Albumin and cytochrome P450 binding characteristics of juvenile hormone and its analogs

PESTICIDE

BIOCHEMISTRY

AND

Albumin

6, 377385

PHYSIOLOGY

(1976)

and Cytochrome P,,, Binding Characteristics of Juvenile Hormone and Its Analogs T. MAYER

RICHARD Veterinary

Toxicology and Entomology Research U. S. Department of Agriculture,

Laboratory, Agricultural College Station, Texas

Research 77840

Service,

AND

M. DANNYBURKE The University Received

Biochemistry of Texas Health September

Department Center,

Science

25, 1975;

accepted

Dallas, January

Texas

76200

8, 1976

Binding data were gathered for the cecropia juvenile hormone (methyl(E, E cis)-lO,llepoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate) and two of its analogs (isopropyl(2E, 4E)(E)-4-C (6,7-epoxy-3,7-dimethyl-2-nonenyl)11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate; oxyl]-1,2-(methylenedioxy)benzene) with bovine serum albumin and rat hepatic microsomal cytochrome P460. The proteins were found to bind the juvenile hormone and juvenile hormone analogs with affinity constants ranging from IO5 to 106 MP. Thermodynamic calculations suggest that the binding of all three compounds is electrostatic in nature and that the size of the ether and ester substituents can greatly influence the binding to proteins. The juvenile hormone and its analogs all formed spectrally apparent Type I complexes with oxidized cytochrome Paso; one of the juvenile hormone analogs formed a spectrally observable product adduct with reduced cytochrome P d50. The product complex may contribute many of the hormonal effects observed for this compound.

Many proteins and subcellular particles bind insect juvenile hormones or JH-like compounds.’ In fact, special hemolymph proteins that transport JH from its site of synthesis in the corpora allata to various sites of action have been reported (1, 2); other proteins such as epoxide hydrases, esterases and oxidases bind JH and juvenile hormone analogs during metabolic processes (3-8). Although we know proteins bind JH and 1 JH = juvenile hormone [methyl(E, E cis)-10, ll-epoxy-7-ethyl-3,1l-dimethyl-2,6-tridecadienoate]; JHA = juvenile hormone analog(s) ; BSA = bovine serum albumin; Ro-203600 = (E)-4-C (6,7-epoxy3,7-dimethyl-2-nonenyl)oxyll-1,2-(methylenedioxy) benzene; K, = association constant; Kd = dissociation constant.

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

MATERIALS

AND METHODS

Reagents. The JH [methyl(E, E, cis)lO,ll-epoxy-7-ethyl-3,11-dimethyl-2,6tridecadienoate] and (E)-4-C (6,7-epoxy-3,7dimethyl-2-nonenyl)oxyl]-1,2-(methylenedioxy)bensene (Hoffmann-LaRoche, Inc., 377

Copyright All rights

JHA, few data exist to describe the binding phenomena. In this article, we report on the binding characteristics of the JH and two JHA to bovine serum albumin and hepatic microsomal cytochrome P450.These proteins represent classes of proteins or enzymes that might be involved in the transport or metabolism of JH-like compounds in insects or other animals.

378

MAYERANDBURKE

Nutley, N.J.)” were 99% pure. Methoprene [isopropyl (WE, 4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate; Ziiecon Corp., Palo Alto, Calif.] was 95yo pure. The fluorescent probe, N-n-butyld(dimethylamino)-1-napthalene-sulfonamide, was prepared according to the method of Mayer and Himel (9). Solutions of these compounds in methanol were used in the following experiments. All other chemicals were of reagent quality or the highest quality available. Removal of lipids from BSA. Fatty acids and other lipids were removed from BSA (4X crystallized; ICN Pharmaceuticals, Inc., Cleveland, Ohio) by an activa,tcd charcoal treatment of albumin at low pH (10). After the albumin solution was neutralized with NaOH, it was dialyzed overnight in the cold against 200 volumes of distilled water. The salt-free solution was lyophiliztld and stored at -20°C. Concentrations of BSA solutions were determined by measuring the absorbance at 280 nm 1 o,11c%’ = 6.6 ; a molecular weight of and E 6.6X104 was assumed (11). Protein solutions for spectral analyses were prepared in 0.05 111Tris-HCl (pH 7.4). Treatment o$ animals. Male rats of the Sprague-Hawley strain, S-10 weeks old (Charles It&r Labs., Wilmington, Mass.), were injected with sodium phenobarbital (80 “g/kg) ip once daily for 4 days; they received their last injection 24 hr before they were sacrificed. The animals were allowed food and water ad lib. except the last 24 hr prior to sacrifice during which they were starved. Preparation. of microsornes. Rat liver microsome suspensions were prepared as described by Remmer et al. (12) and their protein content assayed by the biuret reaction (13). Studies involving microsomes were conducted on the day of preparation. Arzalysis of binding. Binding constants for the JH and JHA with BSA were detcr2 Mention of a commercial or proprietary product in this paper does not constitute an endorsement of this product by the USDA.

