268 Bioc~imica et ~j~phys~a Acfu, 112.5 (1992) 268-273

0 1992 Elsevier Science Publishers B.V. All rights reserved OOOS-2760/92/%05.00

BBALIP 53901

The cyanide insensitive stearoyl-CoA desaturase in the housefly, Musca domestica: possible interactions of cytochrome b,

and cytochrome P-450

Stanley Pierce, Rhonda L. Monroe, Marilyn Kuenzli and Ronald C. Reitz

Department of Biochemistry, University of Neuada, Reno NV (USA)

(Received 20 December 1991)

Key words: Cyanide; Cytochrome b,; Cytochrome P-450; Fatty acid desaturation; (Desaturase, M. domestica)

It is well known that cyanide is toxic to many species of vertebrate animals. However, many different insect groups have a resistance to the effects of cyanide. One of the reactions affected by cyanide is the conversion of stearate to oleate by the acyl-CoA desaturase. At concentrations of cyanide (l-2 mMf which completely inhibit the vertebrate desaturase, we observed that the housefly desaturase was inhibited only 37%. We showed cyanide to be a non-competitive inhibitor of the desaturase. We also have studied the sensitivity of this reaction to other inhibitors, to metal chelators and to free radical scavengers. Data from the free radical scavengers suggested that some form of free radical is involved in desaturation. Piperonyl butoxide and carbon monoxide, inhibitors of cytochrome P-450, resulted in 19% and 29% inhibition, respectively, in the housefly. These compounds did not inhibit the vertebrate desaturase. Combining piperonyi butoxide with cyanide resulted in an additive inhibitory effect. These data suggest that, in the housefly there is an interaction between both cytochrome P-450 and cytochrome b, and the desaturase which may help to explain the insensitivity of the desaturase to cyanide. Another interpretation of our data suggests the possibility of three forms of the desaturase: one sensitive to cytochrome P-450 inhibitors; one sensitive to cyanide; and one insensitive to cyanide.

Introductjon

Brooks [l] has suggested that one form of resistance to the toxicity of certain compounds in insects is the result of target site insensitivity (TSI). This he defines as the failure of a toxicant to bind to its target due to an alteration in the structure or in the accessibility of the target site. Recently, Heisler et al. [2] have demon- strated TSI in the case of mitochondrial oxidation in the southern armyworm, Spodoptera eridania. This in- sect has been shown to be 800-times more tolerant to cyanide than rats 131. Much of this insensitivi~ relates to the metabolism of cyanide via either rhodanese 143 or more probably via its conversion into p-cyanoalanine [.5], but neither of these mechanisms can completely

Abbreviations: TSI, target site insensitivi~; HPLC, high performance liquid chromatography; BSA, bovine serum albumin; PB, piperonyl butoxide; BPA, bathophenanthroline disulfonic acid; DEDTCA, di- ethyldithiocarbamic acid; SOD, superoxide dismutase; Defero, defer- oxamine mesylate; HQ, hydroquinone; CYT C, cytochrome c; VPA, valproic acid,

Correspondence: R.C. Reitz, Department of Biochemistry, Univer- sity of Nevada, Reno, NV 89557, USA.

account for the insect’s tolerance to cyanide. Heisler et al. 121 showed that concentrations of cyanide which completely inhibit mammalian mitochondria resulted in only about 60% inhibition of mitochondrial oxida- tion in the armyworm.

Earlier, we had reported [6] that the acyl-CoA de- saturase in the housefly, Musca domestica, was sensi- tive to cyanide similar to the vertebrate enzyme; how- ever, during our investigations into the chain elonga- tion reactions which occur during the biosynthesis of the sex pheromone [73, we observed little effect of cyanide upon the desaturation of stearoyl-CoA. These later studies used greater substrate concentrations than previously used [6] allowing the inhibition of cyanide to be overcome more effectively. We also used a more sensitive method of analysis, i.e., radio-high perfor- mance liquid chromatography (I-IPLC). These later in vitro studies that showed houseflies to be only partially sensitive to cyanide (Vaz, 1988, unpublished data) im- plied that some insects may use alternate mechanisms for desaturation or possibly the acyl-CoA desaturase represents a case of TSI. The research described in this communication demonstrates the possible interaction of cytochrome P-450 with cytochrome b, as well as

269

with the desaturase. These cytochromes may work in concert in the housefly and in so doing this could allow the desaturation of stearic acid to oleic acid when cyanide is present.

