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A microsomal enzyme inhibitor in the gut contents of the house cricket (Acheta domesticus)
Comp. Biochem. Physiol., 1973, Vol. 45B, pp. 59 to 70. Pergamon Press. Printed in Great Britain
A MICROSOMAL ENZYME I N H I B I T O R IN T H E G U T C O N T E N T S OF T H E HOUSE CRICKET
(ACHETA DOMESTICUS)* L. B. B R A T T S T E N and C. F. W I L K I N S O N Department of Entomology, Cornell University, Ithaca, New York 14850
(Received 26 September 1972) Abstract--1. An inhibitor of microsomal oxidation was isolated and partially purified from the soluble fraction of house cricket (Acheta domesticus) gut contents. 2. Inhibitory activity was greater towards armyworm gut microsomal enzymes than those from rat liver. 3. The material was characterized as a proteolytic enzyme with a molecular weight of approximately 16,500. 4. Proteolytic and inhibitory activity were blocked by Soy trypsin inhibitor and phenylmethanesulfonyl fluoride but not by p-chloromercuribenzoate or reduced glutathione. 5. Inhibition apparently results from solubilization of NADPH cytochrome e reductase from the microsomal membrane.
INTRODUCTION IN ATTEMPTS to improve our understanding of the manner in which insects metabolize insecticides and other foreign compounds emphasis in recent years has been placed on in vitro studies of the microsomal enzymes (Terriere, 1968; Hodgson & Plapp, 1970; Wilkinson & Brattsten, 1973). Although such in vitro studies have clearly established that the mierosomal drug metabolizing enzymes in insects are biochemieally similar to those found in mammalian liver the investigations are often severely hampered by the presence of endogenous inhibitors liberated during the process of enzyme preparation. It is now clear that several endogenous materials with potentially inhibitory effects on one or more components of the microsomal enzyme complex may be released during the homogenization of insect tissues. The chances of encountering such materials are of course greatest when such heterogeneous tissues as whole insects are used as an enzyme source, but they can also constitute important artifacts when specific body regions or individual organs are employed. The insect eye pigment, xanthommatin, has recently been established as an important inhibitory factor in preparations from whole houseflies (Musca * The work was supported by grants from the U.S. Public Health Service (ES-00400) and the Rockefeller Foundation (RF 69073). 59
60
L. B. BRATrSTm~AND C. F. WILKXNSO~
domestica) (Schonbrod & Terriere, 1971, 1972) fruit flies (Drosophila melanogaster) and honey bees (Nakatsugawa, personal communication) and has been shown to act as an electron sink at the flavoprotein, N A D P H cytochrome c reductase of the microsomal electron transport chain (Nakatsugawa, personal communication; Schonbrod & Terriere, 1972; Wilson & Hodgson, 1972). Although it is probable that xanthommatin constitutes the major inhibitory factor associated with the insect head, other inhibitors have been reported to occur in the abdominal region of houseflies (Tsukamoto & Casida, 1967; Jordan et al., 1968; Jordan & Smith, 1970) and cockroaches (Periplaneta americana) (Nakatsugawa & Dahm, 1965; Fukami et al., 1969). A potent inhibitor of microsomal oxidation has been isolated and partially purified from the gut contents of the southern armyworm (Prodenia eridania) (Krieger & Wilkinson, 1970). The material was characterized as a proteolytie enzyme and subsequent studies have established that its inhibitory activity results from an ability to solubilize N A D P H cytochrome c reduetase from the microsomal membrane (Orrenius et al., 1971). Inhibitory activity has also been associated with the gut contents of other species of lepidopterous larvae (Krieger, 1970; Kuhr, 1970, 1971) and a caddisfly larva (Limnephilus sp.) (Krieger & Lee, 1973) suggesting that such materials may have a broad natural distribution. In the course of recent investigations in this laboratory another endogenous microsomal enzyme inhibitor has been encountered in the gut contents of the house cricket (Acheta domesticus) (Benke, 1971; Benke & Wilkinson, 1971). This paper describes the isolation and characterization of this material.
MATERIALS AND METHODS
Animals Larvae of the southern armyworm (Prodenia eridania Cramer) were maintained under greenhouse conditions as reported by Krieger & Wilkinson (1969) and house crickets (Acheta domesticus L.) initially obtained from Fluker's Cricket Farm, Baton Rouge, La., were reared as described by Benke & Wilkinson (1971). Male 7-8-week-old Sprague-Dawley rats were obtained from Blue Spruce Farms, Inc., Altamont, N.Y.
Chemicals Analytical grade aldrin (1,2,3,4,10,10-hexachloro-l,4,4a,5,8,-8a-hexahydro-l,4-endoexo-5,8-dimethanonaphthalene), dieldrin (the 6,7-epoxide of aldrin), dihydroisodrin (1,2, 3,4,10,10 - hexachloro-1,4,4a,5,6,7,8, 8a-octahydro*l, 4-endo--exo-5,8-dimethanonaphthalene) and 6-exo-monohydroxydihydroisodrin were kindly provided by the Shell Development Co., Modesto, Calif. p-Nitroanisole, p-nitrophenol, p-chloro N-methylaniline, p-chloroaniline, glucose-6-phosphate (G6-P), G6-P dehydrogenase, NADP, NADPH, vitamin free casein, bovine serum albumin, ~-chymotrypsinogen, trypsin, Soy trypsin inhibitor, reduced glutathione and phenylmethanesulfonyl fluoride were purchased from Calbiochem, Los Angeles, Calif. Lipase and phosphatase substrates, thymolphthalein indicator solution, ribonuclease B (bovine pancreas) and cytochrome c were obtained from Sigma Chemical Co., St. Louis, Mo., and the sodium salt of 4-chloromercuribenzoic acid was from Aldrich Chemical Co., Milwaukee, Wis. All other solvents and chemicals employed were of analytical reagent grade.