mined via the fluorescent probe technique outlined by Jun et al. (14). The probe has a different quantum yield and different emission and excitation wav&ngths in aqueous solution than when it is bound to BSA in solution. The probe (0.5~.-S.OX lo-” M final concentration) was tit’rated against high (3.5 X 10e4 fi1) and low (7.0 X 1O-G n/r) concentrations of BSA with microsyringes. The samples (2 ml total) w~r(’ c>xcited at 340 nm and the fluorcscencc at; 48~ nm was recorded at, oath point, in t’hc titration curve. The following equation was ustad to calrulatct the amount’ of bound proho : iI,lI,) J- = --~----.f (Jh”I/)

-

1

-

1

whcrcx X = bound fraction ; I,,, If = fluorc+ cencc intensities of prohcb in solutions of low protcGn concentration and without’ protein, rcspcctively ; Ib = fluorescence intc>nxity of probe in solutions of high prot,tlin concentration. Methanol had no effect on thtl binding of the probe in the concentration range used (510.5 pl per 2 ml of protein solution). Det’ermination of X alloFv\-cadthe use of Scatrhard’s equations (15) for the ealeulation of tho binding (#on&ant ()f the probe. The value X can bc d&!rmincltl in the prrsrnce of other compounds or ligands that bind to BSA. The .JH and *JHA were added to 2 ml of BSA at the low concentration in a cuvette and stirred. After a 3-min equilibration time in the thermostatcbd cell holder of the spectrofluorimeter, thr fluorcscence titrat,ion proceeded. A decrease in the binding of the probe to BSA resultIs in a decrease in t,he fluorescence intensity of the titration curve. The multi-equilibria equations derived by Klot’z (16) describing a simple competition between different ligands for the same binding site were used to c*alculate the binding constants of JH and JHA. An unoorrect)ed spectrofluorimcter (Aminco Ratio SPF) was used for all fluorescence measurements. Apparent association con&ants for the

PROTEIN

BINDING

CHARACTERISTICS

test compounds with cytochrome Pd5,, were calculated from double reciprocal plots of absorbance change versus concentration of the compound with an Aminco DW-2 UV-Vis spectrophotometer equipped with a thermostated cell holder (17). With mismall amounts (l-10 ~1) of crosyringes, stock solutions (3 mM) of JH, methoprene, and Ro-203600 were titrated into the sample cuvette containing 3 ml of microsomal suspension (ca. 2 mg/ml) and stirred. Equal volumes of solvent were titrated into the reference cuvette. Absorbance spectra from 350 to 510 nm were recorded for each addition. Water solubility. Water solubility of the test materials was determined turbidimetrically at 400 nm. Solutions of the compounds (30 mM in methanol) were titrated into sample cuvettes containing 3 ml of distilled water; reference cuvettes were titrated with equivalent amounts of methanol. After the solutions were stirred, the turbidity was measured at an absorbance span of 0.05 on the spectrophotometer. Water solubility of the compounds was determined from plots of turbidity versus concentration. RESULTS

