Inhibition of housefly microsomal epoxidase by the eye pigment, xanthommatin

PESTI(‘TIlE

13IO(‘HEMISTRY

.\ND

Inhibition

PHYSIOLOGY

1,

of Housefly Eye

409417

Microsomal

Pigment,

of Entomology,

Received

Oregon

September

by

the

Xanthommatin’

It. II. SCHONBROD Department

Epoxidase

ASD

State

L. C. TERRIERE University,

Corvallis,

2, 1971; accepted November

Oregon

97331

12, 1971

The material present in housefly heads which is known to inhibit the microsomal oxidase system in. vitro has been identified as xanthommatin, an ommochrome pigment of the housefly. This inhibits the activity of housefly microsomal epoxidase at concentrations as low as 5 X lo-’ M. It acts by competing with the system for electrons, probably at) the cytochrome c rednctase site. BSA, which is sometimes nsed to connteract, such inhibitors, has no effect on xanthommatin. TNTKOI)UCTIOK

Heat’ stable, water soluble substances

in housefly homogcnatrs n-crc reported t’o inhibit the oxidation of N-methyl carbamates by housefly and mouse liver microsomps (1). The effect of thrsc inhibitors can

be reduced by using only the abdomen of the fly (2) and by adding bovine serum albumin (BSA) to homogenates during preparation of microsomes (3). Presumably, BSA removes or countclracts the inhibitory substancrs. Since these discoveries it has become customary to prepare housefly microsomes from thr abdomens and to homogenize and/or assay the cnzymrs in the presencr of RSA. Jordan and Smith (4) reported that the heat stable oxidaw inhibitor was absent in a mutant eye color strain. WC have also shown thut

the

inhibitor

is associated

\vit’h

cyr

pigmentation and w haw suggested that the inhibitor may bc xanthommatin, an ommochrome pigment found in insects (5). The eyes of wild-type houseflies contain, in addition to xanthommat’in, kynurcnine, 3-hydroxykynureninr (3-OHK), and other 1 Oregon Agricultural Technical Paper No. 3162.

Experiment

Station, 409

tryptophan metabolites involved in xanthommatin synthesis (6). This worker also reports that kynurenine and 3-hydroxykynurrnine are present in housefly strains with mutant eye colors including ocra (kynurenine) and white (both kynurenine and 3hydroxylrynurenine). Butenandt and Schafer (7) report that 3-hydroxykynurenine is converted to xanthommatin in t’he presence of cytochrome c oxidase, or tyrosinase and dihydroxyphenylalanine (DOPA). We have noted that solutions of 3-OHK arc gradually converted to xanthommatin, presumably by air oxidation. It seems likely that enzymes capable of converting kynurenine to 3hydroxykynurenine might be present in housefly homogenates. Therefore, although the st,rongest evidencr pointed to xanthommatin as the inhibitor, it was necessary to consider the possibility that these intermcdiates could be converted t’o xanthommatin in any housefly homogenate and that the inhibition was act’ually due to some other difference between the wiild-type and mutant-type eye colors. WC now report additional experiments which prove that t,he inhibitory substance is indeed xanthommat’in. Our results indicate

410

SCHONBROD

that it’ acts as an electron acceptor, removing reducing cquivalcnts from the microsomal electron transport’ system. We also show that BSA has no effect on this inhibit)or. METHODS

