooled

active fractions was to allow binding of the enzyme to the

second affinity column since binding is prevented by 2 mM

S-octylglutathione. S-Octylglutathione also apparently

stabilizes glyoxalase II and complete removal of this

inhibitor by dialysis results in substantial loss of

activity.

Figure 8 shows the protein concentration and the glyox-

alase II activity profiles obtained on the second oxidized

glutathione-Sepharose affinity chromatographic column.

Homogeneous glyoxalase II was eluted with 2 mM S-octylgluta-

thione. The fractions which had high specific activities

were pooled and concentrated by ultrafiltration.

Although it is apparent that glyoxalase II binds to

oxidized glutathione-Sepharose (second purification step),

it is possible, because of the reduced glutathione present

in the crude liver preparations (1-2 mM) used in the first

purification step, that the Sepharose-bound oxidized

glutathione can be reduced to reduced glutathione residues.

Consequently, glyoxalase II binding could occur with both

Sepharose-bound reduced and oxidized glutathiones. However,

even after repeated use of the same affinity chromatographic

column material with crude liver preparations, no change in

48

Figure 8

Second Oxidized Glutathione-Sepharose Affinity

Chromatography of Swiss Mouse

Liver Glyoxalase II


described in Experimental Procedure. Pooled and diluted

fractions (920 ml) from the first column were applied to a

2.6 x 40 cm preequilibrated oxidized glutathione-Sepharose

column. Fraction volumes were 18.4 ml. The arrow marks the

application of S-octylglutathione (2 mM). o — o , glyoxalase

II activity, -AA/min (75 pi of each fraction was used in the

assay); • — • , protein concentration, pg/ml.

49

P R O T E I N ( p g / m l )

( u i u i / w )

A 1 I A I O V I I 3 S V 1 V X O J 1 1 9

50

glyoxalase II binding characteristics was observed. Further,

pretreatment of the column material with several volumes of

1 mM dithiothreitol does not change the glyoxalase II

binding or elution patterns. After such usages or pre-

treatments, an increase of Sepharose-bound sulfhydryl groups

cannot be detected by use of Ellman's reagent (85). It

would thus appear that the dithio group of Sepharose-bound

oxidized glutathione is not readily reduced.

Polyacryamide gel electrophoresis and sodium dodecyl

sulfate polyacrylamide gel electrophoresis were conducted on

the purified glyoxalase II. The amount of the enzyme

applied to the gels varied from 8 to 20 pg protein. The

gels exhibited only one band with both electrophoretic

procedures; no minor bands could be detected either visually

or by densitometric tracing of the gels.

Molecular weight determinations. The weight-average

molecular weight of native mouse liver glyoxalase I was

estimated by the meniscus depletion method of sedimentation

equilibrium ultracentrifugation. A plot of log(y-yQ)

2

versus r is shown in Figure 9. The linearity of the plot

is additional evidence for the homogeneity of the enzyme

preparation. The partial specific volume of glyoxalase I

was taken as 0.728, determined from the partial specific

volumes of the component amino acids (55). The molecular

weight determined by sedimentation -equilibrium, 43,000

51

Figure 9

Sedimentation Equilibrium Ultracentrifugation

of Glyoxalase I

Experimental details are given in Experimental Procedure.

Ultracentrifugation was carried out at 14.4°c at 28,000

rpm for 30 hours. The abscissa represents the square of the

distance (cm) from the center of rotation. The ordinate

represents the log of the fringe displacement in mm.

53

daltons, agrees well with the value reported previously for

the enzyme from the same source (55).

Estimation of the molecular weight of mouse liver

glyoxalase I by sodium dodecyl sulfate polyacrylamide gel

electrophoresis using bovine serum albumin, ovalbumin and

ribonuclease A as molecular weight standards, gave a value

of 21,500 daltons (Figure 10). From the sedimentation

equilibrium and sodium dodecyl sulfate polyacrylamide gel

electrophoresis experiments, it has been concluded that

mouse liver glyoxalase I has a molecular weight of approx-

imately 43,000 and is a dimer composed of apparently

identical subunits.

A molecular weight estimation of mouse liver glyoxalase

II was conducted by sodium dodecyl sulfate polyacrylamide

gsl electrophoresis using ovalbumin, glyceraldehyde-

3-phosphate dehydrogenase, triosephosphate isomerase and

hemoglobin as molecular weight standards. The resulting

experiments gave a value of approximately 29,500 daltons.