AND

DISCUSSION

Binding to BSA. The recent purification of the JH-carrier proteins from two insect species has allowed the evaluation of binding of JH to these proteins (1, 2). Kramer et al. (1) reported that the dissociation constant for the JH-JH-carrier protein complex was 3X lo-’ M (equivalent to a K, of 3.3 X lo6 M-1). Also, they described the binding process as a simple thermodynamic equilibrium. However, they reported that the JH did not bind to BSA (1). Ferkovich et al. (18) observed JH binding to BSA via difference spectroscopy. When we investigated the binding of JH to delipidated BSA using the fluorescent probe technique, we found that BSA binds JH, methoprene, and Ro-203600. Figure 1 shows a Scatchard plot used to calculate

OF JUVENILE

379

HORMONE

t 7 6.0 2 Y t

00

O-3

0.6

0.9

1.2

FIQ. 1. Scatchard plots of the probe-BSA complex alone (0) in the preknce of 1.S7sX 1P M ( l ) and 2.76 X I@+ M (0) JH. P is the number of moles of bound probe per mole of protein; A is the concentration of free probe. The slope of the probe-BSA complex is equal to --IL; the ordinate and abscissa intercepts of this line give nK, and n, respectively; n is the number of binding sites on the protein.

the K, of the fluorescent probe and of JH. The decreased slope of the plot observed when JH was added indicated competition between the probe and JH for binding sites on BSA. Identical abscissa intercepts indicated competition for the same binding sites on BSA. Nevertheless, there may be more than one binding site on BSA for JH. Because the data indicated the probe has a single apparent binding site on delipidated BSA, we were able to observe binding at that site alone. We obtained similar results with methoprene and Ro-203600. From the magnitudes of the K,‘s in Table 1, the affinity of BSA for the compounds followed the order Ro-203600 > methoprene > JH. At 25°C BSA bound JH about 30 X less effectively than did the JH-carrier protein from the tobacco hornworm, Manduca sesta (L.). The binding constant for Ro-203600 was within the same order of magnitude as the JH-JHcarrier protein complex (1). Therefore, from the binding constants, the JH-carrier protein has a higher specificity for the JH molecule than does BSA. Binding to cytochrome P,,,. We investigated the binding of these compounds to

380

MATERAND

TABLE; Thermodynamic

Compound

Temperature

(M

BURKE

1

Binding Data for Bovine Serum Albumin Hepatic Microsomal Cytoch,romr I’,,” fLa x 1O-5 f SD)

(“C) Defat,ted

AH’ (kcaI/ mol)

AS” (e.11.)

-7.86 -7.69 -7.60

-13.48 - 13.43 - 13.43

- 19.51 - 19.27 - 18.93

- 6.9.5 -6.83 -6.76

-10.45 - IO.45 - 10.45

-12.1.5 - 1 1 .!)5 - 1 1.96

(kcal/ mol) BHA

- ,j..i(j -w -.,.t4 --- ;i.xs

Sulfonamide

15 25 35

9.00 f 4.26 f 2.42 f

JH

15 25 35

1.84 f 0.50 1.01 rt 0.18 0.62 f 0.18

Met,hoprene

15 25 35

11.98 f 9.20 7.35 f 3.37 3.17 f 1.60

-8.02 -8.01 -7.76

- 13.29 - 13.29 - 13.29

- 1X.2!) -17.72 - 17.9.i

--5.27 -. X2X -- .x.-d

1.5

41.19 25.08 15.91

-8.73 -8.74 -8.7.5

-8.46 -8.46 -X.46

+0.04 f0.94 -j-O.96

3-0,27 t 0.28 t-o.29

Ito-

25 3.5

Hepatic

Methoprene

Ho-203600

F’djo -5.97 -5.97 -5.97

j-2.98 f3.06 +x32

15 2s 35

0.28 f 0.10 0.22 zt 0.08 0.17 * 0.09

-5.87 - r,.!X3 - 5.97

-4.97 -4.97 -4.97

+x10 +3.20 +x23

2.67 2.62 2.28 2.05

-6.91 -7.15 - 7.32 -7.49

- 1.00 -1.00 - 1.00 - I .oo

5

reported

cytochrome -6.83 -6.88 - 6.99

25 36 v&es

microsomal

1.30 f 0.36 1.10 f 0.24 0.90 f 0.20

1.5

h The

f 2.92 f 1.91 * 2.19

25 35

15

JH

0.70 0.51 0.17

AG”

and

are the averages

f 0.41 f 0.42 f 0.30 f 0.27 & SD of 3-6

cytochrome P4b0 in microsome preparations because this CO-sensitive microsomal enzyme plays an important part in the metabolism of many xenobiotics in vertebrates and invertebrates. Others (3, 5, 6) have shown that JH or certain JHA inhibit microsomal oxidases or are themselves metabolized by these cytochrome PlsO-mediated enzymes. Also, when JH is administered to adult house flies, Musca domestica IA, induct)ion of microsomal oxidase follows (5). Figure 3 shows the effect of titrating increasing amounts of JH on the difference