AND

Housefly Strains and

MATERIALS

Preparation

of

Micro-

somes

The inhibitor solutions n-cm prepared from heads of various housefly strains (5)) all with wild-type eye color unless specified. Microsomes were prepared in the usual manner from lo- to 14.day-old headless Orlando-DDT houseflies, a strain with a moderate microsomal oxidasr level. Enzyme Assays The standard assay of microsomal oxidase activity was for aldrin epoxidation. A normal 5-ml assay mixture had the following composition: 3.5 X 10e3 M glucose-6-phosphate; 3.54 X 10-4M NADP; 0.5 unitsof glucose-6phosphate dehydrogenase; 0.1 M phosphate buffer, pH 7.5; 10 housefly equivalents of microsomes; and 60 X 1O-6 M aldrin (in 0.1 ml of double distilled methyl Cellosolve). When BSA was used, it was added to the incubation mixture at the rate of 1 mg per housefly. Incubations were for 15 min at 34°C in an atmosphere of air, and the rcxction was stopped by shaking with 15 ml of 3 : 2 hexane : isopropyl alcohol. After removing and retaining the solvent layer, the incubates were extracted three times with 10 ml of hexane. Dieldrin concentrations were determined on a 6ft X g in. aluminum column of 5 % DC11 and 5 % QFl in a Varian Aerograph gas chromatograph equipped with an electron capture detector. NADPH : O2 oxidoreduct’ase was assayed in a &ml mixture with the following composition: 1 X 1O-3 M NaCN; 0.06 M phosphate buffer, pH 7.8; and five fly equivalents of microsomes. After the addition of 0.1 Mmole of NADPH t’o a 2.0-ml aliquot of this mix-

AND

TERRIERE

t,urc, the change in percent, transmission at 340 nm n-as recorded for 35 min. The linear portion of the curve was extrapolated and t’ht: rate of change in optical densit,y cstimated for each assay. NADPH : cytochromc c oxidorcductase was assayed with either cytochromc c or dichlorophenol indophrnol (DCPIP) as clrctron acceptor. The absorbance maxima of thr reduced forms were at 550 nm and 600 nm, rrspcctivcly. The s-ml mixture con t)ained 1 X 1O-3 A$ NaCS; 0.06 :lir phosphate buffer, pH 7.8; 50 pal electron acceptor; and five fly equivalents of microsomcs. On addition of 0.1 pmole of NADPH to 2.0 ml of the mixt’ure, the change in optical density was recorded as before. oxidoreduct,asc NADPH : ncotctrazolium was assayed under the same conditions as above, using the dye nrotrtrazolium chloride and ten fly cquivalcntx of microsomes. The rate of change in optical density was rccordrd at 55.5 nm. Preparation of Head Inhibitor Housefly hrads wrre homogrnizcd in a motor-driven tissue grinder using 2.5 heads/ ml of distillrd water. This homogrnatr \vas approximatrly 0.5 % (w/v) compared to thr 20% mixture usrd by Matthrns and Hodgson (1). The homogenates were left at room trmprrature for approximatrly 3 hr prior to hrating since this rrsulted in a 50 % incrrasr in inhibitor act#ivity. Thr homogrnat,es were then placed in a boiling bath for 10 min, cooled, filtrrrd and made to volume. Inhibitor prrparations could be stored in a rcfrigcrator for a wrek beforr losing activity. Prepamtion

of Xanthonznzatin,

The met’hods of Umebachi and Uchida (8) and Hiraga (6) werr followrd in the isolation of xanthommatin from housefly heads. litsuspension in acidic methanol and centrifugation werr repeated until thr rxtracts were free of thr dark orange color. The methanol was evaporatrd from the combined rxtracts

INHIBITIOK

OF

\IICROSO~IAL

and tht: product was dissolved in 0.05 M phosphate buffer, pH 7.0. Xanthommatin was recovered from this solution by the drop-wise addition of coned HCl to pH 3.0, st,oragc overnight in the rcfrigcrator and crntrifugation of the> cold solution. The> product was dricxd in ~1vacuum dcssicator over PZO,. Th(t yic>ld of xanthommatin, based on the weight of the fly heads, K:IS about 0.1 o/c. Xrmthommatin wus also prcparcd from :~-I~~droxyliSInurcrlinc by oxidation with potassium fcrricyanide using the m&hod of ButcJnandt and Schafer (7). The spect8ral and chromatogrnphic propclrtiw of both the isolatrd and synthetic matc>rials wrc idrntical to thaw published for xanthommatin (6-S). RESULTS

We have previously rrportc>d (5) that the hc>adinhibitor is associated with rycl color in T$BT,E Cross between associalion

St rain

or cross”

Eye

kdr.O; w5 stw; Did; ocla F‘ Fr F, F2 a (kdr-0; 6 Inhibitor assay.

