Insect glutathione S-transferase: Separation of transferases from fat bodies of American cockroaches active on organophosphorus triesters

PESTICIDE

RIOCHEMISTRY

Insect

AND

7, 249-260

PHYSIOLOGY

(1977)

Glutathione S-Transferase: from Fat Bodies of American on Organophosphorus KENJI

USUI

AND

Separation of Transferases Cockroaches Active Triesters

JUN-ICHI

FUKAMI

Laboratory of Insect Toxicology, The Institute Chemical Research, Wako-shi, Saitama

of Physical 351, Japan

and

AND TAKASHI National

Institute

of Agricultural

Received

June

SHISHIDO Sciences,

8, 1976;

Nishigahara,

accepted

August

Tokyo

114, Japan

3, 1976

Several glutathione S-transferases which catalyze the conjugation of reduced glutathione with organophosphorus triesters were separated from fat bodies of adult female American cockroaches, Periplaneta americana (L.). Two transferases (I, V) were active on diazinon and three transferases (II, III, IV) were active on methyl parathion. The transferase (I) active on the pyrimidinyl moiety of diazinon was distinguishable from the other transferases on the Omethyl portion of methyl parathion, as shown by chromatographic properties, and additionally it was almost inactive or less active on 3,4-dichloronitrobenzene, methyl iodide, p-nitrobenzyl chloride, trans-cinnamaldehyde, and 1,2-epoxy-3-(p-nitrophenoxy)propane. Transferase II had high activities with “aryl” and “aralkyl” compounds, transferase III with “epoxide” and “alkene,” and transferase IV with “alkyl,” “aryl,” and “aralkyl” compounds. Thii indicated that the transferases had overlapping substrate specificities. The molecular weight was 35,00037,000 for both of the enzymes active on methyl parathion and diazinon. The pH optima with methyl parathion and diazinon were about 8.5 and 6.5, respectively. At a glutathione concentration of 5 mM, Michaelis constants were 0.28 and 0.13 mM for methyl parathion and diazinon, respectively.

S-aryl (2, 3), alkyl (4), aralkyl (5), alkene (6), epoxide (7, 8), and phosphoric acid triester alkyltransferase (9). Recently, Habig et al. (10) separated and purified several glutathione transferases from rat liver and showed that each of the enzymes had overlapping substrate specificities, i.e., several enzymes were active on a specific compound, and also that each enzyme was active on several different types of compounds. It was concluded that the enzymes were not classified in accord with the designations described by Boyland and Chasseaud (1). This con-

INTRODUCTION

Glutathione S-transferases (EC 2.5.1.18) play a physiologically important role in initiating the detoxication of foreign compounds, including toxicologically active pesticides. These enzymes are classified (1) on the basis of the transfer group from the substrate molecule to the sulfur atom of reduced glutathione (GSH)’ to glutathione 1 Abbreviations used: GSH, reduced glutathione; DCNB, 3,4-dichloronitrobenzene; OD, optical density; e, molar extinction coefficient; &,,ax, wavelength of maximum absorption; K,, Michaelis constant; velocity. and Max, maximal 249 Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in my form reserved.

