Insect Biochem. Vol. 18, No. 8, pp. 861-866, 1988 0020-1790/88 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1988 Pergamon Press pie

EVIDENCE FOR THE PRESENCE OF GLUTATHIONE PEROXIDASE ACTIVITY TOWARD AN ORGANIC

HYDROPEROXIDE IN LARVAE OF THE CABBAGE LOOPER MOTH, TRICHOPLUSIA NI

SAMI AHMAD and RONALD S. PARDINI Department of Biochemistry, University of Nevada, Reno, NV 89557-0014, U.S.A.

(Received 17 May 1988; revised and accepted 5 August 1988)

Abstract--Using enzyme preparations of mid-fifth instar larvae of the cabbage looper moth, Trichoplusia ni, we have detected glutathione peroxidase activity against a model organic hydroperoxide substrate, cumene hydroperoxide. Specific activity, pmol NADPH oxidized/mg protein/min at 25°C and pH 7.0 was 54,3 for 600g (10 rain) and 57.3 for 850g (15 min) supernatant. Cysteamine and cyanide which inhibit the peroxidase activity of glutathione transferase, significantly inhibited the enzyme activity, while azide, an inhibitor of catalase and peroxidase had no effect. Specific activities associated with the nuclear, microsomal and cytosolic fractions were 74.0, 34.6 and 13.2, respectively. No activity was detected in the mitochondrial fraction. In the absence of a Se-dependent glutathione peroxidase in T. hi, the GSH-dependent peroxidase activity appears to conform to the peroxidase activity analogous to the mammalian non-Se glutathione transferase.

Key Word Index: antioxidant enzymes, glutathione peroxidase, glutathione transferase, hydroperoxides, lipid peroxidation

INTRODUCTION

Many biochemical processes in aerobic cells lead to the production of hydroperoxides by activated forms of dioxygen; namely, superoxide, hydroperoxy and hydroxyl radicals (Fridovich, 1983). Tissues are oxidatively damaged directly from hydroperox- ides, as well as from more reactive free radicals and other chemical species arising from the decomposi- tion of hydroperoxides (Mannervik, 1985). A Se- dependent glutathione peroxidase (GSH-peroxidase; EC 1.11.1.9) well-known from all mammalian species examined (Mannervik, 1985), catalyzes the reduction of hydrogen peroxide (H202) and a wide range of lipid and other organic hydroperoxides (ROOH):

2GSH + H202-,GSSG + 2H20 (1)

2GSH + ROOH--,GSSG + H20 + ROH. (2)

In mammalian species the enzyme is primarily located in the cytosol and the mitochondrial matrix (Mannervik, 1985). The GSSG generated by the catalytic activity of Se-dependent GSH-peroxidase is reduced to GSH by the enzyme glutathione reduc- tase (EC 1.6.4.2). The subcellular distribution of this enzyme is similar in mammalian species to that of the Se-dependent GSH-peroxidase (Chance et al., 1979), and the reaction catalyzed is as follows:

GSSG + 2NAD(P)H-,2GSH + 2NAD(P) +. (3)

The enzymatic mechanisms by which insects defend themselves from oxidative stress have received little attention. Very recently, we demonstrated exis- tence of the antioxidant enzymes, superoxide dis- mutase (EC 1.15.1.1), catalase (EC 1.11.1.6) and

glutathione reductase in larvae of three insect species, the cabbage looper Triehoplusia hi, southern army- worm Spodoptera eridania and the black swallowtail butterfly Papilio polyxenes (Ahmad et al., 1987; Pardini et al., 1989; Pritsos et al., 1988a, b). However, the assays of 850g supernatants (Ahmad et al., 1987; Pardini et al., 1989; Pritsos et al., 1988a, b), sub- cellular localization studies (Ahmad et al., 1988a, b) and induction from pro-oxidant exposure (Pritsos et aL, 1988b, c; also, S. Ahmad et al., unpublished data) have, in all cases investigated in depth, failed to reveal the existence of Se-dependent GSH-peroxidase in these species.