Uotila estimated the molecular weight of human liver

glyoxalase II to be 22,900 using gel filtration (57). In

the light of this study, mouse liver glyoxalase II appears

to be a single polypeptide with a molecular weight of

approximately 29,500 (Figure 11)• Isoelectric focusing

experiments were conducted on the homogeneous mouse liver

glyoxalase II, and a pi value of approximately 8.1 was

54

Figure 10

Molecular Weight Determination of Glyoxalase I by Sodium

Dodecyl Sulfate Polyacrylamide Gel Electrophoresis


Molecular weight on a log scale versus relative distance of

migration is plotted. Standards: Bovine serum albumin

(BSA); ovalbumin (OVAL); ribonuclease A (RNase A). The

migration of sodium dodecyl sulfate-treated glyoxalase I

(G-I) indicates an apparent molecular weight of approx-

imately 21,500 daltons.

56

Figure 11

Molecular Weight Determination of Glyoxalase II by Sodium

Dodecyl Sulfate Polyacrylamide Gel Electrophoresis


Molecular weight on a log scale versus relative distance of

migration is plotted. Standards: Ovalbumin (OVAL);

glyceraldehyde-3-phosphate dehydrogenase (G3PDH); triose-

phosphate isomerase (TPI); and hemoglobin (Hb). The

migration of sodium dodecyl sulfate-treated glyoxalase II

(G-II) indicates an apparent molecular weight of approx-

imately 29,500 daltons.

58

obtained. Uotila reported a pi value of 8.35 for the human

liver enzyme (57).

Inhibition of glyoxalase enzymes by nucleotides, nu-

cleosides and a series of structurally related compounds.

Nucleotides, nucleosides and some structurally related

compounds are potent inhibitors of both glyoxalase enzymes

derived from different sources (86). Figure 12 shows the

activity response obtained for pure mouse liver glyoxalase I

using GTP, ATP, CTP and UTP. Similar results (cooperative

inhibitions) were obtained for pure mouse liver glyoxalase

II. As an example, Figure 13 shows the effect of GTP on

both enzymes; the inhibitions have almost identical

sigmoidicities.

S-Octylglutathione is a linear-competitive inhibitor of

the glyoxalase enzymes, and it has been used as an affinity

ligand for the purification of mouse liver glyoxalase I (54),

as well as an eluant medium for mouse liver glyoxalase II

purification from the affinity chromatography columns (58).

The glyoxalase I inhibition response curve given by S-octyl-

glutathione is quite different from the response curve given

by nucleotides as shown in Figure 14 for GTP. When a Hill

plot, log[%Inh./ (100- %Inh.)] versus log[GTP], (87) is

constructed with the data of Figure 14, a Hill coefficient

(slope) of 3.3 is obtained for the GTP inhibition (Figure

59

Figure 12

Inhibition of Glyoxalase I by GTP, ATP, CTP and UTP

The calculated hemimercaptal concentration is 0.2 mM. The

free glutathione concentration, after equilibration, is 1.55

mM. Glyoxalase I used in this study was the homogeneous

enzyme preparation obtained from mouse liver (54). • •,

GTP; • • , ATP; o o, CTP; • — • , UTP.

60

o> 80

a>

% 40 o a ^ 20

100 200 300 400 500 [ILjjM

61

Figure 13

Inhibition of Glyoxalase Enzymes by GTP



mM. The S-£-lactoylglutathione concentration for the glyox-

alase II assays is 0.4 mM. Both glyoxalases were obtained

from mouse liver (54,58). • — g l y o x a l a s e I; A A ,

glyoxalase II.

62

o> 80

o O

100 150 200 250 [GTPL/jM

63

Figure 14

Inhibition of Glyoxalase I by GTP and

S-Octylglutathione



mM. The glyoxalase I used in this study was the homogeneous

enzyme preparation obtained from mouse liver (54). • •,

GTP; A A, S-octylglutathione.

64

100 150 200 250 [ILpM

65

Figure 15

Hill Plot for the Inhibition of Glyoxalase I by GTP

The data from Figure 14 were used to construct the plot. A

value of 3.3 was obtained from the slope of the line.

66

c o

I o o

CD o

1.2 1.4 1.6 13 2.0 log[colchicine]

67

15). In a binding study the Hill coefficient will theo-

retically establish the minimum number of sites.