ISSN

0948-3575

250

USUI,

PUKAMI

elusion is part~iall~~ wpportcitl 1)5, dat,a report’cd in Rcfs. (11-15). Fukami and Shishido (16, 17), working on organophosphorus tricstcrs, found that methyl parathion, methyl paraoxon, and fenitrothion Tvcrc degraded by soluble fractions from the tissues of mammals and insects, in the presence of GSH, to form the demethyl derivatives. These workers also investigated some propcrtics of these mzymes. Diazinon was found to conjugat,c with GSH at the pyrimidinyl moiety (IS). Many investigations have confirmed that mevinphos (19), azinphosmethyl (20, al), dichlorvos (22), tetrachlorvinphos (9), dimethyl-p- (methyl sulfonyl)phenyl phosphate (23), prophos (24), and parathion (25-27) as well as methyl parathion, methyl (16, 17, 28), and paraoxon, fenitrothion diazinon (18, 27, 29) conjugate with GSH. It has been documented that some compounds conjugate in two ways: Methyl parathion, parathion, and their oxygm analogs conjugate with GSH at both the 0-alkyl portion (16, 17, 25-28) of the molecule and the p-nitrophenyl moiety (25, 30), and diazinon conjugates at the pyrimidinyl moiety (18, 29, 30) and at the O-ethyl portion (27, 30). However, it has not been defined whether these two modes of rcaction arc catalyzed by the same cnzymc or by different enzymes. With insects there have been several investigations on GSH conjugation with halogenated bcnzyl or phenyl compounds and hexachlorocyclohexanes (5, 11, 31, 32). Recently, glutathione X-transfcrases have been implicated as a cont’ributing factor of insecticide resistance by insects (20, 21, 23, 26, 27, 30, 33). However, few enzymological studies on glutathione S-transferases in insects have been conducted (31, 32). Therefore, investigations utilizing the fat bodies of American cockroaches were undertaken t,o characterize the transferases catalyzing the conjugation of GSH with organophosphorus triesters and to compare them with t,he

AND

SHISHIDO

MATEItIALH

AND

METHODS

Itmcfs Adult

American rockroaches (L.)) reared at 26% were used for thcsc st#udics. (Periplaneta

female

americaua

Chemicals

[L4C]nwt~hyl-labelcd methyl parathion (1.S3 mCi/mmol, Sumit,omo Radiochemical Co., Ltd., Osaka, Japan) with a radiochemical purity of 99% and [l-‘“Clethyl-labeled diazinon (0.68 mCi/mmol, Sumitomo Chemical Co., Lt’d., Osaka, Japan) with a radiochemical purity of 99% were kept as stock solutions in n-hexane. Unlabeled insecticides were obtained from the following sources: methyl parathion from Sumitomo Chemical Co., Ltd., Osaka, Japan; diazinon from Nippon Kayaku Co., Ltd., Tokyo, Japan. GSH was purchased from Boehringer Mannheim GmbH, Mannheim, Germany. Srphadex G-100 and G-75 wre obtained from Pharmacia Fine Chemicals, Uppsala, Sweden, and Ampholine carrier ampholyte from LKB Produktrr AB, Stockholm, Sweden. Collodion bags used as membrane filters for ultrafiltration were obtaincad from Sartorius-Membranfilter GmbH, Gijttingcn, Germany. ‘2-Isopropyl-4-methyl-6-chloropyrimidine was prepared from the corresponding 6hydroxypyrimidine and phosphorus oxychloride (34), and 2-chloro-4,6-diphenoxys-triazinc was prepared from cyanuric chloride and sodium phenoxide (35). 1,2Epoxy-3- (p-nitrophcnoxy)propane was prepared by reacting cpichlorohydrin and p-nitrophenol (36). Other chemicals were of guaranteed purity or of the highest purity commercially available. Assay

of Enzyme

Activities

Unless ut’hcrwisc nwntioned, assays wtrc performed at 3O”C, and the wnct.ion mix-