Materials and Methods

Materials [l-14C]Stearic acid was obtained from New England

Nuclear, Boston, MA. The substrate, [l- 14C]stearoyl- CoA, (11063 or 12935 dpm/nmol) was prepared by the method described by Bergstrom and Reitz [81. NADPH, bovine serum albumin (BSA), potassium cyanide (CN-1, piperonyl butoxide (PB), sodium azide (NY), aniline, bathophenanthroline disulfonic acid (BPA), diethyldithiocarbamic acid (DEDTCA), super- oxide dismutase (SOD), deferoxamine mesylate (De- fero) and hydroquinone (HQ) were obtained from Sigma, St. Louis, MO. Cytochrome c (CYT C) was obtained from Nutritional Biochemicals, Cleveland, OH. BF, was obtained from Eastman Kodak, Roch- ester, NY. Other chemicals were analytical grade or the purest available. BSA was defatted according to the procedure of Goodman [9].

Insects Housefly pupae (Fales 1958 Strain T-II) were sup-

plied courtesy of Dr. Ted Shapas, S.C. Johnson and Sons, Racine, WI. Within 24 h after emergence, adult insects were separated according to sex and were main- tained ad libitum on sucrose and low-fat powdered milk (1: 1, w/w) and water. Insects were maintained at 25°C. Before utilization in experiments, insects were immobilized by placing them at - 20°C until movement ceased.

Preparation of microsomes For each experiment, 200-500 female flies were

immobilized at -20°C. The flies were then put in a cheesecloth and homogenized in a chilled No. 2 mortar and pestle with approx. 25 ml cold 0.25 M sucrose solution containing 3.4 mM Tris (pH 7.4). The ho- mogenate was centrifuged at 315 x g for 10 min at 4°C and the pellet was discarded. The supernatant was centrifuged at 9480 X g for 10 min at 4°C. These two centrifugations were in a Beckman JA20 rotor. The pellet was discarded and the supernatant was cen- trifuged again at 175 000 X g in a Beckman Ti 50.2 rotor for 45 min at 4°C. The pellet containing the microsomes was resuspended in the homogenization buffer and assayed for protein using the Bio-Rad, Coomassie blue procedure [ 101.

Desaturase assay The reaction mixture consisted of 50 PM [l-

14C]stearoyl-CoA, 0.5 mM NADPH, 60 mM KH,PO,

(pH 7.21, 2.0 mg BSA in a total volume of 1 ml. The reaction was initiated by adding 2.0 mg microsomal protein. In some incubations, CO was bubbled through the microsomal protein at 20 cc/min for 1 min before the protein was added to the incubation [ll]. The reaction mixture was incubated in a test tube for 8 min at 37°C. Controls consisted of a zero-time and a no protein reaction. The reaction was stopped by the addition of 0.5 ml of 0.5 M KOH in methanol. This mixture was heated in a sand bath for 30 min, acidified with HCl and extracted by the procedure of Bligh and Dyer 1123. The solvent was evaporated under N, and the fatty acids esterified with BF,-methanol (12%) reagent [13]. Fatty acid methyl esters were extracted into chloroform. Individual fatty acid methyl esters were separated according to degree of unsaturation by radio-HPLC [7]. Appropriate standards were used to allow identification of 18:0 and 18: 1. Desaturase ac- tivity was calculated by determining the amount of oleic acid formed using the ratio of the counts in the product to the counts present in the substrate. This ratio was multiplied by the amount of stearate added to determine the nmoles of product formed. This value was divided by the reaction time and the mg of micro- somal protein added to yield the specific activity. In order to normalize the different experiments, this value was then converted to a percentage of the control value.

Results

Fig. 1 clearly demonstrates the cyanide insensitivity of the desaturase enzyme from the housefly compared to that in vertebrates, i.e., mouse liver. The mouse enzyme was inhibited almost 100% with 2 mM cyanide,

Housefly

Mouse 04

Liver

0 1 2 3 4 5 6

Cyanide Concentration (mM)

Fig. 1. Comparison of the effects of cyanide on the desaturase activities from housefly and mouse liver. Percent activity was used in order to normalize the two different activities. The specific activity for the mouse liver at 100% activity was 0.205 f 0.06 nmoles/min per mg and that for the housefly was 0.196kO.041 nmoles/min per mg. The incubation conditions are described in Materials and Methods.

The n for each value was 3.