In numerous studies related to ageing in Musea domestiea, antioxidant enzymes detected were super- oxide dismutase, catalase and glutathione reductase, but not GSH-peroxidase although the oxidation of GSH to GSSG was observed (Allen et al., 1983, 1984a, b). In Drosophila melanogaster the presence of peroxidase activity was reported by Nickla et al. (1983), and discussed along with the essential role of GSH as an antioxidant. Therefore, we cited this study (Ahmad et al., 1987, 1988a) as an indication of the occurrence of a GSH-dependent peroxidase activity in an insect. However, very recently we were able to acquire, hence review the protocol for the assay of peroxidase activity by Nickla et al. (1983) which was according to the method of Armstrong et al. (1978). It is now clear that the activity measured in D. melanogaster was of a non- GSH dependent peroxidase (EC 1.11.1.7). Peroxidase is a dual substrate enzyme which uses as an oxidant H202, and as a hydrogen donor a non-pyridine nucleotide such as catechol. Both catalase and

861

862 SAM1 AHMAD and RONALD S. PARDINI

peroxidase are hemoproteins and are inhibited by N a N 3 . Moreover, the potential role of peroxidase as an antioxidant enzyme has not been investigated, but the enzyme is implicated in the polymerization pro- cess of cuticular proteins; the oxidation of catechols to quinones and semiquinones may provide bridges for linking proteins (Hasson and Sugumaran, 1987). The occurrence of a Se-dependent GSH-peroxidase has been documented only in mammalian species (Larson, 1988). In all other eukaryotes examined, e.g. plant and insect species, and also prokaryotes, no published account exists of this typically mammal ian enzyme.

Recent work on mammalian species has shown that some multiple forms of non-Se glutathione trans- ferase (GSH-transferase; EC 2.5.1.18) catalyze the reduction of a wide range of organic hydroperoxides, but not H202 (Mannervik, 1985). Therefore, we explored the possibility that in insects GSH-trans- ferase may, in large measure, substitute for Se-depen- dent GSH-peroxidase in alleviating oxidative stress from organic hydroperoxides. In this report, we describe GSH-dependent peroxidase activity in the insect, T. ni, together with the enzyme's subcellular profile, and ascribe the activity to the non-Se GSH- transferase.

MATERIALS AND METHODS

Enzyme source

In initial experiments, 850g (15 min) supernatants from crude homogenates of 2 g mid-fifth instar larvae of T. ni homogenized in 8 ml of pH 7.0, 50 mM potassium phos- phate buffer containing I mM EDTA, were obtained by the method of Ahmad et al. (1987).

The subeellular fractions of T. ni were prepared by a procedure which upon electron microscopy was reported to yield nuclear, mitochondrial and microsomal fractions of "high quality and negligible cross-contamination" (Ahmad et al., 1988b). From a crude homogenate of 40 g mid-fifth instar larvae in a buffer of the composition given earlier (Ahmad et al., 1988a), by differential centrifugation the subeellular fractions obtained were: 600 g (10 min) superna- tant; 12,000g (5 s) nuclear; 46,000g (15 s) mitochondrial; 100,900g (45min) microsomal; and 100,900g (45-min supernatant) cytosolic fraction. The pellets of nuclear, mito- chondrial and microsomal fractions were gently resus- pended in the homogenization buffer and centrifuged again to obtain washed pellets. All washed pellets were re- suspended in 2 ml of the pH 7.0 potassium phosphate buffer containing 1 mM EDTA.

Protein concentrations of the enzyme samples were deter- mined according to Lowry et al. (1951).

Sonication

A portion of the 850 g supernatant and membrane-bound fractions, all chilled on ice, were gently sonicated (Ahmad et al., 1988a, b) in order to facilitate the interaction of enzymes internal to subeellular organelles with the exo- genous contents of the incubation mixture.

Enzyme assays

GSH-peroxidase activity was measured spectrophoto- metrically by a modification of the procedure described by Strauss et al. (1980), which is based on coupling the production of GSSG [cf. reaction (2)], to the reaction catalyzed by glutathione reductase [reaction (3)], hence the enzyme activity could be monitored as the amount of NADPH oxidized. The standard incubation mixture (3 ml) contained: 50mM potassium phosphate buffer of

pH 7.0 and containing 1 mM EDTA; I mM GSH; 0.2 mM NADPH; 5 IU glutathione reductase (Sigma Chemical Co., St Louis, Mo.; baker's yeast, 200 U mg-~); 1.2 mM cumene hydroperoxide; and 20 #1 of the enzyme sample.