INSECT

GLvTATHI~NE

ture without enzyme or GSH was used as a control. 1. 14C-labeled methyl parathion at&d diazinon. In a lo-ml stoppered tube, the reaction mixture consisted of 0.3 mM labeled insecticides, 5 mM GSH, 0.05% Triton X-100, 1% ethanol, 0.05 M phosphate buffer, pH 8.0 for methyl parat,hion and pH 6.5 for diazinon, and enzyme solution in a total volume of 2 ml. The mixture was incubated for 60 min and immediately transfered into boiling water for 1 min and then cooled in ice water. Unchanged radioactive insecticide was removed by extraction with chloroform and the radioactivity of the aliquot of the water-soluble fraction was determined as the amount of formed conjugate in 10 ml of scintillation cocktail (3.5 g of 2,5diphenyloxazole, 0.1 g of 1,4-bis[2-(4methyl-5-phenyloxazolyl)]benzene, 50 g of naphthalene, and 1000 ml of dioxane) (16, 18, 41). Less than 5% of the radioactivity of the substrate added was recovered in the water-soluble fraction. 2. Diazinon for the conjugation at the pyrimidin yl moiety. The incubation mixture was the same as for labeled diazinon. The reaction was stopped by the addition of 0.4 ml of 20% trichloroacetic acid. The denatured protein was removed by centrifugation. The optical density (OD) at 296 nm, X,,, of the conjugate S-(2-isopropyl-4-methyl-6-pyrimidinyl)glutathione in an acidic solution (c = 11.4 mM-’ cm-‘), was measured with a Hitachi 124 spectrophotometer (18). The amount of formed conjugate was less than 10% of the substrate added. 3. S,.&Dichloronitrobenzene (DCNB). The reaction method was essentially the same as for diazinon, but the concentration of DCNB was 0.5 mM. The pH of phosphate buffer was 7.4 and the incubation time was 30 min. The reaction was stopped by cooling in ice and the increase in OD at 340 nm was measured (2, 10). 4. Other substrates. Except for N-hetero-

S-TRANSFERASES

251

cycles, the enzyme activity was determined by measuring the disappearance of GSH in the reaction mixture using 5,5’-dithiobis-(2nitrobenzoic acid), according to Ellman (37). With N-heterocycles the enzyme activities were determined by measuring the increase in OD at x,,, of the conjugate : 296 nm for 2-isopropyl-4-methyl-6-chloropyrimidine (38), 254 nm for 2-chloro-4,6diphenoxy-s-triazine (38), and 295 nm for p-nitropyridine N-oxide (10, 39). The reaction method was essentially the same as for DCNB and incubation time was 30 min. The other reaction conditions are shown in Tables 2 and 3. SpeciJic Activity Specific activity was defined as the enzyme activity catalyzing the conjugation of the substrate (nanomoles per minute) with GSH per milligram of protein determined by the method of Lowry et al. (40), with bovine serum albumin as a standard. Preparation

of Enzymes

Unless otherwise mentioned, all procedures were carried out below 5°C. 1. Centrifugation. Fat bodies from adult female American cockroaches were collected by dissection. The tissue was homogenized with a glass homogenizer in 3 vol (v/w) of 0.25 M sucrose-0.01 M EDTA-0.05 M phosphate buffer (pH 7.4) and centrifuged at 105,OOOg for 1 hr. The supernatant obtained was filtered through a plug of absorbent cotton to remove floating fat. 2. Ammonium sulfate fractionation. To this clear supernatant fluid, solid ammonium sulfate was gradually added with stirring to achieve 40yo saturation. The pH was adjusted to 7.4 with aqueous ammonia, and the precipitate was removed by centrifugation. The second precipitate was obtained by the further addition of ammonium sulfate until 80% saturation was reached. The pellet obtained by centrifugation was dissolved in a minimal volume of 0.01 M Tris-HCl buffer, pH 7.4 (Buffer A).