270

TABLE I

Efjcrcts of cyanide on drsuturusr kitwticc

The constants were determined using linear regression analysis ot

the curves shown in Fig. 3.

K, (ILM) V Mslr (nmol/min per mg)

t (‘yanide - (‘yanide

3x.5 X..;

I.40 II.OX

g Whole Flies no KCN

Whole flies + KCN

Abdomen + KCN

0 1 2 3 4 5 6 7

Protein (mg)

Fig. 2. A comparison of the effects of cyanide on the desaturase

activity from whole flies to that from only the abdominal tissue. The

incubation conditions are described in Materials and Methods. Ap-

prox. 200 flies were dissected to get enough abdominal tissue for

assay.

while the housefly enzyme was inhibited to the extent of only 40% even at 5 mM.

Increasing protein concentration tended to provide some protection against cyanide toxicity, and micro- somes from whole flies were less sensitive to cyanide than microsomes prepared from only abdominal tissue (Fig. 2). This may be related to the binding and/or detoxification of cyanide by proteins from other tissues in the fly.

Substrate concentration curves in the presence and absence of cyanide indicate that cyanide may be a non-competitive inhibitor (Fig. 3). From these data we calculated that only the V,,, was influenced by cyanide. It was reduced 51% from 1.40 to 0.68 nmol/min per mg (Table I). The K, was not affected.

15 1

+CN-

-CN-

I I I I I I

-0.200 -0.100 0.000 0.100 0.200 0.300

1 /Substrate (@vi-’ )

In an attempt to try to better understand why micro- somes from the housefly were less sensitive to cyanide than those from mouse liver, we conducted experi- ments in which we compared microsomes from the housefly to those from mouse liver with respect to the effects of various inhibitors. The data in Table I1 compare the effects of several compounds known to specifically inhibit cytochrome P-450 as well as of compounds which inhibit the desaturase directly. Sodium azide reacts with nonheme iron proteins simi- lar to cyanide, and similar inhibitions were observed with cyanide and azide in microsomes from both the housefly and mouse liver. However, piperonyl butoxide (PB) and CO, compounds known to specifically react with and inhibit cytochrome P-450, had essentially no

TABLE II

Effects of rnhihitors on drsaturaw uctir ‘it\

The desaturase was assayed as described in Materials and Methods.

Statistical comparisons were made with controls using the Student‘s

I = test (n = 3). The percent inhibition values were determined using

the activity when no additions were made as 100%.

Additions Housefly

rate

(nmol/mln

per mg)

Mouse ____

C; inhib- rate (; Inhib-

ition (nmol/min ition

per mg)

None 0.253 f 0.025

(0.299 * 0.007)

KCN (I mM) 0.160+0.017 37% I’

NaN, (1 mM) 0.160+0.020 37% ,’

PB (250 PM) 0.206+0.015 19% ‘

co 0.180 f 0.052 29%

(0.218+0.017) 27’: “

co

PB (250 @M) 0.186 k 0.021 27rr, ’

KCN (1 mM)

PB (250 wM) 0.133&0.006 47% ~I

NaN, (1 mM)

PB (250 FM) 0.136+0.025 46% “

KCN (1 mM)

NaN, (1 mM)

PB (250 PM) 0.143 * 0.015 44?4 “

0.27 + 0.015

0.13 -to.025 .i2c; h

0.015 +0.017 44’; “ 0.25 k 0.012 7“f

0.30 (2 assays) O’G

N.D.

0. I6 f 0.006 11% “

(I.16 +0.021 3 I ‘G ,>

N.D.

Fig. 3. Lineweaver-Burk plot of desaturase activities in the presence “ P < 0.001; ” P < 0.01; c P < 0.02; ” P < 0.05; N.D. = not deter-

and absence of 1 mM cyanide. Reaction conditions are described in mined; values in parentheses for the none and CO experiments

Materials and Methods. represent a repeat of this experiment (n = 3).

271

effect on the desaturase from mouse liver, yet they inhibited the desaturase from the housefly by 19% and 29%, respectively. Because there was a 29% decrease as a result of adding CO to the housefly microsomes in the original experiment and yet this decrease was not statistically significant, we repeated this experiment, and we found a 27% decrease which was statistically significant (Table I). Thus, it is clear that CO is in- hibitory to the A’-desaturase in the housefly. Adding CO and PB together resulted in no additive effect in the housefly, but incubations in which either cyanide or azide were added together with PB resulted in additive inhibitory effects. Adding cyanide, azide and PB all together only resulted in the additive effect of either cyanide and PB or azide and PB. Thus, it would appear that the effects of cyanide and azide may be at differ- ent locations than PB or CO. Further, these data implicate cytochrome P-450 in the pathway of electron movement from NADPH to the desaturase in the housefly and confirm its absence in microsomes from mouse liver.