1

eye color mutant of eye color with oxidase inhzhition.

strcins lo show mzcrosornol

color

white ocra wild wild white ocra

w5 X stw; at two

Did; fly

ma). head

C,o Inhibition of epoxidation” $2.53 7.lC.i 56.5 47.6 2.2 +1.0

eqaivaletlts

per

YOOH

FOOH

Hf--NH2

H~-NH;

FIG.

1. Structures

the housefly, whito and ocrawycld mutants containing rwgliblc levels of the inhibitor compared to flies with wild-type cy color. Additional expcrimrnts in \vhich white and ocra-eyc,d mutants \v(w crossed and their FI and Ic2 progrny twted for the inhibit’or confirmed this association. Tlw rtwlts of t,hew cxpc>rimcwts :w shown in Tabk 1. Heads of tllc> wild-type ICI hcitrrozygotrs \vcw inhibitory brcauw t’hr> two cyc coior mutants arc’ rcwssiw. Howcvcr, the mutant homozygotw of the> IT2popul:btion contained no inhibitory substanws showing that the inhibition arises from thcl wild-t\-pc> dye color pigmrnts. Thr inllibition of thcb c>poxid:w system b? xanthommntin and :~-llr-droxvlivnurr~ninc (3OH&J, I’ig. 1, \YELS compared in c~xpcriments using tlw standard microsomal incubation s?-stcxmwith aldrin as substrat,cl. The> inhibitors ww kstrd us t he pure compound and, for :i-OHK, undw conditions which could result’ in its convcwion t’o xunt~hommatin. Somr mixturw ww subjcctrd to thcl proccdurrs uwd in preparing the> hwd inhibitor. This includrd holding tlw dilutci homogrnuto for 3 llr at room tcmpc~rnturc~ follo\vc>d by heating in boiling water for 10 min. Thcl results of thew c~xpwimtwts arc’ summarizc>d in Table 2. It c:m bc ww t’llat’ x:mthomm:~tin conc(w trat,ions as low as 3 X 10-7 AII affcctrd the clpoxidasr syst,cm und t8hat fwshly prepared 3-OHK solut8ions ww noninhibitory. Howrvrr, %OHIi in nqueous solutions was gradually conwrtc>d to an inhibitory subst:tnce, COOH c HI-NH2

(II)

(I)

(yellow-brown) 0).

411

OXII)ASiES

(III)

(red) oj zranthommalin

(I),

tlihydrozanlho,,l?nalin

(II),

and

S-hyilrox~/kyn?Lrenine

(III)

412

The

SCHOKBROD

inhibition qf by xanthommatin

Inhibitor

TABLE 2 houseJly microsomai

AND

epoxiclation

and S-hydroxykynurenine (S-OHK).

Treatment of inhibitor

mixturea

Xanthommatin, 0.5 rM Xanthommatin, 5.0 PM Xanthommatin, 50.0 MM 3-OHK, 44.4 /.&I 3-OHK, 44.4 PM 3-OHK, 44.4 PM 3-OHK, 44.4 PM +

‘jh inhibition of :poxidase

Hynthet,ic

17.5

Synt,hetic

72.5

Synthetic

100.0

Fresh solution “aged” 2 days “aged” 7 days Fresh solution

0 46.6 81.9 100.0

Fresh solution Inhibitor prep

28.4 G.6

&Fe(CN)G , 444 pM K3Fe(CN)s , 444 PM 2 head equiv, white eye 2 head equiv, whit,e eye + tyrosinase, 220 units 2 head equiv, white eye + 3-OHK, 178 pM 2 head equiv, white eye + tyrosinase 220 units + 3OHK, 178 piM DOPA, 274 PM + tyrosinase, 220 units DOPA, 100 PM + 3OHK, 248 @M + tyrosinase, 220 units DOPA, 100 fiM u Indicates ture.