Inhibitions of the mouse liver glyoxalase enzymes by

some nucleic acid bases, nucleosides, nucleotides and a

series of structurally related compounds are summarized in

Table IV. 1 ^ values (the concentrations of the inhib-

itors which give 50% inhibition) for each inhibitor are

given; all compounds listed in the table inhibit both

glyoxalase enzymes in a cooperative manner. Within a given

family of nucleic acid bases, there is no difference in the

extent of inhibitions given by the free base, the corre-

sponding nucleoside, or the nucleoside 5'-mono-, di- or

triphosphates. In addition to the inhibitors listed in

Table IV, pyridine nucleotides, cAMP and cGMP were also

studied. Significant inhibitions (35-60%) of both glyox-

alase enzymes were given by these nucleotides at 150 pM

concentrations. In general, aromatic compounds which

contain heterocyclic nitrogen and/or have amino groups on

the aromatic ring, are effective cooperative inhibitors of

both glyoxalase enzymes. Compounds not having these

structural features (e.g., tyrosine, phenylalanine, histi-

dine, arginine, imidazole) do not inhibit either enzyme,

even at concentrations above 2 mM.

Comparisons of the inhibition by nucleotides of both

glyoxalase enzymes from different sources were undertaken.

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69

There are no significant differences between the inhibition

of mouse liver glyoxalase I and the yeast enzyme. Nucleo-

tides are comparatively better inhibitors of the glyoxalases

from Pseudomonas fluorescens than of these enzymes from

mouse liver. Interestingly, the inhibition of glyoxalase II

from the bacterial source does not exhibit sigmoidal

kinetics.

Since intracellular levels of some of the nucleotides

(88, 89), for example ATP, are significantly higher than the

concentrations needed to inhibit both enzymes completely

(0.5 mM), the inhibitions observed may have physiological

significance. The glyoxalases are either completely in-

active in vivo (there is experimental evidence to the

contrary (90)), or they are active because of isolation from

inhibitors through compartmentalization. An alternate ex-

planation is that there is a variable endogeneous activator

which reverses the inhibition of the enzymes, thereby

regulating glyoxalase activities. The identification of

such an activator would provide much information about the

function of the glyoxalase enzymes.

Inhibition of Glyoxalase Enzymes by Microtubule Poisons.

As previously mentioned, it was shown that nucleotides,

nucleosides and some structurally related compounds were

potent, cooperative inhibitors of both glyoxalase enzymes

(86). The biological significance of these inhibitions is

70

not readily apparent. It was found that certain microtubule

poisons (colchicine, vinblastine, podophyllotoxin and

griseofulvin) also inhibit both glyoxalases in a cooperative

manner. As an example, in Figure 16, the inhibitions of

homogeneous mouse liver glyoxalase I and glyoxalase II by

colchicine are shown, and the inhibitions have almost

identical sigmoidicities. When a Hill plot is constructed

for the glyoxalase I response curve (Figure 17), a value of

3.7 (from the slope) was obtained for the Hill coefficient.

The Hill coefficients were also similarly determined for

glyoxalase I inhibitions by vinblastine, podophyllotoxin and

griseofulvin, and are 3.6, 3.4 and 3.1, respectively.

Figure 18 shows the Lineweaver-Burk plot for the inhibition

of glyoxalase I by colchicine. A Dixon plot of the data

indicated that colchicine is a nonlinear-competitive

inhibitor. Plots derived from data of glyoxalase II inhi-

toition toy colchicine are quite similar to those shown in

Figures 17 and 18 for glyoxalase I inhibition.

After the observation of the inhibition of glyoxalases

by the microtubule poisons, the questions arose: What is the

specificity of the inhibitions observed? Are these micro-

tubule poisons general enzyme inhibitors? As an approach to

answering these questions, the effects of colchicine,

vinblastine, podophyllotoxin and griseofulvin on various

enzymes from a variety of sources were examined, and Table V

71

Figure 16

Inhibition of Glyoxalase Enzymes by Colchicine



mM. The S-D-lactoylglutathione concentration for the glyox-

alase II assays is 0.4 mM. Both glyoxalases were obtained

from mouse liver (54,58). • •, Glyoxalase I; o o,

glyoxalase II.

72

o> c 80 c o £ CD 60 >*

• a m >

73

Figure 17

Hill Plot for the Inhibition of Glyoxalase I by Colchi cine

The data from Figure 16 was used to construct the plot. A

value of 3.7 was obtained from the slope of the line.

74

' 0.5

• a - 0 5 o

12 1.4 1.6 15 log[colchicine]

75

Figure 18

Lineweaver-Burk Plot of the Effect of Colchicine

Concentration on the Glyoxalase I Activity

The assay method is described in Experimental Procedure.