We next added metal chelators such as batho- phenanthroline (BPA) and diethyldithiocarbamic acid (DEDTCA) and tested them in the presence of the cytochrome P-450 inhibitor, PB (Table III). We also added CYT C as a good electron acceptor and aniline as a substrate for cytochrome P-450. CYT C inhibited desaturase activity almost completely in both systems,

TABLE III

EpPects of inhibitors on desaturase actiuity

The desaturase was assayed as described in Materials and Methods. Statistical comparisons were made with controls using the Student’s t-test (n = 3). The Percent Inhibition values were determined using the activity when no additions were made as 100%.

Additions Housefly Mouse

rate % inhib- rate % inhib- (nmol/min ition (nmol/min ition

Control

per mg)

0.200 f 0.010

per mg)

0.253 + 0.025 CYT C 0.002 + 0.000 99% a 0.04 rtO.38 85% a Aniline 0.137*0.015 32% ’ 0.16 f0.017 41% b BPA (1 mM) 0.1~*0.015 47% = 0.21 kO.075 22% DEDTCA

(1 mM) 0.196+0.055 2% 0.23 15%

BPA (1 mM) PB (250 /.LM) 0.123 + 0.025 39% b N.D.

DEDTCA (1 mMf

PB (250 @Ml 0.263kO.038 132% d N.D.

DEDTCA (1 mM1

BPA (1 mM) 0.145 28% N.D.

aP<O.OU1; b PcO.01; ‘P<O.O2; d P~0.05. N.D.=not deter- mined.

TABLE IV

Effects of free radical scavengers, chelators and other inhibitors on desaturase activity

The desaturase was assayed as described in Materials and Methods. Student’s t-test analysis was used for the statistics (n = 3). 300 units of SOD were added to each of the indicated incubations. The Percent Inhibition values were determined using the activity when no additions were made as 100%.

Additions Rate (nmoI/min per mn)

% Inhib- ition

Control 0.134+0.002 KCN (1 mMI 0.070+ 0.002 48% a PB (250 mM) 0.105 + 0.004 22% a BPA (1 mM) 0.102+ 0.007 24% b HQ (1 mM) 0.015~0.~5 89% = SOD 0.138+0.011 0% SOD + KCN (1 mM1 0.080 f 0.008 41% a SOD + PB (2.50 mM) 0.126+0.008 6% SOD + HQ (1 mMI 0.066 f 0.016 51% = Defer0 (0.15 mMI 0.118t0.009 12% Defer0 (0.15 mM)+ KCN (1 mMI 0.047 + 0.006 65% a Defer0 (0. 15 mM)+ PB (250 mM1 0.103~0.~ 23% = Defer0 (0.15 mM1 + BPA (1 mM1 0.109 * 0.003 19% a

a P < 0.001; b P < 0.01.

and aniline resulted in similar inhibition in both sys- tems; 32% and 41% in housefly and mouse liver, respectively. This latter effect of aniline was not ex- pected in microsomes from mouse liver, and unfortu- nately we did not measure aniline hydroxylase directly. BPA, an iron chelator, inhibited the desaturase by 47%, a value more than double that of the mouse. In the housefly, both the P-450 and the b, have iron in their constituents possibly accounting for the 2-fold increase in inhibition. The inhibition by BPA in both systems is consistent with the involvement of nonheme iron as suggested by the inhibition by azide. The lack of inhibition by DEDTCA would suggest that copper is probably not involved. It also should be pointed out that the specificity of the various metal chelators is such that any conclusions regarding the specific metals involved can only be strongly suggestive. There was no additive effect of PB and BPA, in fact, there may have been some slight protection in the housefly. An inter- esting observation in the housefly was the fact that the combination of PB and DEDTCA actually resulted in a stimulation of desaturase activity. DEDTCA also seemed to protect the desaturase somewhat from the effects of BPA. These latter combinations were not tested in microsomes from mouse liver.