We first investigated whether initiating the enzyme assay by addition of the enzyme preparation was advantageous over incubation started with the substrate. Using two replicates from the same enzyme preparation we found that the net activity averaged the same (c. 52-54 pmol NADPH oxidized/mg protein/min). Therefore, there was no indica- tion of a significant interference by endogenous GSSG, presumably, because levels of GSSG in the enzyme prepara- tion were too small to be picked up by our procedure. On the other hand, starting the reaction first by adding the substrate to the incubation mixture had the advantage of correcting for slight changes in absorbance at 340 nm due to the slow oxidation of NADPH to NADP ÷. Procedure for this preliminary experiment was the same as described next for the standard assay, except for the switch in first adding substrate or the enzyme preparation.

The standard assay was as follows. To 2.8 ml of the incubation mixture (all ingredients except cumene hydro- peroxide and the enzyme sample), 0.1 ml of a cumene hydroperoxide solution to yield 1.2 mM final concentration, was added. The reaction was monitored at 340 nm until a linear rate was established followed by addition of the enzyme sample. The difference between the two rates corresponded to the GSH-peroxidase activity. The specific activity was calculated as pmol NADPH oxidation/mg protein/min at 25°C at pH 7.0.

In experiments on the effect of substrate and enzyme concentrations, the levels of cumene hydroperoxide (1.2 and 0.12 mM) and enzyme volume (10-80/H) were varied. In some assays, the enzyme sample was pretreated with N-ethylmaleimide (NEM) for 30 s at 5 (Morgestern and DePierre, 1983) and 20 mM concentrations. Nonenzymatic activity was checked by boiling the insect's 850g super- natant for 1 h (Ahmad et al., 1987).

In the experiment on the effect of enzyme activity by an inhibitor of catalase and peroxidase activity, NaN 3 (Ahmad et al., 1987, 1988b), the incubation was performed according to the standard assay with 5 mM NaN3 added to the incubation mixture.

Because cysteamine is soluble only in acidic solutions, the pH of the incubation mixtures containing cysteamine was lowered to 6.5. On the other hand, in experiments where KCN was added, the pH was raised to 7.5 to increase the CN- concentration. Moreover, the GSH concentration was reduced to 0.2 mM, to enhance the competitive inhibition of the GSH-dependent peroxidase reaction by KCN. These protocols for cysteamine and KCN competitive inhibition experiments are the same as previously reported in detail by Prohaska (1980). Because of alteration in the pH in assays using the two compounds, and only one-fifth the amount of GSH in the KCN experiment, the control (no inhibitor added) values for GSH peroxidase activity were significantly less than in the standard assay conducted at pH 7.0.

RESULTS AND DISCUSSION

At 1 .2mM concentration of cumene hydropero- xide (Fig. 1), the enzyme activity of the 850g super- natant exhibited a significant linear relationship for enzyme samples ranging from 10 to 80/zl. The specific activity averaged 52 pmol N A D P H oxidized/ mg protein/min. At one-tenth concentration of the substrate, cumene hydroperoxide, the specific activity was 13.8, c. 4-fold less than at 1.2 m M cumene hydro- peroxide (data not shown). Therefore, in additional assays 1 .2mM cumene hydroperoxide was used, which is also the concentration employed by Reddy

250

.E E 200 !

1 E ~ 100

z ~ '~0 [

o

Glutathione peroxidase activity 863

strated activation of both transferase and peroxidase activity when the enzyme was pretreated with 5 mM NEM. Our data (Table 1) show no activation of unsonicated or sonicated enzyme preparation; instead at 4-fold higher concentration of NEM, 20 mM, there was a significant decrease in enzyme activity, which could have occurred indirectly by inhibition of the enzyme, glutathione reductase. Another explanation deserving consideration is that the lack of activation, or even inhibition by NEM seems to depend on the subeellular location of the

0 20 40 60 0 1 O0 Sq~.atant V~ (~.)

Fig. 1. The influence of varying the amount of 850g supernatant of mid-fifth instar larvae of T. ni on GSH peroxidase activity against cumene hydroperoxide. The data points are averages of duplicate assays, each assay with 2 replicates (n = 4), and the difference between the values of duplicate determinations did not exceed 10%. A signifi- cantly linear relationship was found for enzyme aliquots in the 10-80/~1 (0.4-3.2 mg protein) range; regressed corre- lation coefficient, r =0.9486, and the P = <0.01. From protein analysis of the enzyme aliquots, the mean specific activity obtained was 52 + 4.3 pmol NADPH oxidized/mg

protein/rain at 25°C and pH 7.0.

et al. (1981) in their demonstration of non-Se GSH- peroxidase activity attributed to GSH-transferase in rat liver microsomes.