Velocity is in terms of pioles of S-g-lactoylglutathione

produced per minute. The free glutathione concentration,

after equilibration, is 1.55 mM, for each point. The

hemimercaptal concentration was varied with the following

constant levels of colchicine: 0 (•), 30 jaM (•), 40 pM (A),

45 JJM (o) , 50 JAM (•) .

76

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78

summarizes the results# The levels of these drugs tested

are the concentrations which gave 50% inhibition (I 50

values) for the glyoxalase enzymes. It was found that only

the glyoxalases were inhibited by these microtubule poisons,

and that no significant inhibition could be detected for any

of the other enzymes investigated at the inhibitor

concentrations employed.

Tubulin is known to have two binding sites for guano-

sine nucleotides (64) and tubulin also binds the drugs

tested. It is therefore, interesting to note that some of

the compounds known to bind tubulin and/or affect micro-

tubule assembly also inhibit both glyoxalase enzymes. It is

also known that lumicolchicine does not bind tubulin (91);

therefore, experiments were conducted to determine the

effects of lumicolchicine on the glyoxalase enzymes. As

shown in Figure 19, the inhibition of glyoxalase I by

lumicolchicine is not sigmoidal, and significantly less

inhibitory activity is exhibited by it as compared to

colchicine. It is apparent that lumicolchicine binds to a

noncatalytic site on dimeric glyoxalase I (54), producing an

altered enzyme, since a maximum of only 50% inhibition is

obtained. Lumicolchicine, on the other hand, does not

inhibit glyoxalase II to any appreciable extent.

On the basis of the inhibitions exhibited by micro-

tubule poisons on glyoxalase I and glyoxalase II, it is

79

Figure 19

Inhibition of Glyoxalase Enzymes by Colchicine

and Lumicolchicine

The calculated hemimercaptal concentration for glyoxalase I

assays is 0.2 mM. The free glutathione concentration, after

®ciuilibration, is 1.55 mM. The S—D— lactoylglutathione

concentration for glyoxalase II assays is 0.4 mM. m • ,

inhibition of glyoxalase I or glyoxalase II by colchicine;

• •, inhibition of glyoxalase I by lumicolchicine;

the effect of lumicolchicine on glyoxalase II.

80

!> 40

20 40 60 80 [Colchicine or LumicolchicineLjjM

81

tempting to speculate that these two enzymes may have some

involvement in the microtubule assembly process. Such spec-

ulations have appeared in the literature (60-62). It has

been suggested that the thioester, S-D-lactoylglutathione

(the product of glyoxalase I and the substrate of glyoxalase

II), may have a significant role in the formation of the

mitotic apparatus of dividing eucaryotic cells (62). In

relation to the postulate that a-ketoaldehydes have a

special function as regulatory molecules in cell division

(32,33,35), concanavalin A, a cell-division stimulator, has

been shown to increase the activity of both glyoxalase

enzymes in lymphocytes and polymorphonuclear leukocytes

(61). It is apparent that the intracellular concentrations

of both glutathione and S-D-lactoylglutathione could well be

affected by such an activation process. There is also

evidence in the literature that the glyoxalase system may

regulate microtubule assembly in vitro (60). The effect of

low levels of S-D-lactoylglutathione on the assembly of

purified tubulin was determined, and it was found in a

number of experiments to increase the rate of microtubule

assembly. In addition, it has been demonstrated (62) that

S-D-lactoylglutathione enhances the rate of anti-IgE-induced

histamine release from human leukocytes - a secretory

process believed to require an intact microtubule assembly

(92).

82

In light of this study, it is concluded that the rather

specific inhibitions of the glyoxalases by microtubule

poisons may have metabolic significance. However, the

evidence for a proposed functional relationship between the

glyoxalase enzymes and microtubule assembly is largely

circumstantial at this point. Further investigations in

this direction may bring into light the biological function

of glyoxalases and a better understanding of the regulation

of microtubule assembly-disassembly processes.

Kinetics. Excessive free glutathione concentrations

were found to be inhibitory to mouse liver glyoxalase I

activity (23). Mannervik and coworkers (24) have subjected

several steady-state kinetic models to linear and nonlinear

regression analyses. It was concluded that the kinetics of

glyoxalase I are best described by a model including alter-

nate one- or two-substrate pathways. Except at very low

hemimercaptal and free glutathione concentrations, the one-

substrate pathway predominates. At higher hemimercaptal and

free glutathione concentrations the one-substrate pathway

model predicts a hemimercaptal-free glutathione competition.

The effects of varying concentrations of free glutathione on

the reaction kinetics of glyoxalase I were studied. As

shown in Figure 20 free glutathione was indeed a competitive

inhibitor. This effect has been previously investigated

(93), and it was concluded that free glutathione was

83

Figure 20

Lineweaver-Burk Plot of the Effect of Glutathione



Velocity is in terms of jamoles of S-D-lactoylglutathione

produced per minute. The hemimercaptal concentration was

varied with the following constant levels of free gluta-

thione: 0.3 mM (•), 3.0 mM (•), 6.0 mM (A), 8.0 mM (o),10.0

mM (A) .