252

I:SVI,

FI’KAMI

3. Xephadcx (;-100 i$ Jiltmtiotl. This solution (20 ml) \vits applied to a c~~lu~nn (3.6 cm in diamctw X 100 cm in hclight) of Scphadex G-100 cquilibratc~d with HufY~r A, and the protein was ci1utc.d with the same buffer. Fractions of 10 ml each \\-crc collected. This procedure was wpeatod. 4. DEAE-cellulose columrl chromatoyraphy. The pooled active fractions (50 ml) from the Scphadcx G-100 column ware applied to a column (1.7 X 40 cm) of DEAE-ccllulosc (Brown) c>quilibratcd wit,h Buffer A. After the column was washrd with about 2 bed vol of the b&w, the enzymes were cluted stcxpwiso with the buffer cont,aining 0.1 M SaCl, 200 ml, and 0.3 M NaCl, 200 ml. Fractions of 10 ml each were collected. Activitiw ww found in nonadsorbcd (A) and 0.1 M NaCl-clutc>d (B) fractions. The lattw fractions wrrc concentrated by ultrafiltration through a collodion bag and dialyzed against 500 ml of 0.01 M Tris-HCl buffer cont.aining 0.5 mM ~-mc~rcaptocthaIlo1, pH 7.4 (Bufff>r U). This proccdurc was rcp(tatcd. 5. Hydroxyapatite columtr chromaioyraphy. The above two active fractions (A, SO ml; B, 20 ml) were scparatcly applied to columns (1.2 X 45 cm) of hydroxyapatite equilibrated with Buff cr B. The column was washed with 0.01 M phosphate buffer containing 0.5 mM 2-mcrcaptocthanol (pH 7.4) and then developed with :I 500-ml linear gradient of 0.01 to 0.25 M potassium phosphate containing 0.5 mM 2-mcrcaptoethanol (pH 7.4). Fractions of 10 ml each were collected. Active fractions wrc pooled, concentrated and dialyzed if wcrssary, and stored below -20°C until required. The five peaks of transferase activities wcrc designated I-V. Electrofocusing Elwtrofocusing was carried out on a llO-ml column (LBB SlOO) using ampholyte (lo/,, w/v) with a pH range of 3.5-10 in a O-50y0 (w/v) sucrose density gradient. The dialyzed cnzymr solution (4.6 ml) was

AND

SHISHIDO

Gel Filtratiott

for Molecular

Tt’eiyht Determi-

The determination of molt~cular n-eights of the enzymes was carried out on a column (l.S X 100 cm) of Sephadex G-75 caquilibrated with 0.01 M Tris-HCl buffer contraining 0.1 M sodium chloride and 0.5 mM %mrrcaptoct~hanol, pH 7.4. The following prot’eins of known molecular weights were used as standards ; bovine swum albumin (MW, SS,OOO), ovalbumin (45,000), a-chymotrypsinogm A (%,OOO), and myoglobin (17,500). IdentiJication

of C’otljuyafes

Enzymatic wact’ion products formed b> the conjugation of organophosphorus tricstcrs with GSH wwc identified with thinlayer, papw, or ion-exchange chromatography bawd on cochromatography with known st’andards, as previously rcaported (16, lS, 41). RESULTS

Separatiorz of b’lutathiorre S-Transferases The glutathione S-t’ransfcrascs in the supernatant fraction of American cockroach fat bodies were fractionated by several procedures and their activities towards methyl parathion, diazinon, DCNB, and methyl iodide were examined. As methyl parathion and diazinon arc conjugated with GSH at two positions on the molecule, their conjugation products were identified with the methods described in Materials and Methods. Under these cxperimental conditions [14C]demethyl parathion and S-[14C]mcthy1 glutathionc wwc mainly obtained from [‘4C]mcthyl para-

INSECT

GLUTATHIONE

253

S-TRANSFERASES Activity nmc

O.D-280nm

1.0

-

pooled -

I

nir

-1 0

! 5 .f .-: 0

0.5

5

-

i

0 -“FL. 40

FIG. 1. El&ion with

methyl

parathion;

patterns (

l

of glutathione ), diazinon;

Fraction

S-transferases DCNB;

(A),

thion. [14C]dimethyl phusphorothioic acid, which was found by the conjugation of GSH with the p-nitrophenyl moiety of methyl parathion, was less than 2~5% among the degradation products. And at pH 6..5, pyrimidinyl glutathione from diaeinon or [14C]diethyl phosphorothioic acid from [ethyV4C]diazinon was found only as the mctabolite of GSH-dependent metabolism. The elution pattern of the enzymes from the ammonium sulfate fractionation step of the Sephadex G-100 column is shown in o.D.280

No.

from Sephadex G-100 (X), OD at 280 nm.