MICROSOMAL ENZYME I N H I B I T O R I N THE CRICKET
61
Preparation of cricket gut contents inhibitor T h e guts were removed from a large number of adult crickets and the gut contents were collected in ice-cold glass distilled water by gentle agitation so as not to disrupt the gut tissues. T h e resulting suspension was centrifuged at 10,000 g for 30 min and the supernatant recentrifuged at 150,000 g for 2 hr at 4 ° C in an International Equipment Co. (IEC) B-60 preparative ultracentrifuge with a fixed angle rotor (A-211). T h e high-speed supernatant was freeze dried and yielded a powder (Fraction I) which was stored at 0 ° C. Enzyme preparations T h e guts were removed from sixth instar armyworms and after removal of the gut contents the tissues were rinsed and homogenized in 1"15 % KCI as described by Krieger & Wilkinson (1969). T h e crude homogenate was centrifuged for 2 rain at 1000 g in an IEC clinical centrifuge and the supernatant, containing approximately 0.6-0.8 mg protein/ml was routinely used to measure oxidase activities. Armyworm microsomes were obtained by centrifugation at 4°C of a 10,000g (10 rain) supernatant at 105,000g for 1 hr and the resuiting pellet was resuspended in 1 "15 % KC1. Rat liver microsomes were prepared according to Ernster et al. (1962) and the final pellet was suspended in 1.15% KCI to give approximately 0-8 mg protein/ml. Protein determinations were carried out by the Biuret method (Robinson & Hogden, 1940) or a modified Folin-Ciocaheau method (Lowry et al., 1951). Enzyme assays T h e epoxidation of aldrin, hydroxylation of dihydroisodrin, N-demethylation ofp-chloro N-methylaniline and O-demethylation of p-nitroanisole were used as indicators of microsomal enzyme activity. T h e incubation mixtures and conditions as well as product analyses were as described by Krieger & Wilkinson (1969). In the case of the O-demethylation of p-nitroanisole the reaction was terminated by the addition of 1"0 ml 5% K O H and the absorption of the 10,000 g (10 re_in) supernatant was measured at 400 nm against appropriate blanks in a Norelco ® Unicam SP-800 double beam spectrophotometer (Netter & Seidel, 1964). Is0 values (the amount required to cause 50 per cent inhibition) were determined graphically from plots of percentage inhibition vs. log of dry weight of Fraction I. Proteolytic enzyme activity was measured by a method similar to that described by Kunitz (1947) in incubations containing 2"5 ml 0"5% casein in 0"05 M T r i s - H C l buffer, p H 7"4. Assays were for 30 rain at 30°C. T h e proteolytic activity was estimated as AO.D.2s0, per 30 rain measured against appropriate blanks. Lipase activity was measured titrimetrically using N a O H and thymolphthalein indicator after incubation with 3"0 ml Sigma lipase substrate and 1"0 ml 0"1 M Tris-HC1 buffer, p H 7-8 for 2 hr at 30°C. T h e incubation system was modified from the method described in Sigma Technical Bulletin No. 800 (1963). Phosphatase activity was measured after incubation with 0"5 mg p-nitrophenylphosphate (Sigma 104) in 2"5 ml 0-1 M T r i s - H C l buffer ranging in p H from 5"9 to 8"0 for 30 min at 30°C as the increase in absorbance at 400 nm (the p-nitrophenolate ion) against appropriate blanks. N A D P H - c y t o c h r o m e c reductase activity was determined from the increase in the band of reduced cytochrome c (550 nm) during the first 2 min of reaction following the addition of 0"1 pmoles N A D P H to the enzyme suspended in 0"05 M potassium phosphate buffer, p H 7.8, m M with respect to cyanide and containing 0"5 mg cytochrome c/ml. Reductase activity is expressed as units per mg protein, 1 unit being defined as that amount of enzyme which causes an increase in optical density of 1"0 per rain with 1 cm light path (Wilson, 1970). Cytochrome P--450 was measured in microsomal suspension by the method described by Omura & Sato (1964) and expressed as AO.D.490-450/mg protein.
62
L . B . BRATTSTENAND Co F. WILKINSON
Partial purification of gut contents material
Aqueous solutions containing 30 mg/ml of Fraction I were applied to a 2"8 × 30 cm Sephadex G-100 column equilibrated at 4°C with 0.05 M Tris-HCl buffer, pH 7"8 at a flow rate of 20 ml/hr. The eluate was continuously monitored for protein at 280 nm by a Uvicord II spectrophotometric scanner connected to the LKB Ultrorac ® refrigerated fraction collector. Consecutive 2.4-ml fractions were collected and assayed for enzymatic and inhibitory activities. Fractions containing inhibitory material were combined and are referred to as Fraction II. The molecular weight of this fraction was estimated by reference to the retention times of standards including ribonuclease B, ~-chymotrypsinogen and bovine serum albumin chromatographed on the same column under identical conditions. The void volume was determined by blue dextran. RESULTS T h e inhibitory material in the cricket gut contents was associated with the soluble fraction (105,000 g, 2 hr supernatant) and in the freeze-dried state could be stored for periods of up to 18 months without significant loss of activity. T h e initial freeze-dried material (Fraction I) proved to be a potent inhibitor of aldrin epoxidation, dihydroisodrin hydroxylation, N - and O-demethylation in crude enzyme preparations and microsomes from armyworm gut. Each of the reactions was equally susceptible to inhibition (I60 0.45-0.49 mg) in the crude preparations (Table 1) although an approximately threefold increase in inhibition of epoxidation (I5o 0.17 rag) was observed when armyworm gut microsomes were employed. In contrast, suspensions of rat liver microsomes at approximately the same protein concentration appeared considerably more refractory to inhibitory attack (I50 7.0 mg). TABLE 1--INHIBITION OF MIXED FUNCTION OXIDASEACTIVITYBY FRACTION I OF THE CRICKETGUT CONTENTSINHIBITOR
Is0 (rag/incubation) Crude armyworm gut homogenate Epoxidation Hydroxylation N-demethylation O-demethylation Armyworm gut microsome Epoxidation Rat liver microsome Epoxidation Hydroxylation
0'49 0'49 0'45 0"45 0.17 7"0 7"0
Aqueous solutions of Fraction I retained full activity when stored at 4°C for up to 3 weeks whereas at 30°C 10 per cent of the inhibitory activity was lost in 2 hr and at 60°C only 50 per cent of the initial activity remained after 20 rain. One min at 100°C completely eliminated activity. Solutions of Fraction I were unaffected by p H over a fairly broad range (5.9-8.5) but exhibited a 17 and 34 per cent reduction in inhibitory activity after being held for 30 rain at p H values
63
MICROSOMAL ]~qZY1VI~ I N H I B I T O R I N THE CRICKET
of 12 and 2 respectively. Inhibition was also decreased by 12 per cent when solutions of Fraction I were subjected to an increasing ionic strength by addition of KC1 up to 0.2 M. Experiments in which both armyworm gut and rat liver enzyme preparations were preincubated with Fraction I for varying periods of time at 30°C established that inhibition was progressive with time (Fig. 1). Thus an inhibitor concentration which without preincubation resulted in a 10 per cent inhibition of epoxidation in the armyworm gut preparation caused 100 per cent inhibition after a 10min preincubation period and a similar increase in inhibitory activity occurred over a period of 18.5 min with rat liver microsomes. The progressive nature of the inhibition and its previously established heat lability suggested that the inhibitory factor in the gut contents material might be associated with some kind of enzymatic activity. A preliminary examination of Fraction I revealed that it contained substantial levels of proteolytic (caseinolytic) activity as well as acid and alkaline phosphatase and lipase activities and an attempt was made to determine which if any of these was associated with inhibitory activity.
1S so u
I 5
I 10
An~wonn
! 15
gut
I
20
Time (min)
FIG. 1. Progressive inhibition of aldrin epoxidation by Fraction I. The passage of Fraction I through a Sephadex G-100 gel column in 0.05 M Tris-HC1 buffer, pH 7.8, revealed the presence of one major (A) and two incompletely resolved minor (B and C) protein peaks (Fig. 2). The major protein peak (A) eluted with the void volume and although it exhibited lipase and acid and alkaline phosphatase activities it showed no inhibition towards microsomal enzyme activity. Of the two smaller incompletely resolved protein peaks which were retained by the column peak B (Fraction II) exhibited both proteolytic and inhibitory activity (Fig. 2). The I50 value of Fraction II towards aldrin epoxidation in the crude armyworm gut preparation (0.12 mg protein per incubation) indicated an approximately fourfold purification of the inhibitory material. The molecular weight of Fraction II as determined by direct comparison with a calibration curve of several standard proteins of known molecular weight (Fig. 3) was 16,500. It is, however, probable that in solutions of distilled water the material exists in an aggregated form since the inhibitory activity was associated with the
64
L.B. BRATTSTENAND C. F. WILKINSON
void volume fraction when eluted from the same Sephadex G-100 column with distilled water. 10 -
• Protein .............. Lipase • .... Phosphata~
A
• ......
Casei~lysisinhibition Microsornal
A -
C
0.5
-
.-'#~
t
il
'
!/
<
o.o
~
. ................