+21.30 +21.30 +21.20 +21.10

-+0.X!) +0.9.s +o.s9 +.X,2 +(;.I:: +6.X? +6.49 - -. _

experiments.

spectrum of oxidized hcpatic cyt’ochromr P1,, (Fe3+). ,411of the compounds produced difference specka wit*h absorption maxima at about 350 nm and minima at about4 420 nm. Spectra such as these havn been t’ermc>d Type I by Remmcr et al. (17) and have been interpreted as manifestat,ions of the binding of substrates to cytochromc> for RoP d5,,. Type I difference> spc&ra 203600 with rat hepatic microsomcs were previously recorded by Mayer et al. (3). Table 1 lists the spectrally apparent. KU’s of the compounds ; the values w;llrc’ all

PROTEIN

BINDING

CHARACTERISTICS

within the same range (105-lo6 M-r) and are much closer to each other than those we observed for the BSA binding. One of the compounds, Ro-203600, was found to be aerobically metabolized by hepatic microsomes to form a stable, spectrally observable complex with reduced cytochrome P4bo (Fe*+) (see Fig. 3). We also confirmed the aerobic formation of this complex with NADPH-reduced cytochrome Pd5,, from a diazinon-resistant strain of house flies. We did not detect similar product complexes with methoprene or JH. Figure 3 is a repetitive scan of the formation of the complex by hepatic microsomes. It shows that the complex is similar

OF JUVENILE

HORMONE

381

1 FIG. 3. Repetitive scanning of the difference spectra of hepatic microsomal pigments during the oxidative metabolism of Ro-20~600, The microsomes were diluted to 2.3 mg of protein/ml (2.0 nmol of cytochrome PUO/ mg protein). To this suspension was added a NADPHgenerating system (8 mM sodium isocitrate and 0.96 units of isocitrate dehydrogenase/ml) and Z?Oopl of Ro-.?ZOSGOO solution (100 PM Jinal concentration). Three milliliters of this suspension were delivered to the sample and reference cuvettes. AJter an optical baseline was established, the reaction was initiated by the addition of NADPH (416.~MjinaL concentration). Time-dependent changes oj the difference spectrum were recorded by repetitive scanning at a speed of 6 nm/sec (ea. 1 cycle/min). The direction of absorbance changes is indicated by the arrows.

FIG. 2. Titration of oxidized hepatic microsomal cytochrome P,,o with JH at ,%FC. The microsomes used contained 2.0 nmol OF cytochrome P&mg of protein. The microsomes were diluted to a &al concentration of 2.3 mg of protein/ml. Three milliliters of this suspension were delivered into reference and sample cuvettes. AJter temperature equilibration, a baseline of equal light absorbance was established (0). Subsequently, microliter amounts of JH (8.07 mM stock solution in methanol) were added to the sample cuvette and an equal volume of methanol to the reference cuvette. After each addition, the cuvette contents were stirred, and the difference spectrum was recorded.

to those formed by other substrates such as ethyl isocyanide (19) and piperonyl butoxide (20-22) and that there are two absorption maxima at 427 and 455 nm, respectively. The 427-nm peak seen in Fig. 3 is the result of reduced cytochrome bs absorbance and the absorbance contributed by the product complex. The formation of the 455-nm product complex absorbance peak was essentially complete by about 51 min after the addition of NADPH to the sample. Thereafter, there was a rapid increase in absorbance at 452 nm, which we believe results from the solution becoming anaerobic and the reduced cytochrome P450complexing a small amount of CO that arises in the solution.

382

Fro. 4. Structures o/ the juvenile methoprene (If), and Ro-,WSGOO (ZZZ).