Measurements of the enzyme activity with sonica- tion of the 850 g supernatant showed an inexplicable decline in activity (Table 1). In previous studies of S. eridania and T. ni (Ahmad et al., 1988a, b), sonica- tion did not affect assays of the membrane-enclosed antioxidant enzymes, instead enrichment of activities were detected in some fractions. Similarly, soni- cation apparently did not affect the assays of GSH- peroxidase activity toward cumene hydroperoxide as evidenced by data presented later in the text. More- over, Friedberg et al. (1979) used sonication as the initial step in the purification of GSH-transferase from rat liver microsomes.

In a study of rat microsomal GSH-transferase activity against l-chloro-2,4-dinitrobenzene (CDNB) and peroxidase activity against cumene hydro- peroxide, Morgenstern and DePierre (1983) demon-

Table 1. The effect o f mild sonication and N E M treatment on glutathione peroxidase activity of T. ni

Sonication N E M Specific activity ( + ) (mM) mean + SD

- - - 57.3 +_ 2.23a - 5 59.5 + 4.02a - 20 39.9 + 4.95b + - - 31.9 _+ 5.32b + 5 33.9 + 3.89bc + 20 21.3 + 2.84c

Activities were determined at pH 7.0 and 25°C, using standard incubation mixture. The enzyme source was 850 g supernatant of T. hi. Means are derived from a pool of 2 separate determina- tions, each determination with 2 replicates (n = 4). The specific activity equals pmol of N A D P H oxidized/rag protein/min at 25°C and pH7.0 . Following A N O V A (d . f .=5,18; F = 4.99; P > F = 0.0001), Duncan ' s multiple range test was used to discern significant differences among the means; means not accom- panied by the same letter are significantly different (~ = 0.05).

enzyme. According to Reddy et al. (1981), GSH- transferase's peroxidative activity against cumene hydroperoxide for the cytosolic enzyme decreased when treated with NEM. They also reported that while a nearly 2-fold higher activity occurred with NEM-treated microsomes, the activity was 5% less in NEM-treated solubilized microsomal enzyme than the control (untreated enzyme). This aspect clearly requires clarification with the enzyme that has been purified and its isozymes characterized.

Results presented in Table 2 indicate that, (1) the peroxidase activity against cumene hydroperoxide was enzymatic; (2) treatment with CO shows that the activity was not subject to erroneous estimation from enzymes such as cytochrome P450, which in an uncoupled reaction without the added substrate, can cause oxidation of NADPH to NADP ÷ (Ahmad et al., 1988a); and (3) the activity requires GSH. The crucial GSH requirement for the reaction further rules out any meaningful interference in the assays from direct NADPH oxidation by oxidoreductases such as NAD(P) ÷ transhydrogenase (EC 1.6.1.1), and NAD(P)H dehydrogenase (quinone-acceptor; EC 1.6.99.2). The lack of inhibition by azide indi- cated no interference from the enzyme, catalase, a finding consistent with the observation that this enzyme has no reactivity against organic hydroperox- ides more complex than ter t-butyl hydroperoxide (Chance et al., 1979). Moreover, this lack of inhibi- tion indicated no interference from peroxidase. Data in Table 2 also show that both cysteamine and KCN markedly suppressed the GSH-dependent peroxidase activity against cumene hydroperoxide. Significant 17.2 and 37% inhibition from cysteamine occurred at 1 and 10 mM concentrations, respectively. Inhibition by KCN was not apparent at 1 mM concentration, but at 10 mM a significant inhibition of 45.4% was recorded.

According to Prohaska (1980), unlike the Se-de- pendent GSH-peroxidase catalysis of hydroperoxides [reaction (2)], the catalysis by the non-Se GSH-trans- ferase proceeds in two steps:

ROOH + GSH--.[GSOH] + ROH (4)

[GSOH] + GSH--,GSSG + H20. (5)

While reaction (4) is enzymatic, reaction (5) of the unstable sulfenic acid of glutathione (GSOH) with GSH is nonenzymatic. Both eysteamine and CN- inhibit the reaction (5) by competing with GSH for the sulfenic acid. This, in turn, decreases the amount of GSSG formed, and results in concomitant formation of a mixed disulfide of GSH and cyste- amine (GSSR) and glutathione thiocyanate (GSCN), respectively (Prohaska, 1980). Furthermore, the yeast