I
"-,,:,:.,2..~--.-..~,.?*.~...
I
I
I
30
I
I
60
I
90
Elution v o l u m e
[
i
120
(ml)
FIG. 2. Fractionation of cricket gut contents material on S e p h a d e x G-100.
120
g
RNa
Cricket gut c~ntents inhibitor
100 a Chymotrypsinogen
o o_: L~ 70
A I
2 Molecular
4 weight
6
8
x l O" s
FIG. 3. M o l e c u l a r w e i g h t estimation o f cricket gut contents material.
In view of the apparent association between the proteolytic and inhibitory activities of the gut content material, the proteolytic enzyme properties were studied in greater detail using casein as a substrate. Fraction I showed substantial caseinolytic activity and the activity of Fraction II was approximately equal to that of commercial trypsin. This represents a similar degree of purification to that obtained with respect to inhibitory activity. The pH optimum for the caseinolytic activity of Fraction I was 7.4-7.5 in either 0.05 M Tris-HC1 or 0.05 M phosphate buffers (Fig. 4). Activity increased with increasing amounts of Fraction I (Fig. 5) and the reaction was a linear function of time for periods of up to 90 min (Fig. 6). The addition of several metal ions to the incubations established that
MICROSOMAL ENZYME INHIBITOR IN THE CRICKET
65
m M concentrations of Mg ~+, Ca 2+, Mn 2+, Co 2+, Cu 2+, Ag+ and Cr a+ had little or no effect on caseinolytic activity whereas the same concentrations of Fe 3+, Zn 2+, Hg ~+, Sn 2+ and Ni 2+ caused 85, 43, 33, 28 and 21 per cent inhibition respectively. Activity was enhanced in the presence of Cu + and Fe ~+ ions. Proteolytic activity was reduced by 14 and 9 per cent respectively in the presence of mM concentrations of p-chloromercuribenzoate, and reduced glutathione but the addition of 1 mg of Soy trypsin inhibitor resulted in a 50 per cent decrease in proteolysis. The most effective inhibitor was phenylmethanesulfonyl fluoride which caused a 61 per cent reduction in caseinolytic activity at 10 -6 M. 100
.>
//
"~ U D.,
~
~--~.
0.05 M Tris41CI
I
I
I
6
7'
8
pH
FIe. 4. Effect of pH and buffers on e~einolytic activity of Fraction I.
==
50
.>_
10 i
2.0 '
Fraction I ( r a g )
FIG. 5. Effect of concentration of cricket gut contents material (Fraction I) on caseinolytic activity. The effect of these and other reagents on the inhibitory properties of the gut contents material towards aldrin epoxidase in the crude armyworm gut preparation were also determined. The results of Table 2 showed that the addition of 0.05
66
L . B . B ~ T r s ' r ~ Arm C. F. Wz~nqSON
o so
I
I
25
.SO Time
I 75
(min)
FIG. 6. Rate of caseinolysis of cricket gut contents material. m-moles of p-chloromercuribenzoate or reduced glutathione had no effect on the level of inhibition caused by 0.5 m g of Fraction I. I n contrast, the addition of a similar a m o u n t of phenylmethanesulfonyl fluoride resulted in a 41 per cent reversal of inhibition. L o w concentrations of bovine s e r u m albumin had no effect on the level of inhibition observed although at the high concentration of 12.5 m g per incubation it reversed the inhibitory action of Fraction I by 34 per cent in crude a r m y w o r m gut homogenates (Table 2). T h e addition of BSA or TABLE 2--EWEcT WORM
OF V A R I O U S A D D I T I O N S
O N EPOXIDASE ACTIVITY IN CRUDE A R M Y -
GUT PREPARATION I N THE ABSENCE AND PRESENCE OF FRACTION I
Activity (%) Additions to the complete incubation mixture (5 ml) None 0"25/zmoles NADPH (replacing the NADPH-generating system) 0"05 m-moles p-chloromercuribenzoate 0"5 m-mole reduced glutathione 100/zg BSA 12"5 mg BSA 0"5 m-mole phenylmethanesulfonyl fluoride None 2 mg trypsin + 2 mg Soy trypsin inhibitor* 2 mg trypsin+ 2 mg Soy trypsin inhibitort 1 mg Soy trypsin inhibitor
Without Fraction I
With Fraction I
(mg)
100
50
(0"5)
92 100 100 97 98 98 100 93 93 96
50 51 50 50 84 91 16 62 55 85
(0"5) (0"5) (0"5) (0"5) (0"5) (0-5) (1 "0) (1 "0) (1"0) (1"0)
*Fraction I preincubated for 5 min at 30°C with trypsin. Soy trypsin inhibitor added to inactivate trypsin and terminate preincubation. ~Fraction I preincubated for 5 n'tin at 30°C with both trypsin and Soy trypsin inhibitor (inactivated trypsin).
67
MICROSOMAL ENZYME I N H I B I T O R I N THE CRICKET
casein to armyworm gut microsomes at 200/~g per incubation caused no reversal of inhibition. Experiments in which Fraction I was preincubated with trypsin prior to addition to the microsomes initially suggested that the inhibitor was being partially destroyed by tryptic action (Table 2). However, the established sensitivity of microsomes to trypsin proteolysis (Orrenius et al., 1969) dictated that after the preincubation period all trypsin be deactivated by addition of Soy trypsin inhibitor and subsequent experiments established that it was the latter material and not trypsin which was responsible for the observed reversal of inhibition (Table 2). In the presence of 1 mg of Soy trypsin inhibitor per incubation the inhibitory activity of Fraction I (1 mg per incubation) was reversed by 60 per cent. Soy trypsin inhibitor alone at this concentration had no effect on microsomal enzyme activity. Replacement of the NADPH-generating system with N A D P H in the microsomal incubation medium did not change the inhibitory effect of Fraction I (Table 2) strongly suggesting that the inhibition involved direct proteolytic attack on some component of the microsomal enzyme complex. The effect of Fraction I on components of the microsomal electron transport chain was determined by incubating the 10,000 g mitochondrial supernatant with the inhibitor TABLE
3--EFFECT
OF CRICKET GUT CONTENTS I N H I B I T O R ON COMPONENTS OF MICROSOMAL ELECTRON TRANSPORT CHAIN
Inhibition of microsomal aldrin epoxidase after preincubation (%)* 30 (0"7)t 13 (0.5) 24 (50)
33 (1) 24 (50)
NADPH cytochrome c reductase activity (units per mg protein) Treatment
Microsome
Supematant
Control Preincubated (10 min) Control Preincubated (5 min) Control Preincubated (20 rain)
1"247 0.462
0.235 0.787
1"509 Armyworm gut 1"249
1-454 1.242
0-327 0.517
1-781 1"759
0"982 0"662
0.056 0.095
1"038 Rat liver 0"757
Control Preincubated (5 min) Control Preineubated (20 rain)
Total
Microsomal cytochrome P-450 (AO.D.,~o-oo per mg protein) 0"032 0"030 0-075 0"071
Enzyme source
Armyworm gut
Armyworm gut Rat liver
* These experiments were performed with a batch of inhibitor less active than that employed in other parts of this investigation. t mg of Fraction I added to 10,000 g supernatant.