FIG. 2. El&ion patterns are the same as those in Fig.

of glutathione 1.

(O),

Bctivity

Activity nmc 31,/min/ml

t-B-i :\

Fraction

columns.

Fig. 1. Peaks of transferase activity with methyl parathion, diazinon, and DCNB were almost at the same position. The peaks of transferase activity with methyl iodide (not shown in Figs. l-3) was at the same position as methyl parathion. The fractions active with former four compounds were combined and applied to the DEAE-cellulose column. Figure 2 shows the elution pattern from the DEAEcellulose column developed with stepwise elution with NaCl. When the column was

nm +A+

0

60

il.5

30-150

No.

S-transferases

from

DEL4E-cellulose

columns.

Symbols

254

USUI,

FUKAMI

AND

YHISHIDO

0.D.zsonm

Fract,on

No.

FIG. 3. Elution patterns of glutathione S-transferases A ; bottom, fraction B from DEAE-cellulose columns. The other symbols are the same as those in Fig. 1.

developed with a linear NaCl gradient, the enzymes adsorbed on DEAE-cellulose were separated into two active peaks at NaCl concentrations of 0.05-0.1 M; however, the separation was not complete. Therefore, elution was carried out stepwise. Two major peaks of transferase activity were observed and designated A and B. Fraction A, in which activities with the four compounds were found, was not adsorbed on DEAE-cellulose, and fraction B, in which activity with diazinon was fairly weak, was eluted with 0.1 M NaCl. Fraction A and B were applied separately to hydroxyapatite columns. The elution patterns from the hydroxyapatite columns, developed with the linear potassium phosphate gradient, displayed two clearly separated peaks of transferase activity with diazinon and three peaks of activity with methyl parathion, methyl iodide, and DCNB which were not distinguishable from each other (Fig. 3). These five peaks were designated transferases I-V. The results of purification of the transferases catalyzing the conjugation of methyl

from (-),

hydroxyapatite Concentration

columns. Top, fraction of potassium phosphate.

parathion and diazinon with GSH are summarized in Table 1. The purificat.ion factor for the hydroxyapatite column chromatography step with diazinon was about 40-fold, while methyl parathion was purified only S-fold. Transferase I was further purified using electrofocusing, and the activity with diazinon was eluted at pH 9.3-9.5 (Fig. 4). The specific activity was about 200-fold higher than that of the supernatant step, but the yield was very low (Table 1). Optimum pH As shown in Fig. 5, the optima1 pH for the conjugation of GSH with diazinon by transferases I and V was about 6.5. The optimal pHs of transferases II, III, and 1V with methyl parathion were broad, ranging between pH 8 and 9. Substrate

Specificity

Glutathione X-transferase activity with some typical substrates of transferascs, DCNB for “aryl’‘-transferasc,lbutyl iodide

INSECT

GLUTATHIONE

TABLE Partial

PuriJication

Purification step

1

of Glutathione

Total protein bd

1. 2. 3. 4.

Supernatant Ammonium sulfate Sephadex G-100 DEAE-cellulose A B 5. Hydroxyapatite I II III IV V 6. Electrofocusing I6

a Not determined. * Purified from hydroxyapatite

255

S-TRANSFERASES

Methyl Total activity (nmol/ min)

2930 1540 190 36 52 8.0 1.7 2.3 1.9 1.3 0.76

S-Transferases parathion

Diazinon

Specific activity (nmol/ min/mg)

405 273 101 14.3 28.0 1.8 1.8 2.7 1.9 -a 0.1

Total activity (nmol/ min)