MAYER

hormone

AND

(I),

The resulting peak (452 nm) is an intermediate of the one usually seen for the reduced carbonmonoxy-cytochrome P4s0 (450 nm) and the product complex (455 nm). We found that bubbling O2 into the sample cuvette would cause the spectrum to revert to its previous product complex form. Franklin (22) noted CO formation as a result of sonication of piperonyl butoxide in aqueous solution. However, in our experiments, in which the difference spectrum was recorded between microsomal suspensions mixed with Ro-203600 (160 PM) and without Ro-203600 and both subsequently reduced with dithionite, no CO formation occurred, and there never was an absorption increase in the 450-nm region of the spectrum. Such an increase would be expected if CO was generated upon mixing (18). Only when NADPH was added simultaneously or subsequently to the addition of Ro-203600 to microsomes was CO ever observed. The CO probably derived from heme catabolism resulting from NADPHsupported lipid peroxidation (23). The discovery of the product adduct with reduced cytochrome P450 may be important in understanding the mode of action of many JHA. Werringloer and Estabrook (24) showed that compounds forming such a complex will do so with particular types or pools of cytochrome PdbO. We suspect that the pools of cytochrome P4s0 that form a product adduct from Ro-203600 or other JHA are also the

BURKE

ones responsible for the nntabolism of endogenous JH in vivo. This hypothesis is being tested in our laboratories and will be the subject of future communications. If proven, it would be good support of the synergistic t.heory of ,JHA action in insects (25). Thermodynamics. Thermodynamic data related to the binding of small ligands to proteins can provide information on the nature of the binding processes themselves, hydrophobic or hydrophilic e.g., whether forces play the major role in binding. We expanded our studies on binding to cover a temperature range of 5 to 35°C. Thcrmodynamic functions can bc calculated from the association constants drttrmincd for the compounds over this tempcrat~ure range. Because the K, describes an equilibrium for the binding process the following equations can be derived : AG:”

=

-

AS”

=

(AH”

RT In k’,, -

A(;=‘)/T,

where AG”, AH’, AS”, T, and R correspond to the free energy change, rnthalpy change, entropy change, absolute temperature (OK), and the universal gas constant,. Enthalpy was determined graphically from van’t Hoff plots where In K, is plot>ted against l/T. The slope of the straight line graph is equal Do - AH”/R. Initially, Ko’s were determined at intervals of 5°C and plott,ed as described above ; after establishing that the graph was a straight line, K,‘s were determined at intervals of 10°C. The results are listed in Table 1. The thermodynamic data were difficult to interpret for the binding experiments involving BSA. This is because the only apparent trend in the binding of the three compounds to BSA is an inverse rclationship between K, and temperature. This is not an uncommon relationship and has been reported to exist for the binding of progesterone to human serum albumin and for corticosteroid binding to corticostctroidbinding globulin (26). Thermodynamic

PROTEIN

BINDING

CHARACTERISTICS

functions calculated for the latter are not unlike the data for JH and methoprene. The large negative free energy functions, indicating that the ligand-protein complex is favored, is comprised of a large negative enthalpy in association with a negative entropy. This can be interpreted as a high affinity of the interacting components which would reduce the enthalpy and make the system less random so that a negative entropy results (28). The binding of Ro-203600 to BSA can be interpreted in the same manner except for the fact of the positive entropy. Frank and Evans introduced the term “iceberg formation” to describe the effect of water molecules on nonpolar solutes (27). Water molecules are conceived to cluster around nonpolar solutes in some fashion through the action of van der Waals forces. If enough of these water molecules are displaced upon binding and return to solvent the net effect would be a higher randomness within the system, hence a positive entropy (28). The thermodynamic data calculated for the binding of the JH and JHA to cytochrome P4s0 are consistent in that they all follow the same trends. An inverse relationship also exists between K, and temperature. The data are like those recorded for Ro-203600 binding to BSA and can be interpreted similarly. One question we puzzled over was: “What is the meaning of the inverse relationship between K, and temperature?” We believed initially that there would be a direct relationship of K, to temperature because we assumed the binding would be hydrophobic in nature. Characteristically, hydrophobic binding tends to become stronger with increasing temperature which is in accord with its endothermic nature and the breakdown of the “iceberg” (26). Because of this, we can only conclude that electrostatic (i.e., polar) forces play a larger part in the binding than first thought. Stereochemical and polarity considerations. If the compounds are arranged in order of