68
L . B . BRATTSTEN AND C. F. WILKINSON
and subsequently centrifuging the incubate at 100,000 g for 1 hr to obtain the microsomes. Measurement of the cytochrome P-450 content of microsomes treated in this manner indicated no change from controls with either armyworm gut or rat liver enzyme preparations (Table 3). It was, however, established that NADPH cytochrome c reductase activity in the armyworm gut microsomal pellet was decreased as a result of preincubation with Fraction I and that this was accompanied by an increase of reductase activity in the soluble (100,000 g supernatant) fraction (Table 3). The shift of NADPH cytochrome c reductase from the microsomal to the soluble fractions was increased with increasing degree of exposure of the enzyme to Fraction I (concentration or preincubation time). The total NADPH cytochrome c reductase activity recovered (Table 3) (microsomal plus soluble) was observed to decrease with increasing action of Fraction I probably indicating some destruction of enzymatic activity (Table 3). At much higher levels of exposure to Fraction I a similar effect was observed with rat liver microsomes.
DISCUSSION The inhibition of microsomal enzyme activity by the gut contents material of the house cricket appears to be closely associated with a proteolytic enzyme. The inhibitory factor is a soluble, heat labile material with a molecular weight of 16,500 and its inhibition by phenylmethanesulfonyl fluoride and Soy trypsin inhibitor suggests that it is closely related to trypsin. The mechanism by which it inhibits microsomal enzyme activity is probably related to its ability to solubilize the flavoprotein, NADPH cytochrome c reductase from the microsomal membrane and consequently to disrupt the flow of electrons from NADPH to cytochrome P-450. The activity is similar to that previously reported to occur with trypsin (Orrenius et al., 1969) as well as the microsomal enzyme inhibitor present in the gut contents of southern armyworm (P. eridania) larvae (Orrenius et al., 1971). Unlike the armyworm gut inhibitor which exhibits a similar degree of activity towards microsomes from both mammalian liver and insect tissues the cricket gut contents material is approximately fortyfold more active towards insect microsomes than those from rat liver. The higher resistance to inhibition by rat liver microsomes is reflected by both the lower rate of progressive inhibition and inferior ability of the inhibitor to solubilize NADPH cytochrome c reductase in this preparation. The inhibitory factor in the cricket gut contents has a lower molecular weight (16,500) than that isolated from armyworm larvae (26,000) and is considerably more heat labile (Krieger & Wilkinson, 1970). In addition, the microsomal enzyme inhibition resulting from the cricket gut contents is much less susceptible to reversal by BSA than that from the armyworm gut contents. With the latter material substantial reversal of inhibition was obtained with 80tzg BSA per incubation whereas even in the presence of 200/zg BSA inhibition by the cricket gut contents was not reversed. Some reversal of inhibition was, however, observed in crude armyworm gut homogenates at extremely high levels (12.5 mg per
MICROSOMAL ENZYME I N H I B I T O R I N THE CRICKET
69
incubation). Another difference between these two materials is that the action of the cricket gut contents material, but not that from the armyworm gut is susceptible to reversal by Soy trypsin inhibitor. Although the armyworm and cricket gut contents materials appear to have a similar action on the microsomes it is evident therefore that they are not identical. It is probable that many other kinds of proteolytic enzymes could cause similar effects and that such materials could be of general significance in in vitro studies of insect microsomes. T h e importance of these inhibitors will depend largely on their localization in the insect relative to that of microsomal activity. T h u s the fact that maximum microsomal enzyme activity in the cricket is localized in the Malpighian tubules (Benke, 1971; Benke & Wilkinson, 1971) decreases the practical importance of the cricket gut contents inhibitor. In the armyworm, however, maximum enzyme activity is associated with the mid-gut tissues (Krieger & Wilkinson, 1969) and contamination of these preparations with the gut contents present a more serious problem. REFERENCES
ANONYMOUS (1963) The titrimetic determination of serum lipase. Sigma Technical Bull. No. 800. BENI~ G. M. (1971) Microsomal oxidases in selected orthopteran insects, with special reference to the house cricket, Acheta domesticus, and the Madagascar roach, Gromphadorhina portentosa. M. S. thesis, Cornell University, Ithaca, New York. BENK.EG. M. & WILKINSONC. F. (1971) Microsomal oxidation in the house cricket, Acheta domesticus (L.). Pestic. Biochem. Physiol. 1, 19-31.
ErU~STER L., SIE~VITZ P. & PALADEG. E. (1962) Enzyme-structure relationships in the endoplasmic reticulum of rat liver, ft. Cell Biol. 15, 541-562. FUK~MI J. I., SnISHIDO T., FtrKUNAGAK. & CASlDAJ. E. (1969) Oxidative metabolism of rotenone in mammals, fish and insects and its relation to selective toxicity. J. agric. Fd Chem. 17, 1217-1226. HODOSON E. & PLAPP F. W. (1970) Biochemical characteristics of insect microsomes. J. agric. Fd Chem. 18, 1048-1055. JORDA~ T. W. & SMITHJ. N. (1970) Oxidation inhibitors in homogenates of houseflies and blowflies. Int. ff. Biochem. 1, 139-149. JORDAN T. W., SMITH J. N. & WHITEHEADN. (1968) Preparation of housefly oxidation enzymes. Australas. ft. Pharm. 49, Suppl. 66. KRIEGER R. I. (1970) Microsomal oxidases in selected lepidopterous larvae, primarily the southern armyworm, Prodenia eridania. Ph.D. thesis, Cornell University, Ithaca, New York. KRIECER R. I. & LEE P. W. (1973) Properties of the aldrin epoxidase system in the gut and fat body of a caddisfly larva. J. econ. Ent. (In press.) KRIECERR. I. & WILKINSONC. F. (1969) Microsomal mixed-function oxidases in insects--I. Localization and properties of an enzyme system effecting aldrin epoxidation in larvae of the southern armyworm (Prodenia eridania). Biochem. Pharmac. 18, 1403-1415. KRIECER R. I. & WILKINSON C. F. (1970) An endogenous inhibitor of mixed-function oxidases in homogenates of the southern armyworm (Prodenia eridania). Biochem. ft. 116, 781-789. KUHR R. J. (1970) Metabolism of carbamate insecticide chemicals in plants and insects. J. agric. Fd Chem. 18, 1023-1030. KUHR R. J. (1971) Comparative metabolism of carbaryl resistant and susceptible strains of the cabbage looper. J. econ. Ent. 64, 1373-1378.