0.14 0.18 0.51 0.40 0.54 0.23 1.0 1.2 1.0 -a 0.1

3010 2200 1500 800 127 331 11.7 0.9 3.2 10.4 150

1.0 1.4 7.6 22 2.5 41 6.9 0.4 1.7 8.0 197

I fraction.

or methyl iodide for “alkyl’‘-transferase, pnitrobenzyl chloride for “aralkyl’‘-transferase, trans-cinnamaldehyde for “alkene”transferase, and 1,2-epoxy-3- (p-nitrophenoxy)propane for “epoxide’‘-transfer&se were tested (Table 2). Data with methyl iodide are not shown in Table 2. As the volatility of methyl iodide is high (bp, 42.5”C), the exact activities of the transferases could not be obtained. Transferase I, which was active with the pyrimidinyl moiety of diazinon, was virtually inactive or less active with these compounds. Transferasc

II was highly active with DCNB and p-nitrobenzyl chloride. Activities with DCNB and p-nitrobenzyl chloride by transferase I and with diazinon by transferase II were observed to some extent. This might be due to the insufficient separat’ion of transferases I and II (Fig. 3). Transferase 111 was highly active with 1 ,‘&epoxy-3- (p-nitrophenoxy)propane and trans-cinnamaldehyde, while transferasc IV was highly active with butyl iodide or methyl iodide, DCNB, and p-nitrobenzyl chloride. The act.ivities of the transferases

Q.D~ZlOnm

P’-’

0.6 1

412

Activity nmol/min/ml -116

I Fraction

FIG. 4. Electrojocusing

Specific activity (nmol/ min/mg)

of transjerase

1. (

l

6

No.

), Activity

with diazinon;

(O),

pH;

(X),

OD at 280 nm.

256

tJSU1,

FUKAMI

AND

iodidr), DCNB, and p-nitrobrnzyl chloride J?-err overlapping. When transferasc activities \\-(‘r(’ tcstcd with 2-isopropyl-4-methyl-6-chloropyrimidine (38), 2,4-diphenoxy-6-chloro-s-triazine X-oxide (39) (38)) and p-nitropyridine which were known as transfwasc substrates with the soluble fraction of rat liver, transferase I showed high activity with 2isopropyl-4-methyl-6-chloropyrimidine and 2,4-diphenoxy-6-chloro-s-triazinc but’ no activiby with p-nitropyridine N-oxide (Table 3). Substrate specificity of transferase V was not examined because insufficient enzyme protein was obtained.

Activity Diazinon

Methyl

parathmn

2

1

0’1

8

6

10

PH

FIG. 5. Optimum pH. ( l ), Activity with diazinon by transferases I and V; (0), activity with methyl parathion by transferases II, III, and IV.

Kirletic

TABLE

of Glutathione

Substrate

Studies

Lineweaver-Burk plots of transferase activity at optimal pH with diazinon by transferase I and with methyl parathion by transferase II, III, or IV are shown in Figs. 6 and 7. The initial reaction rates were determined on varying substrate concentrations at a constant concentration of GSH (1 or 5 mM), and vice versa. These activities were linear for the first 20 min. From intercepts of the axes of the graphs, Michaelis constants (K,) were calculated to be 0.28 mM for methyl parathion and 0.13 mlM for diazinon (Fig. 6). At a GSH concen-

with the various substrates were as follows : With DCNB and p-nitrobenzyl chloride the transferase activity decreased in the order of transferase II > IV > III ; with butyl iodide or methyl iodide transferase activity decreased IV > III > II; and with trans-cinnamaldehyde and 1,2-epoxy3- (p-nitrophenoxy)propane the transferase activity decreased III > IV ‘v II. These results suggested that the properties of t,ransferases II, III, and IV were different from each other, even though activities with methyl parathion, methyl or butyl

Activities

HHISHIDO

2

S-Transferases

with

Concentration

(mM)

Se?ected Substrates*

Activity (nmol/min/mg)