OF JUVENILE

HORMONE

383

the magnitude of their K,‘s there is a difference in the order that they bind to the two proteins. Obviously, this means either that the compounds bind to the proteins in completely different fashions, which seems unlikely, or that there are other influencing factors such as stereochemistry, bulk and polarity to consider. To resolve these questions, we first studied the structures of the compounds. A major difference between the three is the size of the ester or ether moieties, i.e., the methylenedioxybenzene > isopropyl > methyl (see Fig. 4). When the compounds are arranged accordingly, they follow the order Ro-203600 > methoprene > JH, the exact pattern of binding so BSA. Next, we studied the polarities of the compounds on the basis of water solubility. We chose the turbidimetric method because we wanted to measure water solubility of the three compounds in the same manner. Methoprene and Ro-203600 have high extinction coefficients and their water solubility could have been measured otherwise. The fact that the solubilities obtained turbidimetrically are close to reported values indicates that it is valid for limiting amounts of methanol. We report solubilities of 4.8 PM for methoprene, 30 PM for JH, and 55 PM for Ro-203600. Kramer et al. (1) reported the water solubility of JH as 50 PM and a Zijecon technical bulletin on methoprene reports its solubility as 4.5 PM (1.39 ppm; 29). The order of water solubility, hence polarity, parallels the magnitudes of the apparent K,‘s for cytochrome P450 binding. Examination of the data reveals it is likely that the ether or ester moieties are major influencing factors in the binding of these compounds to BSA and cytochrome P 450. We make the assumptions that: (i) overall, the contributions of the hydrocarbon side chains to the binding processes would appear to be equivalent; (ii) the ester and ether substituents (this includes the ester and ether bonds as well) are the strongest polarity centers in the molecules;

384

MAYER

AND

(iii) t,he great’est change in bulk occurs in t’he ester and rthcr moieties. One can argue t’hat the methylenedioxybenzene ether substituent is more nonpolar than its estrlr counterparts, but it must be remembwrd t,hat, this group is very similar to furans which arc largely water soluble. We suggest that,, in regard to BSA, size and polarity of the &cr and ether groups work in conjunction with each other to influence the order of binding; in this instance, size or bulk would be an overriding factor. In regard to cytochromc I’dsO, it appears that polarity is a major binding factor and t,he size differencw between the ester and ether groups have little if any effect on tho binding to this protein. ACKNOWLEDGMENTS

We are grateful to Mrs. J. Durrant of the Veterinary Toxicology and Entomology Research Laboratory, U. 8. Department of Agriculture, ARS, College Station, Tex., for technical assistance. This work was supported in part, by USPHS Chant 2 PI1 GA4 16488.

BURKE

7. (;. T. Brooks, Insect epoxide hytlr:tsr itlhibitiott by jtrvenile hormone artalttgttes and metabolic inhibitors, ~Vutur? (~olkdork) 245, :$x2 (1!)73). 8. 11. Slade and C. II. Zibitt. hIei:~bc~lisnt of cecropia juvenile hormone in insect anti itt mammals, in “Insect, Jttvettile f Iormottes” (J. h\Lenn and 11. Beroza, Eds.), 11. 1.55. Academic Press, New York, 197 I. 9. 1:. 7’. 1Iayer attd C. >I. Himel, l)ynatnics of Rttorescettt, plebe-~holirtest,erase rr:\c+ic,tts, Niorhr~rn. 10.

Il. 12.

1:s.

14.