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L. B. BRATTST~ AND C. F. WILKINSON
KUNITZ M. (1947) Crystalline soybean trypsin inhibitor~ t. gen. Physiol. 20, 291-310. LOWRY O. H., ROSI~BROUGHN. J., FARRA. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. 3. biol. Chem. 193, 265-275. NAKATSUCAWAT. & DAHM P. A. (1965) Parathion activation in the fat body microsomes of the American cockroach..~, econ. Ent. 58, 500-509. NETamR K. J. & SEID•L G. (1964) An adaptively stimulated O-demethylating system in rat liver microsomes and its kinetic properties..7. Pharmac. exp. Ther. 146, 61--65. OMURA T. & SATO R. (1964) The carbon-monoxide-binding pigment of liver microsomes. •. biol. Chem. 239, 2370-2378. OmmNtUS S., BERG A. & EaNSaXR L. (1969) Effects of trypsin on the electron transport systems of liver microsomes. Eur..7. Biochem. 11, 193-200. ORm~IUS S., B ~ G G ~ M., MOLOEUSP. & KatEGER R. I. (1971) Mechanism of inhibition of microsomal mixed-function oxidation by the gut-contents inhibitor of the southern armyworm (Prodenia eridania). Biochem..7. 124, 427-430. ROBINSONH. W. & HOODEN C. C. (1940) The biuret reaction in the determination of serum proteins..~, biol. Chem. 135, 727-731. SCHONnRODR. D. & TEimIEI~ L. C. (1971) Eye pigments as inhibitors of microsomal aldrin epoxidase in the housefly. J. econ. Ent. 64, aa A5" SCHONBRODR. D. & TEmaml~ L. C. (1973) Inhibition of housefly microsomal epoxidase by the eye pigment xanthommatin. Pestic. Biochem. Physiol. (In press.) TEmaiFa~ L. C. (1968) Insecticide-cytoplasmic interactions in insects and vertebrates. .4. Rev. Ent. 13, 75-98. TSUKAMOTO M. & CASlDAJ. E. (1967) Metabolism of methylcarbamate insecticides by the N A D P H s requiring enzyme system from houseflies. Nature Lond. 213, 49-51. WILKINSON C. F. & BRATTSTEN L. B. (1973) Microsomal drug metabolizing enzymes in insects. Drug Metab. Rev. 1, 153-228. WmsoN T. G. (1970) NADPH-cytochrome c reductase in housefly microsomes. M. S. thesis, North Carolina State University, Raleigh, North Carolina. WILSON T. G. & HODGSON E. (1972) Mechanism of a microsomal mixed-function oxidase inhibitor from the housefly, Musca domestica L. Pestle. Bioehem. Physiol. 2, 64-71. Key Word Index--Microsomal enzyme inhibitor; Acheta domesticus; NADPH-cytochrome c reductase.
A MICROSOMAL ENZYME I N H I B I T O R IN T H E G U T C O N T E N T S OF T H E HOUSE CRICKET
(ACHETA DOMESTICUS)* L. B. B R A T T S T E N and C. F. W I L K I N S O N Department of Entomology, Cornell University, Ithaca, New York 14850
(Received 26 September 1972) Abstract--1. An inhibitor of microsomal oxidation was isolated and partially purified from the soluble fraction of house cricket (Acheta domesticus) gut contents. 2. Inhibitory activity was greater towards armyworm gut microsomal enzymes than those from rat liver. 3. The material was characterized as a proteolytic enzyme with a molecular weight of approximately 16,500. 4. Proteolytic and inhibitory activity were blocked by Soy trypsin inhibitor and phenylmethanesulfonyl fluoride but not by p-chloromercuribenzoate or reduced glutathione. 5. Inhibition apparently results from solubilization of NADPH cytochrome e reductase from the microsomal membrane.
INTRODUCTION IN ATTEMPTS to improve our understanding of the manner in which insects metabolize insecticides and other foreign compounds emphasis in recent years has been placed on in vitro studies of the microsomal enzymes (Terriere, 1968; Hodgson & Plapp, 1970; Wilkinson & Brattsten, 1973). Although such in vitro studies have clearly established that the mierosomal drug metabolizing enzymes in insects are biochemieally similar to those found in mammalian liver the investigations are often severely hampered by the presence of endogenous inhibitors liberated during the process of enzyme preparation. It is now clear that several endogenous materials with potentially inhibitory effects on one or more components of the microsomal enzyme complex may be released during the homogenization of insect tissues. The chances of encountering such materials are of course greatest when such heterogeneous tissues as whole insects are used as an enzyme source, but they can also constitute important artifacts when specific body regions or individual organs are employed. The insect eye pigment, xanthommatin, has recently been established as an important inhibitory factor in preparations from whole houseflies (Musca * The work was supported by grants from the U.S. Public Health Service (ES-00400) and the Rockefeller Foundation (RF 69073). 59
60
L. B. BRATrSTm~AND C. F. WILKXNSO~
domestica) (Schonbrod & Terriere, 1971, 1972) fruit flies (Drosophila melanogaster) and honey bees (Nakatsugawa, personal communication) and has been shown to act as an electron sink at the flavoprotein, N A D P H cytochrome c reductase of the microsomal electron transport chain (Nakatsugawa, personal communication; Schonbrod & Terriere, 1972; Wilson & Hodgson, 1972). Although it is probable that xanthommatin constitutes the major inhibitory factor associated with the insect head, other inhibitors have been reported to occur in the abdominal region of houseflies (Tsukamoto & Casida, 1967; Jordan et al., 1968; Jordan & Smith, 1970) and cockroaches (Periplaneta americana) (Nakatsugawa & Dahm, 1965; Fukami et al., 1969). A potent inhibitor of microsomal oxidation has been isolated and partially purified from the gut contents of the southern armyworm (Prodenia eridania) (Krieger & Wilkinson, 1970). The material was characterized as a proteolytie enzyme and subsequent studies have established that its inhibitory activity results from an ability to solubilize N A D P H cytochrome c reduetase from the microsomal membrane (Orrenius et al., 1971). Inhibitory activity has also been associated with the gut contents of other species of lepidopterous larvae (Krieger, 1970; Kuhr, 1970, 1971) and a caddisfly larva (Limnephilus sp.) (Krieger & Lee, 1973) suggesting that such materials may have a broad natural distribution. In the course of recent investigations in this laboratory another endogenous microsomal enzyme inhibitor has been encountered in the gut contents of the house cricket (Acheta domesticus) (Benke, 1971; Benke & Wilkinson, 1971). This paper describes the isolation and characterization of this material.
MATERIALS AND METHODS
Animals Larvae of the southern armyworm (Prodenia eridania Cramer) were maintained under greenhouse conditions as reported by Krieger & Wilkinson (1969) and house crickets (Acheta domesticus L.) initially obtained from Fluker's Cricket Farm, Baton Rouge, La., were reared as described by Benke & Wilkinson (1971). Male 7-8-week-old Sprague-Dawley rats were obtained from Blue Spruce Farms, Inc., Altamont, N.Y.
Chemicals Analytical grade aldrin (1,2,3,4,10,10-hexachloro-l,4,4a,5,8,-8a-hexahydro-l,4-endoexo-5,8-dimethanonaphthalene), dieldrin (the 6,7-epoxide of aldrin), dihydroisodrin (1,2, 3,4,10,10 - hexachloro-1,4,4a,5,6,7,8, 8a-octahydro*l, 4-endo--exo-5,8-dimethanonaphthalene) and 6-exo-monohydroxydihydroisodrin were kindly provided by the Shell Development Co., Modesto, Calif. p-Nitroanisole, p-nitrophenol, p-chloro N-methylaniline, p-chloroaniline, glucose-6-phosphate (G6-P), G6-P dehydrogenase, NADP, NADPH, vitamin free casein, bovine serum albumin, ~-chymotrypsinogen, trypsin, Soy trypsin inhibitor, reduced glutathione and phenylmethanesulfonyl fluoride were purchased from Calbiochem, Los Angeles, Calif. Lipase and phosphatase substrates, thymolphthalein indicator solution, ribonuclease B (bovine pancreas) and cytochrome c were obtained from Sigma Chemical Co., St. Louis, Mo., and the sodium salt of 4-chloromercuribenzoic acid was from Aldrich Chemical Co., Milwaukee, Wis. All other solvents and chemicals employed were of analytical reagent grade.