-

Inhibitor

prep

+O.F

Inhibitor

prep

20.5

Inhibitor

prep

24.5

Inhibitor

prep

3.6

Inhibitor

prep

56.4

Fresh solut,ion

23.0

concentration

in incubation

mix-

and, as indicated by the t’ests with potassium ferricyanide, this substance is xanthommatin (7). Although white-eyed flies contain no xanthommatin, they do contain 3-OHIZ (B), Fig. 1, which can be converted into xanthommatin in the presence of tyrosinase and DOPA (7). It was necessary to determine

TERRIERE

whcthcr this convwsion could occur under our methods of preparing the inhibitor. Head homogenates of white-eyed flies were supplement’ed with tryosinase (1100 units), 3-OHT< (to provide 178 ,.& in the final incubat’ion), or 3-OHK plus tyrosinase. After treatment in the usual manner (3 hr standing at room temperature>, boiling and filtrring) aliquots c>quivalcnt to two fly heads wch wr(A incubated wi-ith the st’andard aldrin cpoxidasr system. Thr results show- that considerable amounts of exogmous 3-OHII arc required to achicvc significant production of the inhibitor and that the level of endogenous tyrosinase is sufficient for the conversion. In another cxpcriment DOPA, 3-OHK and tyrosinase, treated as an inhibitor preparation but without fly tissue present, produced considerable quantities of the inhibitor as indicated by t#hc 56.4% inhibition of the epoxidase. DOPA and tyrosinase, as inhibitor preparat’ions, were not inhibitory but DOPA used directly inhibited the enzyme by 23.0 9’. This suggests that heating of the inhibitor mixture destroys DOI’A. These results, showing that DOPA and tyrosinase may convert 3-OHK to xant’hommatin during tht preparation of the inhibit#or, are consist’ent with the observations of Butenandt and Schafer (7). Howcwr, thr amount of additional 3-OHK required to achiwe sufficient, product’ion of inhibitor indicates that this is not a significant reaction. It’ is concluded from these cxpctriments that cndogcnous xanthommat,in is the inhibitor of consequence and that its prrcursor, 3-OHT<, is of minor import~ancc. E$ect of Inhibitors Transport

on dlicrosomal

Electron

Several easily reducible dyes were compared with xanthommatin as inhibitors of the aldrin epoxidasc and NADPH oxidase system, Table 3. FAD was included because of its role as a coenzymc in the microsomal syst,em. The other compounds wwe: cyto-

INHIBITION

TABLE The

kIIC!ROSOMAL

3

effect of xanthommatin and some electron ceptors on the activity of housejly microsomal NADPH:O? oradoreductase and aldrin epoxidase. Electron acceptor

none xanthommatin xanthommatin FAL) DCPIP cyt,ochrome c neotetrazolium Cl methylene blue brilliant cresyl blue

five

OF

ct 50 NM NADPH flies for spectral

Concentration a

5% Inhibition of epoxidasea

AOD

X

103/min/fly, 340 nm”

20 50 50 50 50 50”

100 0 100 100 81.3

0.7 1.9 3.4 0.7 4.5 1.4 1.4

50 50

100 100

6.0 7.2

0

ac-

and microsomes equivalent to assays; ten flies for epoxidase

assay. 6 Concentration

= 20 PM

for

spect,ral

assay.