84

2 4 6 8 10 12

1 / [ H e m i m e r c a p t a l ] , m M

85

essentially competitive with the hemimercaptal for mouse

liver glyoxalase I.

S-Octylglutathione, which was used as the affinity

ligand in the purification of mouse liver glyoxalase I, was

shown to be a potent inhibitor of glyoxalase I (41).

Examination of the Lineweaver-Burk plot shown in Figure 21

reveals that S-octylglutathione is a linear-competitive

inhibitor of glyoxalase I. By using the graphical method of

Dixon (94), the for S-octylglutathione was determined

to be 0.06 mM.

Experiments were carried out with varying free gluta-

thione and S-octylglutathione concentrations at constant

levels of hemimercaptal to determine whether kinetically

distinguishable binding sites are available simultaneously

for glutathione and S-octylglutathione. The parallel lines

obtained in the plot of l/v versus S-octylglutathione

concentrations (Figure 22) indicate that these two compounds

are mutually exclusive as effectors of the enzymatic activ-

ity (95). The fact that glutathione and its S-substituted

derivative are not only mutually exclusive, but also indi-

vidually compete with the hemimercaptal, suggests that the

site of binding is the active site of glyoxalase I. Similar

experiments were conducted to determine the effects of

various fixed concentrations of S-octylglutathione on the

glyoxalase I activity at increasing levels of free

86

Figure 21

Lineweaver-Burk Plot of the Effect of S-Octylglutathione



Velocity is in terms of pmoles of S-p-lactoylglutathione


varied with the following constant levels of S-octylglu-

tathione: 0 (•), 50 pM (•), 100 pM (A), 150 pM (o), 200 pM

(•).

87

l/[Hemimercaptal],mM

88

Figure 22

Effect of Various Fixed Concentrations of S-Octylglutathione

on the Glyoxalase I Activity at Increasing Levels of Free

Glutathione with a Constant Level of Hemimercaptal


Velocity is in terms of pmoles of S-D-lactoylglutathione


constant at 0.3 mM for all points. S-Octylglutathione

concentrations were: 0 (•), 25 pM (•), 50 pM (A), 75 pM (o),

100 >iM (A).

89

[GSHf],mM

90

methylglyoxal in the presence of a constant level of hemi-

mercaptal. From the plot of l/v versus free methylglyoxal

concentration (Figure 23), which results in parallel lines,

it is concluded that these two compounds are mutually

exclusive as effectors of the enzymatic activity.

As shown in Figure 24, parallel lines were obtained

when l/v versus colchicine concentration were plotted with

varying levels of S-octylglutathione in the presence of a

constant level of hemimercaptal. This indicates that col-

chicine is also mutually exclusive with S-octylglutathione.

Glutathione, methylglyoxal, S-octylglutathione and

colchicine appear to bind the active site of glyoxalase I,

and this conclusion can be substantiated by the observations

that both glutathione and S-octylglutathione compete with

the hemimercaptal, and that both free methylglyoxal and

colchicine are mutually exclusive with respect to S-octyl-

glutathione. When free glutathione is tested with various

fixed concentrations of colchicine at a constant level of

hemimercaptal (Figure 25), the lines are not parallel, but

they cross on the vertical axis. The first interpretation

of the data would be that colchicine and S-octylglutathione

are not mutually exclusive. When two inhibitors can bind to

the same enzyme molecule (when they are not mutually

exclusive) the lines would be expected to cross somewhere to

the left of the vertical axis (96). One way of interpreting

91

Figure 23


on the Glyoxalase I Activity at Increasing Levels of Free

Methylglyoxal with a Constant Level of Hemimercaptal


Velocity is in terms of pinoles of S-D-lactoylglutathione



concentrations were: 0 (•) , 25 jjM (A), 50 pM (•), 100 pM

(A).

93

Figure 24


on the Glyoxalase I Activity at Increasing Levels of

Colchicine with a Constant Level of Hemimercaptal


Velocity is in terms of pmoles of S-g-lactoylglutathione



concentrations were: 0 (•) , 25 juM (•) , 50 pM (A), 75 pM (o) ,

100 pM (•).

94

[Colchicine] , j jM

95

Figure 25

Effect of Various Fixed Concentrations of Colchicine on

the Glyoxalase I Activity at Increasing Levels of Free

Glutathione with a Constant Level of Hemimercaptal


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