Sub- GSH skate Diazinon

0.3 0.3 0.5 0.5 0.5 0.5 0.5

Methyl parathion DCNB Butyl iodide pNitrobenzy1 chloride trams-Cinnamaldehyde 1,2-Epoxy-3-(p-nitrophenoxy)propane a pH

6.5 for diazinon,

pH

8.0 for methyl

parathion,

5 3 5 2 2 2 2 and pH

Transferase I

II

III

IV

41 0.2 8.5 0.8 7.1 1.0 0.4

6.9 1.0 272 3.6 93 4.x 6.9

0.4 1.2 63 7.1 44 14 38

1.7 1.0 140 38 77 4.8 8.9

7.4 for the other

substrates.

INSECT

GLUTATHIONE

TABLE Activities

with

N-Heterocycles

N-Heterocycle

concentration

of 5 mM

3 by Transferase

I0 Activity

Concentration (mM) 0.3 0.3 0.1 0.1

Diazinon 2-Isopropyl-4-methyl-6-chloropyrimidine 2-Chloro4,6-diphenoxy-s-triazine p-Nitropyridine N-oxide a GSH

257

S-TRANSFERASES

and pH

11.8 6.9 58 0

(AOD (AOD (AOD (AOD

~6 nm/hr/nyd

W,,Jhr/mg) 254 dhr/mg) 296.&r/w4

6.5.

tration of 5 mM, T’,,, with diazinon was much higher than with methyl parathion. At this time, lines obtained by transferase I with diazinon at GSH concentrations of 1 and 5 mM had an intersection on the z-axis (Fig. 6a) which was compatible with an ordered sequential bi-bi mechanism for the enzyme reaction (15,42). But when the concentration of GSH was

varied while the diazinon concentration was kept constant (0.3 and 0.1 mM), the curves in double reciprocal plots were not linear (Fig. 71, suggesting a biphasic kinetic mechanism (42). Molecular Weight The molecular weights of transferases I, II, III, and IV estimated by gel filtration on Sephadex G-75 were 35,000-37,000 and almost indistinguishable from each other (Fig. 8). DISCUSSION

Two glutathione S-transferases active on an organophosphorus triester, diazinon, and three other transferases active on methyl parathion were chromatographimirvmg/p

mol

t

I

I

2

4

l/mM

FIG. 7. Lineweaver-Burk plots ,with transferase I. (a), Dia.zinon concentration mM; (0), 0.1 mM.

GSH

‘Y

I 0

?4 FIQ. 6. Lineweaver-Burk plots. a, With diazinon by transferuse I: ( l ), GSH concentration of 6 mM; (0), 1 mM. b, With methyl parathion by transferases II, III and IV at a GSH concentration of 6 mM.