864 SAMI AHMAD and RONALD S. PARDINI

Table 2. Effects of some treatments on the giutathione peroxidase activity of T. ni

Concentration Specific activity % Decrease relative to Treatment (mM) mean + SD the control activity

None (control) 59.5 __. 4.01a - - NaN3 5 58.6 __. 3.70ab - - +CO 54.1 + 6.11b - - - GSH* 3.9 + 0.90c 93.4 Boiling 0.9 + 0.85c 98.5

ANOVA: F4.15 = 293.00, P > F = 0.0001

None (control) 47.7 ___ 1.74a - - + Cysteamine 1 39.5 + 3.56b 17.2

10 30.1 + 4.86c 36.9 ANOVA: F2. 9 = 17.64, P > F = 0.0008

None (control) 27.3 + 3.58a - - + KCN 1 28.9 + 3.51a - -

10 14.9 + 3.36b 45.4 ANOVA: F2, 9 = 17.87, P > F = 0.0007

The source of enzyme was 850 g supernatant of T. ni. The specific activity equals pmol NADPH oxidized/mg protein/min at 25°C. Means are derived from 4 replicates, 2 replicates per determination (n = 4). Following ANOVA means were separated into statistically different groups by the Duncan's multiple range test; means not accompanied by the same letter in each data set are significantly different (~t = 0.05).

*The marginal 6.6% activity in the absence of GSH most likely depended on some GSH endogenous to the 850g supernatant.

g lu ta th ione reduc tase was f o u n d to have negligible act ivi ty ( < 0 .5%) t o w a r d the mixed disulfide o f G S H and cys teamine c o m p a r e d wi th G S S G (P rohaska , 1980). These results , in c o n j u n c t i o n wi th those o f P r o h a s k a (1980), indica te the i nvo lvemen t o f a n o n - Se G S H - t r a n s f e r a s e in ca ta lyz ing the r educ t ion o f o rgan ic h y d r o p e r o x i d e s in insects such as T. ni. In this con tex t , it deserves m e n t i o n tha t the Se-depen- den t G S H - p e r o x i d a s e is no t inh ib i ted by cys teamine o r C N - (P rohaska , 1980).

The subcel lu lar d i s t r ibu t ion o f the G S H pe rox idase act ivi ty aga ins t c umene h y d r o p e r o x i d e is s h o w n in Table 3. The specific act ivi ty o f the 6 0 0 g super- n a t a n t f r o m which the subcel lu lar f rac t ions were o b t a i n e d was the same as desc r ibed above for the 8 5 0 g supe rna t an t . The h ighes t specific activity, 74 p m o l N A D P H ox id i zed /mg p ro t e in /min , wh ich signif icantly exceeds tha t o f the 6 0 0 g supe rna t an t , was local ized in the nuc lea r f ract ion. The specific activit ies assoc ia ted wi th the m i c r o s o m e s and cy tosol were 34.6 and 13, respectively. N o act ivi ty was f o u n d in the m i t o c h o n d r i a l f ract ion. Judg ing f rom the relative specific activit ies and relative p ro t e in a b u n d a n c e (pro te in ra t io for nuclear , micro-

somal a n d cytosol ic f rac t ions = 1 : 1.4: 3.1), the cyto- solic G S H - t r a n s f e r a s e ' s pe rox idase act ivi ty is likely to con t r i bu t e subs tan t ia l ly to the tota l cel lular act ivi ty m e a s u r e d wi th cumene hyd rope rox ide . In m a m m a l i a n species, G S H - t r a n s f e r a s e exhib i t ing t rans fe rase a n d pe rox idase activi t ies has been f o u n d in the cytosol , m i c r o s o m e s a n d in the ou te r mi to - chondr i a l m e m b r a n e (Manne rv ik , 1985). G S H - t r ans fe rase ' s pe rox ida t ive act ivi ty has been s h o w n to play a s ignif icant role in p ro t ec t ing m a m m a l i a n m i c r o s o m a l m e m b r a n e f r o m d e g r a d a t i o n due to lipid p e r o x i d a t i o n (Tan et al., 1984). A c c o r d i n g to M a n n e r v i k (1985) o the r cel lular m e m b r a n e s m a y also be p ro t ec t ed in this m a n n e r . F r i edbe rg et al. (1979) s tud ied G S H - t r a n s f e r a s e ' s subcel lu lar distr i- bu t i on extensively a n d c o n c l u d e d tha t the act ivi ty in nuclei a n d m i t o c h o n d r i a was less t h a n in mic rosomes . In the p resen t s tudy, the f inding tha t the nuc lear f rac t ion had the h ighes t specific act ivi ty is surpr i s ing bu t ra t iona l ized on the p remise tha t there is the need for a repa i r m e c h a n i s m for " p e r o x i d i z e d " D N A . O n the o t h e r h a n d , lower m i t o c h o n d r i a l enzyme levels in m a m m a l i a n species and no act ivi ty in the insect ' s m i t o c h o n d r i a deserve c o m m e n t . In m a m m a l s ,