chrome c and dichlorophenolindophenol (DCPIP), both of which interact with the flavoprotein component of the microsomal clcctron transport system; ncotctrazolium chloride, which interacts with the nonheme iron component of the system; and methylene blue and brilliant cresyl blue whose sites of interaction are not know-n. The latter two compounds were used because they, and dihydroxanthommatin, Fig. 1, are rapidly autooxidized. Only FAD failed to inhibit the epoxidase system although neotetrazolium chloride was less inhibitory than the other act,ivc compounds. All of the compounds except FAD accelerated the oxidation of KADPH, thcx rate being drpcndcnt on corw&ration in the cast of xanthommatin. The results with the dyes arc consistent with their electron-accepting properties and suggwts a mode of action for xanthommatin. Anaerobic condit.ions wrc rquircd to show that dihydroxanthommatin is the product of the> microsomal rclduction of xan-

413

OXIDASES

thommat’in. Under such conditions and after prior reduction of the inhibitor with ascorbic -acid, the xanthommatin dependent oxidation of NADPH was completclly blocked. These observations point out, the rolth of xanthommatin as an acceptor for t,he clcctron transport’ syst’em and suggest its most likely mode of action. The rapid rroxidation of dihydroxanthommatin (7) increases the effectivcncss of the inhibitor by providing a continuing supply of clrctron acccpt’or. Xanthommat’in increased the ratr of VIIzymatic reduction of cytochromc> c and DCPIP, which arc substrates for cytochrome c reductaw, and appctared t’o mediat.r their noncnzymatic reduction by KADPH, Table 4. This increase avrragcd about 50 %’ for cytochromc 2: and %zRO% for DCI’IP. l\Iet’hylenc blue and brilliant crcsyl blue also exhibited t,hesr c$fects. In the cast of cytochrome c, thr failure of enzymatic plus ILOI~TABLE The

4

effect of Xanthommatin and other inhibilors housefly microsomal BADPH:c?ltochrome oxidoreductase.

on c

-

Substrate”

1Microjomes, fly eqmv

‘A OD

None

2.5

35.2

14.08

xanthommatin xanthommatin none xanthommatin santhommatin FAl) methylene blue brilliant cresyl

2.5

54.0

21.60

none

5.8

-

2.5 2.5

27.0 35.2~

10.80 14.08

none

7.4#

Inhibitor

X

10“”

/min /il>

I-

Cytochrome c Cytochrome c Cytochrome DiPIP DCPIP DCPIP DCPIP DCPIP DCPIP

-

5.0 5.0

53. 3i 10.66 70.5~ 14.10

5.0

72.2~

14.44

blue

a L‘oncentrations of substrate, inhibitors and NADPH were all 50 pM. b Cytochrome c at 550 nm; DCPIP at 600 nm.

414

SCHONBROI)

AM)

enzymatic reaction rates to account for all of the increase (41.0 vs. 54.0 AOD X 103/min) may indicate that dihydroxanthommatin, produced enzymatically, also reduces cytochrome c. Support for this is seen in Fig. 2 which shows an increased rate of cytochrome c reduction when xanthommatin is present. A reversible reaction bctwcn dihydroxant,hommatin and cytochromc c would clxplain t’he decrease in concentrations of reduced cytochrome c as the reaction proceeds. An explanation of these results is t’hat both dyes and xanthommatin are reduced by the microsomal system. Some dihydroxanthommatin then reduces additional cyto-

TERHIERE

chrome> c or DCPIP. Concurrently, tlw rapid rcoxidation of dihydroxant~l~ommat~in rcwlts in a rclcycling of the inhibitor. Keotc%razolium chloride will accept clw trons from bhr nonhrme iron protein moiety of the c+ct’ron transfer system. Its reduction was inhibited 45-50 7% by xanthommatin (Table 5), and t)hrre was no measurable interaction bctwcen NADPH, neotetrazolium, and xant8hommat’in in the abwnce of microsomes. The probable site of action of xanthommatin ~VLLS indicated in double reciprocal plots of waction velocity at different substrate TABLE

5

The effect of xanthommatin on the neotetrazolium by house$y microsomal neotetrazolium oxidoreductase.

reduction NADPH:

of

-

“.C” r

Substrate and cone, j& f

“P

Time,

Microsomes! fly equw

Inhibitor and cone, fiM

AODX 103/ min/fly (555 nm)

xanthommatin

min

FIG. 2. The rate of the microsomal reduction of cytochrome c with added xanthommatin. Assay described Pcnder methods.