of

by 0.3

458

USUI,

FUKAMI

AND

SH18111DO

Using .1incric:tn cwckroach t r:uisf(r:wd :tt cwh optimal pH, the glutathione Bovine serum albumin con,jugatc~of methyl parathion was mainly met8hyl glutathionc and that of diazinon was mainly pyrimidinyl glutxthione. Therefore, neither comparisons of alkyl conjuTransferase I - IV gation with aryl conjugation of methyl parathion nor comparisons of ethyl conju\ 3 gation with pyrimidinyl conjugat,ion of di\ Chymotrypsinogen \ azinon were made. In this expwimcnt, it was found that the transftvase which catalyzed the conjugation wit#h the pyrimidinyl moiety of diazinon TWS distinguishable from the enzymes which catalyzed the conjugat’ion , II-I-lI - I 100 150 ml with t#hemethyl group of methyl parathion. Elution volume It, is suggested that t,here are at least three FIG. 8. Geljiltration on Sephadex G-75 for molecular transfcrases active with methyl parathion wei.qht determination. which have overlapping substrates as described by Habig et al. (10). Transfcrase I seems rather specific to S-hetcrocyclcs, tally separated from the fat bodies of adult such as pyrimidinrs and s-t.riazines, and female American cockroaches. It is strongly suggested that there are multiple forms of probably catalyzes the conjugation of glutathione S-transfcrase active on organo- s-triazine herbicides (43) which are known t’o conjugate with GSH by enzymes in phosphorus triesters, DCNB, and methyl iodide, although immunochemical studies higher plant’s and mammals. and amino acid analysis, as described by A few experiments have been conducted Habig et al. (10) in his mammal work, have studying the kinetics of glutat,hione Stransferase with organophosphorus trinot been conducted at this time. Ohkawa et al. (13) examined GSH- esters as the substrates. The K, value of dependent metabolism of fenitrothion by purified pig “alkyl’‘-transferase was cstimouse livers and house flies. They sepa- mated by Hutson et al. (9). The K, for naphthyl phosphate was 0.16 mM at a rated three enzyme peaks by DEAEcellulose column chromatography. Re- GSH concentration of 5 mM, pH 7.4. Similar K, values were obtained with cently, Motoyama et al. (30) conjectured from their work on the difference in t’he methyl parat’hion and diazinon. In the metabolic activity in several strains of study of the kinetic mechanism of rat liver resistant house flies that the enzyme glutathione X-transferase, Pabst it al. (42) responsible for the conjugation of the reported that, when high concentrations of pyrimidinyl moiety of diazinon was not GSH wcrr evaluated, an ordered sequential the same one that catalyzed the conjugation pathway predominated in a biphasic kinetic of Lhe p-nitrophenyl moiety of parathion. mechanism and, when lower concentrations Hollingworth et al. (2.5) suggested that the of GSH were evaluated, a ping-pong pathalkyl and aryl conjugation of parathion way predominated. It has been suggested was catalyzed by two diffcwnt enzymes. in the present st.udy that the insect glutaThe same theory was published by Motothione S-transfcrasrs have a kin&! mechayama et al. (30), but these workers were nism similar to that in mammals, although investigations at low c~ollcc~ntr:ltio~~sllf unable to scpnratc these two c~nzymc~s with GSH were not conducted. CM-cellulose column chromatography. Molecular x104

weight

INSECTGLUTATHI~NE

S-TRANSFERASES

It has been reported that the molecular weights of glutathione X-transferases in mammals are 40,00&50,000 (8, 10, 13, 15) and that the enzymes consist of two subunits each of which has a molecular weight of 23,000-27,000 (8, 10, 13). In insects molecular weights of 36,00&38,000 (1 l), slightly smaller than in mammals, have been reported. Habig et al. (10) reported that three transferases (A-C) obtained from rat livers had essentially the same molecular weight, 45,000. The transferases from the American cockroach fat bodies had almost the same molecular weight of 35,000-37,000. The optimal pH of the transferase with methyl parathion ranged from 8 to 9. This is different from the mouse liver enzyme data described by Hutson et aE. (9) but is similar to the “aryl’‘-transferase data reported by Hollingworth et al. (2rj). The transferases, especially those catalyzing t,he conjugation with methyl parathion, could not be highly purified. This might be due to the instability of the enzymes, especially to dialysis (4). Sulfhydryl group stabilizers, 2-mercaptoethanol and dithiothreitol, were virtually ineffective on these enzymes. Future trials will include a buffer containing 30y0 glycerin-5 mM GSH-1 mM EDTA (8,lO). Compared to mammals, insect glutathione S-transferases acting on organophosphorus triesters have some different properties. Further investigations are necessary to determine the role played by glutathione S-transferases in selective toxicology. ACKNOWLEDGMENTS The authors are grateful to Dr. K. Fukunaga, Chief of the Laboratory of Insect Toxicology, The Institute of Physical and Chemical Research, for his encouragement and to Dr. W. E. Allison, Walnut Creek Center, Dow Chemical USA, for his critical reading of the manuscript. REFERENCES 1. E. Boyland glutathione

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