Table 3. Subcellular distribution of glutathione peroxidase activity of T. ni

Enzyme activity

Specific activity Total activity Subcellular fraction mean + SD per larva

Supernatant (600g x 10 min) 54.3 + 0.04b 2170 Nuclear (12,000g x 5 s) 74.0 + 4.36a 500 Mitochondrial (46,000 g x 15 s)* 0 ND Mierosomal (100,900g x 45 rain) 34.6 __ 2.12c 700 Cytosolic (100,900 g supernatant) 13.0 +_ 0.84d 1400

Means are derived from a pool of values of triplicate determinations; first deter- mination was with 2 replicates, and the second with only 1 replicate (n = 3). The specific activity equals pmnl of NADPH oxidized/mg protein/min at 25°C and pH 7.0. Following ANOVA (F3. s = 225.30, P = 0.0001), Duncan's multiple range test was used to discern significant differences among the means; means not accompanied by the same letter are significantly different (~ ~ 0.05). Total enzyme actb'ity was calculated from the specific activity and the total amount of protein per larva, associated with each subcellular fraction.

*In all assays 20/~l o f the enzyme sample was used, except that in the mitochondrial fraction, rates were recorded with 20, 40 and 80~1 (0.12, 0.24 and 0.48mg protein) samples.

Glutathione peroxidas¢ activity 865

the mitochondrial membrane is protected by the Se-dependent GSH-peroxidase and vitamin E. In insects, protection from H202 in mitochondria would be mainly from catalase, but tocopherols may be important antioxidant defense against organic hy- droperoxides (Ahmad et al., 1988a). However, at this time, we have not excluded the possible existence of other peroxidases.

The simplest hydroperoxide, H202, is detoxified at a rate comparable to organic hydroperoxides derived from polyunsaturated fatty acids, cholesterol, steroid hormones, thymine and peroxidized DNA (Mannervik, 1985). Therefore, the assay for the Se-dependent GSH-peroxidase frequently utilizes H202 as substrate. However, our studies on three insects, T. hi, S. eridania and P. polyxenes, suggest the absence of this enzyme (Ahmad et al., 1987, 1988a, b; Pardini et al., 1989; Pritsos et al., 1988a, b), although in all mammalian species examined the crucial role of Se-dependent GSH-peroxidase to detoxify H202 and repair other hydroperoxides is indisputable (Mannervik, 1985). Catalase, which also decomposes H202, is primarily localized in mammalian perox- isomes, whereas the Se-dependent GSH-peroxidase is found in the cytosol and the mitochondrial matrix. Therefore, in mammalian systems these two enzymes not only have "complementary intracellular localiza- tion", but also "complementary catalytic activities against H202" (Mannervik, 1985; Chance et al., 1979). Thus, it is not surprising that the absence of Se-dependent GSH-peroxidase is accompanied by an unusually high activity (Ahmad et al., 1987; Pardini et al., 1989; Pritsos et al., 1988a, b) and wide sub- cellular distribution of catalase in T. ni and other insects; the high activity is nearly-equally distributed in the cytosol, mitochondria and microsomes, and very little activity in the nuclei (Ahmad et al., 1988a, b).

Using a model substrate, cumene hydroperoxide, we have demonstrated that in T. ni a GSH peroxidase activity exists against organic hydroperoxide but not H202. Based on work with mammalian species, this pattern suggests that the activity is associated with the non-Se GSH-transferase which catalyzes the reduction of organic hydroperoxides, but has no activity against H202 (Jakoby, i985). That GSH- transferase unlike Se-dependent GSH-peroxidase has no reactivity against H202 reinforces the need for a wide intraceUular distribution of catalase, as has already been documented.