Neotetr, Neotetr,

508 50a

Neotetr, Neotetr, Neotetr, Neotetr, Neotetr, Neotetr,

50 50 50 50 100 100

a NADPH

-

none xantho, 100 none xantho, none xantho, none xantho,

at 100 pM;

0

,

50 50 50

none

0

10 10 10 10 10 10

8.14 4.38 8.75 3.52 10.69 5.40

all other

assays

at 50 I.~M

403530-

-2

25-

+xonthommotin

I 60

50

40

30

20

IO

0

IO 20

30 40

50 60

70

80 90 100

‘3

FIG. 3. Double reciprocal plot of the enhancement scribed under methods. Substrate concentration as mM,

of DCPIP reaction rate

reduction by xanthommatin. as AOD/min.

Assay

de-

IKHIBITIOK

OF

~IICROSOBIAL

levc~lsin the reactions bctwcn cytochrome c and ncotetrasolium rcductases and the inhibitor. The pattern of inhibition shown for cytochromr c rcductasc (Fig. 3) was of t,he mixed t,ype, while t’liat of ncotet~razolium reduction appeared to bc noncompetitive (Fig. 4). Evidence of mixed inhibition was also found for xanthommatin in aldrin epoxidation and in the IVADPH requirement for cpoxidation (dat’a not shown). The inhibitor

did not affect the activity of the NADI’H generating system. BSA was essentially inc+fcctive as a prot’cctive agent against’ the inhibitory action of TABLE l’he

2 head alentb 2 head alentb 10 pM matin 10 PM matin none

~ Microsome condition9

equivequivxanthomxanthom-

none none none Teciprocal

reduction methods. reaction

plot of the inhibition by xanthommatin. dssay Substrate concentration rate as AOD/min.

of BS-4

and

xanthommatin

Enzyme NADPH oxidase NADPH oxidase NADPH oxidase cyto. c red. cyto. c red. cyto. c red. cyto. c red. neotetr. red. neotetr. red.

on the activity

50 /& 50 pM 50 /JM 50 &I 50 &IJ~ 50 &f 50 /M Cl, 50 PM Cl, 50 fiLM

a BSA added at 10 mg/incubation (5 ml). b Wavelengths-340 nm for NADPH oxidase; tetrazolium reductase.

“/o re-

I’duction in eporidation

none

55

10

41

none

72

10

72

none

38

10

0

none

62

10

38

equiv-

7 of the microsomal

electron

BSA xantho, xantho, none BSA xantho, xantho, none BSA

600 nm

20 ELM 20 &I +

BSA

50 PM 50 PM +

BSB

for

transport

Microsomes, fly equiv

Additives”

Substrate NADPH, NADPH, NADPH, DCPIP, DCPIP, DCPIP, DCPIP, neotetr. neotetr.

freshly prepared freshly prepared freshly prepared freshly prepared 2-day pellet 2-day pellet 2-day suspension 2.day suspension

BSA ng/incubation

housejly

a Each incubation contained microsomes alent to ten flies (1 mg protein/ml). b Wild-type eye color.

TABLE Effect

6

effect of BSA on the inhibition of microsomal epoxidation by xanthommatin and on the activify of stored inicrosomes.