MICROSOMAL ENZYME I N H I B I T O R I N THE CRICKET
61
Preparation of cricket gut contents inhibitor T h e guts were removed from a large number of adult crickets and the gut contents were collected in ice-cold glass distilled water by gentle agitation so as not to disrupt the gut tissues. T h e resulting suspension was centrifuged at 10,000 g for 30 min and the supernatant recentrifuged at 150,000 g for 2 hr at 4 ° C in an International Equipment Co. (IEC) B-60 preparative ultracentrifuge with a fixed angle rotor (A-211). T h e high-speed supernatant was freeze dried and yielded a powder (Fraction I) which was stored at 0 ° C. Enzyme preparations T h e guts were removed from sixth instar armyworms and after removal of the gut contents the tissues were rinsed and homogenized in 1"15 % KCI as described by Krieger & Wilkinson (1969). T h e crude homogenate was centrifuged for 2 rain at 1000 g in an IEC clinical centrifuge and the supernatant, containing approximately 0.6-0.8 mg protein/ml was routinely used to measure oxidase activities. Armyworm microsomes were obtained by centrifugation at 4°C of a 10,000g (10 rain) supernatant at 105,000g for 1 hr and the resuiting pellet was resuspended in 1 "15 % KC1. Rat liver microsomes were prepared according to Ernster et al. (1962) and the final pellet was suspended in 1.15% KCI to give approximately 0-8 mg protein/ml. Protein determinations were carried out by the Biuret method (Robinson & Hogden, 1940) or a modified Folin-Ciocaheau method (Lowry et al., 1951). Enzyme assays T h e epoxidation of aldrin, hydroxylation of dihydroisodrin, N-demethylation ofp-chloro N-methylaniline and O-demethylation of p-nitroanisole were used as indicators of microsomal enzyme activity. T h e incubation mixtures and conditions as well as product analyses were as described by Krieger & Wilkinson (1969). In the case of the O-demethylation of p-nitroanisole the reaction was terminated by the addition of 1"0 ml 5% K O H and the absorption of the 10,000 g (10 re_in) supernatant was measured at 400 nm against appropriate blanks in a Norelco ® Unicam SP-800 double beam spectrophotometer (Netter & Seidel, 1964). Is0 values (the amount required to cause 50 per cent inhibition) were determined graphically from plots of percentage inhibition vs. log of dry weight of Fraction I. Proteolytic enzyme activity was measured by a method similar to that described by Kunitz (1947) in incubations containing 2"5 ml 0"5% casein in 0"05 M T r i s - H C l buffer, p H 7"4. Assays were for 30 rain at 30°C. T h e proteolytic activity was estimated as AO.D.2s0, per 30 rain measured against appropriate blanks. Lipase activity was measured titrimetrically using N a O H and thymolphthalein indicator after incubation with 3"0 ml Sigma lipase substrate and 1"0 ml 0"1 M Tris-HC1 buffer, p H 7-8 for 2 hr at 30°C. T h e incubation system was modified from the method described in Sigma Technical Bulletin No. 800 (1963). Phosphatase activity was measured after incubation with 0"5 mg p-nitrophenylphosphate (Sigma 104) in 2"5 ml 0-1 M T r i s - H C l buffer ranging in p H from 5"9 to 8"0 for 30 min at 30°C as the increase in absorbance at 400 nm (the p-nitrophenolate ion) against appropriate blanks. N A D P H - c y t o c h r o m e c reductase activity was determined from the increase in the band of reduced cytochrome c (550 nm) during the first 2 min of reaction following the addition of 0"1 pmoles N A D P H to the enzyme suspended in 0"05 M potassium phosphate buffer, p H 7.8, m M with respect to cyanide and containing 0"5 mg cytochrome c/ml. Reductase activity is expressed as units per mg protein, 1 unit being defined as that amount of enzyme which causes an increase in optical density of 1"0 per rain with 1 cm light path (Wilson, 1970). Cytochrome P--450 was measured in microsomal suspension by the method described by Omura & Sato (1964) and expressed as AO.D.490-450/mg protein.
62
L . B . BRATTSTENAND Co F. WILKINSON
Partial purification of gut contents material
Aqueous solutions containing 30 mg/ml of Fraction I were applied to a 2"8 × 30 cm Sephadex G-100 column equilibrated at 4°C with 0.05 M Tris-HCl buffer, pH 7"8 at a flow rate of 20 ml/hr. The eluate was continuously monitored for protein at 280 nm by a Uvicord II spectrophotometric scanner connected to the LKB Ultrorac ® refrigerated fraction collector. Consecutive 2.4-ml fractions were collected and assayed for enzymatic and inhibitory activities. Fractions containing inhibitory material were combined and are referred to as Fraction II. The molecular weight of this fraction was estimated by reference to the retention times of standards including ribonuclease B, ~-chymotrypsinogen and bovine serum albumin chromatographed on the same column under identical conditions. The void volume was determined by blue dextran. RESULTS T h e inhibitory material in the cricket gut contents was associated with the soluble fraction (105,000 g, 2 hr supernatant) and in the freeze-dried state could be stored for periods of up to 18 months without significant loss of activity. T h e initial freeze-dried material (Fraction I) proved to be a potent inhibitor of aldrin epoxidation, dihydroisodrin hydroxylation, N - and O-demethylation in crude enzyme preparations and microsomes from armyworm gut. Each of the reactions was equally susceptible to inhibition (I60 0.45-0.49 mg) in the crude preparations (Table 1) although an approximately threefold increase in inhibition of epoxidation (I5o 0.17 rag) was observed when armyworm gut microsomes were employed. In contrast, suspensions of rat liver microsomes at approximately the same protein concentration appeared considerably more refractory to inhibitory attack (I50 7.0 mg). TABLE 1--INHIBITION OF MIXED FUNCTION OXIDASEACTIVITYBY FRACTION I OF THE CRICKETGUT CONTENTSINHIBITOR
Is0 (rag/incubation) Crude armyworm gut homogenate Epoxidation Hydroxylation N-demethylation O-demethylation Armyworm gut microsome Epoxidation Rat liver microsome Epoxidation Hydroxylation
0'49 0'49 0'45 0"45 0.17 7"0 7"0
Aqueous solutions of Fraction I retained full activity when stored at 4°C for up to 3 weeks whereas at 30°C 10 per cent of the inhibitory activity was lost in 2 hr and at 60°C only 50 per cent of the initial activity remained after 20 rain. One min at 100°C completely eliminated activity. Solutions of Fraction I were unaffected by p H over a fairly broad range (5.9-8.5) but exhibited a 17 and 34 per cent reduction in inhibitory activity after being held for 30 rain at p H values
63
MICROSOMAL ]~qZY1VI~ I N H I B I T O R I N THE CRICKET
of 12 and 2 respectively. Inhibition was also decreased by 12 per cent when solutions of Fraction I were subjected to an increasing ionic strength by addition of KC1 up to 0.2 M. Experiments in which both armyworm gut and rat liver enzyme preparations were preincubated with Fraction I for varying periods of time at 30°C established that inhibition was progressive with time (Fig. 1). Thus an inhibitor concentration which without preincubation resulted in a 10 per cent inhibition of epoxidation in the armyworm gut preparation caused 100 per cent inhibition after a 10min preincubation period and a similar increase in inhibitory activity occurred over a period of 18.5 min with rat liver microsomes. The progressive nature of the inhibition and its previously established heat lability suggested that the inhibitory factor in the gut contents material might be associated with some kind of enzymatic activity. A preliminary examination of Fraction I revealed that it contained substantial levels of proteolytic (caseinolytic) activity as well as acid and alkaline phosphatase and lipase activities and an attempt was made to determine which if any of these was associated with inhibitory activity.