REFERENCES

1. K. J. Kramer, L. L. Sanburg, F. J. Kezdy, and J. H. Law, The juvenile hormone binding protein in the hemolymph of Manduca sfxta .Johannson (Lepidoptera: Sphingidae), R-or. Nat. Acad. Sci. USA, 71, 493 (1974). 2. S. M. Ferkovich, I>. L. Silhacek, and R. I<. liut,ter, Juvenile hormone binding proteins in the haemolymph of the Indian meal mot,h, Insrct Biochem. 5, 141 (1975). 3. It. T. Mayer, A. E. Wade, and XI. R. 1. Soliman, Juvenile hormone analogs as in vitro inhibitsor:: of rat liver microsomal oxidaaes, J. Ayric. Food Chenk. 21, 360 (1973). 4. 6. J. Yu and L. C. Terriere, A possible role for microsomal oxidases in metamorphosis and reproduction in the housefly, J. Insect Physiol. 20, 1901 (1974). 5. L. C. Terriere and H. J. Yu, Insect juvenile hormones: Induction of detoxifying enzymes in the housefly and detoxication by housefly Pcstic. Riochun. Physiol. 3, 96 enz.ymen, (1973). 6. B. 1). Hammock, S. S. Gill, L. Hammock, and J. E. Casida, Metabolic o-dealkylation of I -(4’-ethylphenoxy)-3,7-dimethyl-7-methoxy or et,hoxy-trans-2-octerle, pot,ent jttvenoids, I’mtic. Riochcm. Physiol. 5, 12 (197.5).

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17.

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J.

f:emoval of fatty :tc*ids front sertm nlbttmitt by charcoal treat rttettt , J. Rio(. C’hrm. 242, 173 (I 967). H. A. Sober, Ed., “Hatrdbc,ok of Bit)l~hrmist,ry,” p. C-71, CRC Press, Cleveland, Ohio, 1970. H. ltemmer, II. (irein, J. B. Schettkmart, and 1:. W. Estabrook, hlet,hods fat, the elevation of heparir microsomal mixed fttttct iott clxidasr levels and cytochrome P-4,50. in “>Iei hods in Enzymology” (I(. W. Estabrook, Ed.), Vol. IO, p. 703, Academic Press, New \I.ork, 1967. E. I,aytte, Spect,rophot,otnetrit~ and tttrhitlitnetric methods for nteasttrittg proteins. Irr “Rlethods in Etrz)-mologg” (S. P. (‘olowick atttl N. 0. Kaplan, Eds.), Vol. :I, p. 447, :\~:t~l~mic Press, New York, 19~7. H. W. Jt~tt, It. ‘I’. hlayer, C. .\I. fIitrtr1. a~td I,. A. Lttzzi, Binding si ttdies of ~-)i!:dr”xyt~ett~oit: acid esters to twvitie senim :rltnuuin bj Htttrrescettt probe techtriqttrr, .I I’horrrrrrcol. Sri. 60, lH21 (1971). (i. Scat chard, The attract,ittnh of prott~itts fat small molecules a~td iotas, rl nn. .\ 1‘. .4 mrl. It.

51,

660

(194!)).

I. R2. K1ot.z. fI. ‘I’riwrtsh, ttttd I”. .\I. Walker, The binding of organit’ ions I)>, proteins. Competit,iott phettctmetta and tlr~lnt urat,iotr effects, ./. A tticr. C/z(~t/r. 80~~. 70, 2!l:i5 (1948). H. Remmer, J. St*hettkmatt, I<. W. I+:s/:tbrook, H. Saaame, J. (+ille(ie, 8. Narasinthrtlu, I). Y. (:ooper, and 0. I
PROTEIN

22.

23.

24.

25.

BINDING

CHARACTERISTICS

of methylenedioxyphenyl [1,3-benzodioxole] compounds with enzymes and their effects on mammals, Drug Metab. Rev. 3, 231 (1974). M. R. Franklin, Inhibition of hepatic oxidative xenobiotic met,abolism by piperonyl butoxide, Biochcrn. PharmacoZ. 2 1, 3287 (1972). B. A. Schacter, H. S. Marver, and U. A. Meyer, Hemoprotein catabolism during stimulation of microsomal lipid peroxidation, Rioche,n. Biophys. Acta, 279, 221 (1972). J. Werringloer and R. W. Estabrook, Heterogenity of liver microsomal cytochrome PGO: The spectral charact,erization of reactants with reduced cytochrome Plso, Arch. Riochcjm. Biophys. 167, 270 (1975). M. Slade and C. F. Wilkinson, Juvenile hormone analogs : A possible case of mistaken identity?, Science 181, 672 (1973).

OF JUVENILE

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