According to Jones et al. (1981), in mammalian species such as the rat, the Se-dependent GSH- peroxidase activity is in far excess of the rate of GSH synthesis, glutathione reductase mediated reduction of GSSG and the rate of NADPH genera- tion; c. 1300, 4-5 and 37 times, respectively. The activities of the glutathione reductase and of the NADPH-generating system apparently limit the rate of hydroperoxide reduction and, also, cause GSSG accumulation. As reviewed recently, the thiol status of eukaryotic cells is essential because GSSG accumulation in excess of 12-15% is harmful (Ahmad et al., 1988a, b). Consequently, in the mammalian species, there is GSSG efflux in the form of GS-conjugates into the bile. In insects, at least in T. hi, this situation appears to be opposite of that

in mammals. The glutathione reductase activity in the 850g supernatant of mid-fifth instar homogenate is 1.17 units, or 0.564nmol NADPH oxidized/mg protein/rain (Ahmad et ai., 1987). On the other hand, the rate of NADPH oxidation concomitant with the rate of cumene hydroperoxide reduction is 0.057 nmol NADPH oxidized/rag protein/min. The glutathione reductase activity, therefore, is in excess of 10 times that of the activity of the non-Se GSH- peroxidase. The data on specific activities of T. ni's subcellular compartments' glutathione reductase (Ahmad et al., 1988b) and GSH-transferase's perox- idase activity, as per this report, further provide support to this contention. Thus, from the well-estab- lished stoichiometry of the coupled GSH-peroxidase [reaction (2); Se-dependent, as well as non-Se- dependent] and glutathione reductase [reaction (3)] reactions, the insect's repair mechanism against hydroperoxides may be more efficient than mam- malian systems in that excess GSSG accumulation seems less likely. Nevertheless, this conclusion repre- sents a hypothesis which needs clarification based on in-depth future investigations along the model study of Jones et al. (1981).

The enzyme GSH-transferase occurs in all insects examined, but most work so far has focused on its transferase activity important in xenobiotic metabolism (Ahmad et al., 1986). Also, all published work to date has used the soluble cytosolic enzyme and no authentic information has been available on its subeellular distribution or its peroxidative activity (Ahmad et al., 1986). Recently, GSH-transferase activity toward CDNB as substrate was examined and found in all three species comprising our insect model (Wang et al., 1988). The GSH-transferase activity toward CDNB as substrate in the 850g supernatants of early, mid and late instar larvae of T. ni and S. eridania ranges from 0.1 to 0.2, while in P. polyxenes the levels are 1.3-2.4 nmol CDNB conjugation/mg protein/rain (Wang et al., 1988). In species related to P. polyxenes, the tiger swallowtail subspecies, P. glaucus glaucus and P. g. canadensis exhibited GSH-transferase activity toward CDNB of 403 and 578 (cytosolic transferase) and 103 and 197nmol CDNB conjugation/rag protein/min (microsomal transferase), respectively (R. L. Lindroth, personal communication, 1988). The observed high GSH-transferase activity vs non-Se GSH-peroxidase activity is consistent with the observed ratio of activities in mammalian species. For example, in the rat there are nine isozymes of GSH-transferase, each has high GSH conjugative activity, while only two isozymes possess significant levels of peroxidase activity (Tan et al., 1984; Jakoby, 1985). We are extending these studies to distinguish GSH-transferase's conjugative vs per- oxidative activity in other insects, and to resolve isozymes which may overlap in both types of activity, but some may be more specific for one or the other reaction.

In summary, to the extent a generalization can be made at this time, it appears that GSH-transferase with its peroxidase activity, together with glutathione reductase, may form a line of defense against delete- rious organic hydroperoxides in T. ni and possibly other insects.

866 SAMI AHMAD and RONALD S. PARDINI

Acknowledgements--This work was supported by USDA competitive research grants 86-CRCR-1-2038 awarded to R. S. Pardini, S. Ahmad and G. J. Blomquist and 88-37153- 3457 awarded to S. Ahmand and R. S. Pardini, and is a contribution of the Nevada Agricultural Experiment Station. The authors thank Dr Gary J. Blomquist, Dr Charles R. Heisler (Department of Biochemistry, University of Nevada, Reno) and Dr Richard L. Lindroth (Department of Entomology, University of Wisconsin, Madison) for their helpful comments. The authors acknowledge with gratitude Dr R. L. Lindroth for sharing his unpublished data on glutathione transferase activities of the two subspecies of tiger swallowtail butterflies.

REFERENCES

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