Inhibitor added

FIG. 4. Double of neoletrazolium described ltnder expressed a,s mM,

415

OXIDSSES

cytochrome

components. A OD X lo”*/ n&/fly

5 5 5 5 5 5 5 10 10

c reductase;

1.0 2.9 2.9 11.2 8.2 16.8 12.6 6.0 6.1

555 nm

for

neo-

416

WHOA-BROD

thrh head inhibitor or xanthommatin (Table 6). The same level of BSA restored the decreased enzyme activit’y of microsomes stored for 2 days as either a centrifugal pellet or a resuspension in buffer. Furthermow, we have observed that stored microsomes with decreased enzyme activity do not’ inhibit the activity of freshly prepared microsomes. These observations show that xanthommatin is not the cause of reduced enzyme activities in stored microsomes. BSA did not affect’ the overall t#iciency of the electron transport system (NADPH oxidase) or the microsomal reduction of neotetrazolium (Table 7). Howvcr, the reduction of cytochrome c, which is accelerated in the presence of xanthommatin, was inhibited by 10 mg of BSA even when xanthommatin was present. This apparently does not limit the system, howwer, because the epoxidation of aldrin is enhanced by BSA. DISCUSSION

The fact that xanthommatin inhibits t,he epoxidation of aldrin and enhances the microsomal oxidation of NADPH suggests that it acts as an “electron sink.” The rate of reduction of xanthommatin by the microsomal system and the rate of reoxidation of dihydroxanthommatin are both sufficient to permit a cyclic process which increases the overall effect of the pigment. Our experimental results point to cytochrome c reduct,ase as the site of action of the inhibitor. These indicators are (a) the similarity in action bebween xanthommatin and methylene blw, a known acceptor for flavoproteins; (b) thr chemical interaction between xanthommatin and either cytochromc c or DCPIP, indicating a similarit’y of redox potential; (c) the mixed inhibition kinetics of xanthommatin again& aldrin cpoxidation, NADPH oxidase and DCPIP reduction, and (d) the noncompetitive inhibition of neotetrazolium reduction, indicating a restriction in the supply of reducing equivalents to thr nonhcmc~ iron component.

AND

TERRIERE

Our conclusions arc’ bawd on the> assumption that the microsomal clect,ron t’ransport, system of insects is the samcl as that of mammalian liver. This assumption is validated by recent studies which demonstrate the simlarity of the systems from the two classes of animals (9, 10). In their study of the head inhibitor, -Jordan and Smith (4) found that cyanide counteracted t’he inhibitory action when homogenates of fly heads with wild-typr c’yt’ color were used. We haw confirmed this effect of cyanide (1O-3 ,V) and we belicw it is due t’o the prevention of the oxidation of dihydroxanthommatin by cytochrome c oxidaso and tyrosinaw. Cyanide may also provent the format,ion of xanthommatin from rndogenous 3-OHK. In their first report that housefly abdomens wre a good source of microsomal oxidases, Tsukamoto and Casida (2) noted that bot,h head and thorax contained inhibit’ory substances. Jordan and Smith (4) also mentioned a thoracic subst,ancc with inhibitory properties similar to t’hosr: of the head inhibitor. According to But’cnandt and Schafer (7) xanthommatin is found only in the cycs of the housefly. We have obscrvcd that pigmentation of the ctyes occurs in the late pupal stage which is the only pupal stage from which the head inhibitor can be prcpared. Alt’hough w: did not examine the thorax as a source of xant8hommatin, these considerations lead us to conclude that t’he thoracic inhibitor mentioned by others is not xanthommatin. In t,he various reports of inhibitory substances, at least tlvo are attributed to the abdomen (4), others arc found in the debris fraction (3, 4), and still others have bwn detected in the soluble fraction of housefly homogenates (1, 3). Xanthommatin is not readily freed from rye tissue and can be seen in the dark red lO,OOOy-sediment from homogenates of whole flies. It also forms colloidal particles which sediment at the lower g forces. Howcvcr, the inhibitor is soluble in dilute buffers to the rxtent that] cffwtiw