1S so u
I 5
I 10
An~wonn
! 15
gut
I
20
Time (min)
FIG. 1. Progressive inhibition of aldrin epoxidation by Fraction I. The passage of Fraction I through a Sephadex G-100 gel column in 0.05 M Tris-HC1 buffer, pH 7.8, revealed the presence of one major (A) and two incompletely resolved minor (B and C) protein peaks (Fig. 2). The major protein peak (A) eluted with the void volume and although it exhibited lipase and acid and alkaline phosphatase activities it showed no inhibition towards microsomal enzyme activity. Of the two smaller incompletely resolved protein peaks which were retained by the column peak B (Fraction II) exhibited both proteolytic and inhibitory activity (Fig. 2). The I50 value of Fraction II towards aldrin epoxidation in the crude armyworm gut preparation (0.12 mg protein per incubation) indicated an approximately fourfold purification of the inhibitory material. The molecular weight of Fraction II as determined by direct comparison with a calibration curve of several standard proteins of known molecular weight (Fig. 3) was 16,500. It is, however, probable that in solutions of distilled water the material exists in an aggregated form since the inhibitory activity was associated with the
64
L.B. BRATTSTENAND C. F. WILKINSON
void volume fraction when eluted from the same Sephadex G-100 column with distilled water. 10 -
• Protein .............. Lipase • .... Phosphata~
A
• ......
Casei~lysisinhibition Microsornal
A -
C
0.5
-
.-'#~
t
il
'
!/
<
o.o
~
. ................
I
"-,,:,:.,2..~--.-..~,.?*.~...
I
I
I
30
I
I
60
I
90
Elution v o l u m e
[
i
120
(ml)
FIG. 2. Fractionation of cricket gut contents material on S e p h a d e x G-100.
120
g
RNa
Cricket gut c~ntents inhibitor
100 a Chymotrypsinogen
o o_: L~ 70
A I
2 Molecular
4 weight
6
8
x l O" s
FIG. 3. M o l e c u l a r w e i g h t estimation o f cricket gut contents material.
In view of the apparent association between the proteolytic and inhibitory activities of the gut content material, the proteolytic enzyme properties were studied in greater detail using casein as a substrate. Fraction I showed substantial caseinolytic activity and the activity of Fraction II was approximately equal to that of commercial trypsin. This represents a similar degree of purification to that obtained with respect to inhibitory activity. The pH optimum for the caseinolytic activity of Fraction I was 7.4-7.5 in either 0.05 M Tris-HC1 or 0.05 M phosphate buffers (Fig. 4). Activity increased with increasing amounts of Fraction I (Fig. 5) and the reaction was a linear function of time for periods of up to 90 min (Fig. 6). The addition of several metal ions to the incubations established that
MICROSOMAL ENZYME INHIBITOR IN THE CRICKET
65
m M concentrations of Mg ~+, Ca 2+, Mn 2+, Co 2+, Cu 2+, Ag+ and Cr a+ had little or no effect on caseinolytic activity whereas the same concentrations of Fe 3+, Zn 2+, Hg ~+, Sn 2+ and Ni 2+ caused 85, 43, 33, 28 and 21 per cent inhibition respectively. Activity was enhanced in the presence of Cu + and Fe ~+ ions. Proteolytic activity was reduced by 14 and 9 per cent respectively in the presence of mM concentrations of p-chloromercuribenzoate, and reduced glutathione but the addition of 1 mg of Soy trypsin inhibitor resulted in a 50 per cent decrease in proteolysis. The most effective inhibitor was phenylmethanesulfonyl fluoride which caused a 61 per cent reduction in caseinolytic activity at 10 -6 M. 100
.>
//
"~ U D.,
~
~--~.
0.05 M Tris41CI
I
I
I
6
7'
8
pH
FIe. 4. Effect of pH and buffers on e~einolytic activity of Fraction I.
==
50
.>_
10 i
2.0 '
Fraction I ( r a g )
FIG. 5. Effect of concentration of cricket gut contents material (Fraction I) on caseinolytic activity. The effect of these and other reagents on the inhibitory properties of the gut contents material towards aldrin epoxidase in the crude armyworm gut preparation were also determined. The results of Table 2 showed that the addition of 0.05
66
L . B . B ~ T r s ' r ~ Arm C. F. Wz~nqSON
o so
I
I
25
.SO Time
I 75
(min)
FIG. 6. Rate of caseinolysis of cricket gut contents material. m-moles of p-chloromercuribenzoate or reduced glutathione had no effect on the level of inhibition caused by 0.5 m g of Fraction I. I n contrast, the addition of a similar a m o u n t of phenylmethanesulfonyl fluoride resulted in a 41 per cent reversal of inhibition. L o w concentrations of bovine s e r u m albumin had no effect on the level of inhibition observed although at the high concentration of 12.5 m g per incubation it reversed the inhibitory action of Fraction I by 34 per cent in crude a r m y w o r m gut homogenates (Table 2). T h e addition of BSA or TABLE 2--EWEcT WORM
OF V A R I O U S A D D I T I O N S
O N EPOXIDASE ACTIVITY IN CRUDE A R M Y -
GUT PREPARATION I N THE ABSENCE AND PRESENCE OF FRACTION I
Activity (%) Additions to the complete incubation mixture (5 ml) None 0"25/zmoles NADPH (replacing the NADPH-generating system) 0"05 m-moles p-chloromercuribenzoate 0"5 m-mole reduced glutathione 100/zg BSA 12"5 mg BSA 0"5 m-mole phenylmethanesulfonyl fluoride None 2 mg trypsin + 2 mg Soy trypsin inhibitor* 2 mg trypsin+ 2 mg Soy trypsin inhibitort 1 mg Soy trypsin inhibitor
Without Fraction I
With Fraction I
(mg)
100
50
(0"5)
92 100 100 97 98 98 100 93 93 96
50 51 50 50 84 91 16 62 55 85
(0"5) (0"5) (0"5) (0"5) (0"5) (0-5) (1 "0) (1 "0) (1"0) (1"0)
*Fraction I preincubated for 5 min at 30°C with trypsin. Soy trypsin inhibitor added to inactivate trypsin and terminate preincubation. ~Fraction I preincubated for 5 n'tin at 30°C with both trypsin and Soy trypsin inhibitor (inactivated trypsin).
67
MICROSOMAL ENZYME I N H I B I T O R I N THE CRICKET
casein to armyworm gut microsomes at 200/~g per incubation caused no reversal of inhibition. Experiments in which Fraction I was preincubated with trypsin prior to addition to the microsomes initially suggested that the inhibitor was being partially destroyed by tryptic action (Table 2). However, the established sensitivity of microsomes to trypsin proteolysis (Orrenius et al., 1969) dictated that after the preincubation period all trypsin be deactivated by addition of Soy trypsin inhibitor and subsequent experiments established that it was the latter material and not trypsin which was responsible for the observed reversal of inhibition (Table 2). In the presence of 1 mg of Soy trypsin inhibitor per incubation the inhibitory activity of Fraction I (1 mg per incubation) was reversed by 60 per cent. Soy trypsin inhibitor alone at this concentration had no effect on microsomal enzyme activity. Replacement of the NADPH-generating system with N A D P H in the microsomal incubation medium did not change the inhibitory effect of Fraction I (Table 2) strongly suggesting that the inhibition involved direct proteolytic attack on some component of the microsomal enzyme complex. The effect of Fraction I on components of the microsomal electron transport chain was determined by incubating the 10,000 g mitochondrial supernatant with the inhibitor TABLE
3--EFFECT
OF CRICKET GUT CONTENTS I N H I B I T O R ON COMPONENTS OF MICROSOMAL ELECTRON TRANSPORT CHAIN
Inhibition of microsomal aldrin epoxidase after preincubation (%)* 30 (0"7)t 13 (0.5) 24 (50)
33 (1) 24 (50)
NADPH cytochrome c reductase activity (units per mg protein) Treatment
Microsome
Supematant
Control Preincubated (10 min) Control Preincubated (5 min) Control Preincubated (20 rain)
1"247 0.462
0.235 0.787
1"509 Armyworm gut 1"249
1-454 1.242
0-327 0.517
1-781 1"759
0"982 0"662
0.056 0.095
1"038 Rat liver 0"757
Control Preincubated (5 min) Control Preineubated (20 rain)
Total
Microsomal cytochrome P-450 (AO.D.,~o-oo per mg protein) 0"032 0"030 0-075 0"071
Enzyme source
Armyworm gut
Armyworm gut Rat liver
* These experiments were performed with a batch of inhibitor less active than that employed in other parts of this investigation. t mg of Fraction I added to 10,000 g supernatant.
68
L . B . BRATTSTEN AND C. F. WILKINSON
and subsequently centrifuging the incubate at 100,000 g for 1 hr to obtain the microsomes. Measurement of the cytochrome P-450 content of microsomes treated in this manner indicated no change from controls with either armyworm gut or rat liver enzyme preparations (Table 3). It was, however, established that NADPH cytochrome c reductase activity in the armyworm gut microsomal pellet was decreased as a result of preincubation with Fraction I and that this was accompanied by an increase of reductase activity in the soluble (100,000 g supernatant) fraction (Table 3). The shift of NADPH cytochrome c reductase from the microsomal to the soluble fractions was increased with increasing degree of exposure of the enzyme to Fraction I (concentration or preincubation time). The total NADPH cytochrome c reductase activity recovered (Table 3) (microsomal plus soluble) was observed to decrease with increasing action of Fraction I probably indicating some destruction of enzymatic activity (Table 3). At much higher levels of exposure to Fraction I a similar effect was observed with rat liver microsomes.
DISCUSSION The inhibition of microsomal enzyme activity by the gut contents material of the house cricket appears to be closely associated with a proteolytic enzyme. The inhibitory factor is a soluble, heat labile material with a molecular weight of 16,500 and its inhibition by phenylmethanesulfonyl fluoride and Soy trypsin inhibitor suggests that it is closely related to trypsin. The mechanism by which it inhibits microsomal enzyme activity is probably related to its ability to solubilize the flavoprotein, NADPH cytochrome c reductase from the microsomal membrane and consequently to disrupt the flow of electrons from NADPH to cytochrome P-450. The activity is similar to that previously reported to occur with trypsin (Orrenius et al., 1969) as well as the microsomal enzyme inhibitor present in the gut contents of southern armyworm (P. eridania) larvae (Orrenius et al., 1971). Unlike the armyworm gut inhibitor which exhibits a similar degree of activity towards microsomes from both mammalian liver and insect tissues the cricket gut contents material is approximately fortyfold more active towards insect microsomes than those from rat liver. The higher resistance to inhibition by rat liver microsomes is reflected by both the lower rate of progressive inhibition and inferior ability of the inhibitor to solubilize NADPH cytochrome c reductase in this preparation. The inhibitory factor in the cricket gut contents has a lower molecular weight (16,500) than that isolated from armyworm larvae (26,000) and is considerably more heat labile (Krieger & Wilkinson, 1970). In addition, the microsomal enzyme inhibition resulting from the cricket gut contents is much less susceptible to reversal by BSA than that from the armyworm gut contents. With the latter material substantial reversal of inhibition was obtained with 80tzg BSA per incubation whereas even in the presence of 200/zg BSA inhibition by the cricket gut contents was not reversed. Some reversal of inhibition was, however, observed in crude armyworm gut homogenates at extremely high levels (12.5 mg per
MICROSOMAL ENZYME I N H I B I T O R I N THE CRICKET
69
incubation). Another difference between these two materials is that the action of the cricket gut contents material, but not that from the armyworm gut is susceptible to reversal by Soy trypsin inhibitor. Although the armyworm and cricket gut contents materials appear to have a similar action on the microsomes it is evident therefore that they are not identical. It is probable that many other kinds of proteolytic enzymes could cause similar effects and that such materials could be of general significance in in vitro studies of insect microsomes. T h e importance of these inhibitors will depend largely on their localization in the insect relative to that of microsomal activity. T h u s the fact that maximum microsomal enzyme activity in the cricket is localized in the Malpighian tubules (Benke, 1971; Benke & Wilkinson, 1971) decreases the practical importance of the cricket gut contents inhibitor. In the armyworm, however, maximum enzyme activity is associated with the mid-gut tissues (Krieger & Wilkinson, 1969) and contamination of these preparations with the gut contents present a more serious problem. REFERENCES
ANONYMOUS (1963) The titrimetic determination of serum lipase. Sigma Technical Bull. No. 800. BENI~ G. M. (1971) Microsomal oxidases in selected orthopteran insects, with special reference to the house cricket, Acheta domesticus, and the Madagascar roach, Gromphadorhina portentosa. M. S. thesis, Cornell University, Ithaca, New York. BENK.EG. M. & WILKINSONC. F. (1971) Microsomal oxidation in the house cricket, Acheta domesticus (L.). Pestic. Biochem. Physiol. 1, 19-31.
ErU~STER L., SIE~VITZ P. & PALADEG. E. (1962) Enzyme-structure relationships in the endoplasmic reticulum of rat liver, ft. Cell Biol. 15, 541-562. FUK~MI J. I., SnISHIDO T., FtrKUNAGAK. & CASlDAJ. E. (1969) Oxidative metabolism of rotenone in mammals, fish and insects and its relation to selective toxicity. J. agric. Fd Chem. 17, 1217-1226. HODOSON E. & PLAPP F. W. (1970) Biochemical characteristics of insect microsomes. J. agric. Fd Chem. 18, 1048-1055. JORDA~ T. W. & SMITHJ. N. (1970) Oxidation inhibitors in homogenates of houseflies and blowflies. Int. ff. Biochem. 1, 139-149. JORDAN T. W., SMITH J. N. & WHITEHEADN. (1968) Preparation of housefly oxidation enzymes. Australas. ft. Pharm. 49, Suppl. 66. KRIEGER R. I. (1970) Microsomal oxidases in selected lepidopterous larvae, primarily the southern armyworm, Prodenia eridania. Ph.D. thesis, Cornell University, Ithaca, New York. KRIECER R. I. & LEE P. W. (1973) Properties of the aldrin epoxidase system in the gut and fat body of a caddisfly larva. J. econ. Ent. (In press.) KRIECERR. I. & WILKINSONC. F. (1969) Microsomal mixed-function oxidases in insects--I. Localization and properties of an enzyme system effecting aldrin epoxidation in larvae of the southern armyworm (Prodenia eridania). Biochem. Pharmac. 18, 1403-1415. KRIECER R. I. & WILKINSON C. F. (1970) An endogenous inhibitor of mixed-function oxidases in homogenates of the southern armyworm (Prodenia eridania). Biochem. ft. 116, 781-789. KUHR R. J. (1970) Metabolism of carbamate insecticide chemicals in plants and insects. J. agric. Fd Chem. 18, 1023-1030. KUHR R. J. (1971) Comparative metabolism of carbaryl resistant and susceptible strains of the cabbage looper. J. econ. Ent. 64, 1373-1378.
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L. B. BRATTST~ AND C. F. WILKINSON
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