INHIBITION

OF

YICROSOJIAL

concentrations could be present in the postmicrosomal soluble fraction. These propcrties could explain the reports of an inhibitor in both debris and soluble fraction from housefly homogenates (1, 3, 4). Xanthommatin may not be the only insect pigment capable of accepting elect’rons from the microsomal oxidase syskm. Rhodommatin and ommatin D are the glucose and sulfate conjugates of xanthommatin and may function directly as acceptors or after hydrolysis, as xanthommatin. These pigments have bwn found in tissues of at least, 32 insect species (7). Xanthommatin is commonly found in the epidermis of lepidoptera larvae and has been reported in the testis of one species (S). This could explain low microsomal oxidasc activitiw in preparations from some insect species. BSA has bcrn used in preparing microsomcs and in their assays to increase oxidase activities. It has been suggested t’hat this protein countwacts cndogcnous inhibitors in housefly homogenates (3), but, this has been proven in only one CBSC(11). As w have shown, it has no rffect on the inhibition by xant.hommatin. BS9 shows optimum enhancement of aldrin epoxidation whrn includrd in the microsomc incubation mixt’urc at 1.0-1.5 mg/ml. The same or higher concentration of BSA causw a 30 ‘5 decreaw in the activity of cytochromc c reductasc. Since these effects would scem to oppose one anot’her, and sincr t’hey occur at the same BSA concentration, their libcly cause is an altered configuration of the microsomal particle. This would explain the fact that stored microsomcs can br reactivated by BSA. Therefore it is suggested t,hat B&4 influences the configuration of thr microsomc by providing an environment more closely rrsembling that of the cell. A conformational diffcrCI~CC in a rate limiting factor, possible cytochrome PJjO, has bwn suggcstcd (13).

417

OXIDBSE::

ACKSO\VLEDGJIENT

This work was 5-ROl-ESOO352-13.

supported

by USPHS

grant

No.

REFERENCES

1. H.

2.

3.

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B. Matthews and E. Hodgson, Naturally occurring inhibitor(s) of microsomal oxidations from the housefly, J. Econ. Entomol. 59, 1286 (1966). M. Tsukamoto and J. E. Casida, Metabolism of methylcarbamate insecticides by the NAI>PHz requiring enzyme system from houseflies, Xature 213,49 (1967). M. Tsukamoto and J. E. Casida, Albumin enhancement of oxidative metabolism of methylcarbamate insecticide chemicals by the housefly microsome-NAI)PHn system, J. Econ. Entomol. 60,617 (1967). T. W. Jordan and J. N. Smith, Oxidation inhibitors in homogenates of houseflies and blowflies, Znt. J. Biochem. 1, 139 (1970). R. D. Schonbrod and L. C. Terriere, Eye pigments as inhibitors of microsomal aldrin epoxidase in the housefly, J. Econ. Entomol. 64, 44 (1971). S. Hiraga, Tryptophan metabolism in eyecolor mutants of the housefly, Jap. J. Gen. 39, 240 (1964). A. But’enandt and W. Schafer, Ommochromes, in “Recent Progress in the Chemistry of Natural and Synthetic Colouring Matters and Related Fields,” (T. S. Gore el al. Eds.), p. 13, Academic Press, New York, 1962. Y. Umebachi and T. Uchida, Ommochromes of the testis and eye of Pap&o zuthus, J. Insect Physiol. 16, 1797 (1970). M. I). Folsom and IX. Hodgson, Biochemical characteristics of insect microsomes : NADPH oxidation by intact microsomes from the housefly, Musca domestica, Comp. Biochem. Physiol. 37,301 (1970). T. G. Wilson and R. Hodgson, Microsomal NAT)PH-cytochrome c reduct)ase from the housefly, Musca domestica: propert,ies of the purified enzyme. Insect Biochem. 1, 171, (1970). B. I. Krieger and C. F. Wilkinson, Microsomal mixed function oxidases in insects-I: Localization and properties of an enzyme syst,em effecting aldrin epoxidation in larvae of southern armyworm (Proclenia eridania), Biochem. Pharmacol. 18, 1403 (1969). L. G. Hansen and R. Hodgson, Inhibit,ion of microsomal oxidases from the housefly, Pest. Biochem. Physiol. 1, 109, (1971)