In the final experiments, we compared the effects of various free radical scavengers and metal chelators on the desaturase from houseflies (Table IV). In the con- trols for this experiment, cyanide, PB and BPA pro- duced results similar to those previously noted in Ta- bles II and III. The free radical scavenger, hydro-

272

quinone (HQ), inhibited the desaturase by almost 90% while SOD had essentially no effect. SOD did seem to protect against the marked inhibition observed with HQ. These results implicate free radicals, but probably not superoxide directly except possibly in the presence of HQ. Further, the hydroxyl radical scavenger, defer- oxamine, had essentially no effect, thus, ruling out any effects attributable to the hydroxide radical.

Discussion

Vertebrates are typically quite susceptible to cyanide inhibition of fatty acid desaturation [14]; although, Hiwatashi et al. [16] have reported different responses for desaturases from several different vertebrate and their tissues. In our studies, we observed cyanide inhi- bition of mouse microsomal desaturation to be nearly 4-fold that of the housefly (Fig. 1). These results typify that of other enzymes and enzyme systems from several insect species [2,15]. Our studies clearly show that cyanide affects the rate of the reaction and not the binding of the substrate to the enzyme. This strongly supports the idea of a TSI type of effect.

The presence of two electron transporting systems in the endoplasmic reticulum has been well docu- mented. One system transports electrons from NADH for fatty acid desaturation [20,21], and the other uses NADPH with cytochrome P-450 for hydroxylations and a variety of reductions [22,23]. Additional information has clearly shown that there is considerable interaction between these two electron transporting systems espe- cially with respect to interactions between cytochromes b, and P-450 [17,18,24-261. In general, the interaction proposed is such that usually cytochrome b, supplies a second electron to P-450. Because we have observed several cytochrome P-450 inhibitors to inhibit fatty acid desaturation, our data may be suggesting that the flow of electrons may be from P-450 to b, in the housefly microsomes.

In addition to an interaction between the two cy- tochromes, another interpretation of our data would be to suggest that cytochrome P-450 may interact directly with the desaturase. This raises the possibility that the housefly may have three forms of desaturase activity: one that reacts with cytochrome P-450 and two that react with cytochrome b,. Further, of the two desat- urase forms that would react with cytochrome b,, one must be cyanide sensitive, while the other is insensitive. This would explain the activity remaining after the addition of cyanide together with piperonyl butoxide or CO. If it assumed that CO and/or piperonyl butoxide completely inhibit cytochrome P-450 (Table I), then 75% of the desaturation occurs via cytochrome b,. Further, the data in this table suggest that CN- can only inhibit 28%-38% of the cytochrome b, related desaturase. Thus, there would be about 30-40% of the

total desaturase activity which is not sensitive to cyanide, and this would represent a situation of TSI.

A cytochrome P-450 with a strong affinity for cy- tochrome b, has been purified from rabbit liver [17,18]. Capdevila et al. [19] purified two different forms of cytochrome P-450 proteins from the housefly. One of the cytochrome P-450 preparations contained small amounts of cytochrome b, tightly bound to the P-450. This suggests the possibility of a strong interaction between cytochromes P-450 and b,. The fact that we get additive effects with PB and either cyanide or azide supports this idea of an interaction between b, and P-450. Further, the fact that we cannot totally inhibit the desaturase with cyanide or azide or mixtures of the two suggests that electrons must somehow bypass the cytochrome b,-desaturase interaction. The inhibition of desaturation by inhibitors of cytochrome P-450 strongly implicates this hemoprotein in the overall in- sensitivity of the desaturase to cyanide. The data in Tables II and III also suggest that cytochrome P-450 and cytochrome b, interact in such a fashion as to allow the exchange of electrons between them. This interaction and/or exchange of electrons between P- 450 and b, could account for our earlier observation that either NADPH or NADH can serve almost equally as substrates for desaturation in the housefly [6]. That the presence of the cytochrome P-450 substrate, ani- line, can inhibit the desaturase also supports the idea of an interaction between the two cytochromes.

Cytochrome P-450 has been implicated in the desat- uration of valproic acid WPA) in liver microsomes [27], and another report has appeared relating cytochrome P-450 to the catalysis of the desaturation of testos- terone to 17-P-hydroxy-4,6-androstadien-3-one [28]. The mechanism and the possible involvement of an additional enzyme or protein with cytochrome P-450 have not been determined, but this literature provides evidence to support the idea that cytochrome P-450 may be involved with the removal of protons from adjacent carbon atoms and, thus, in desaturation. Our data are the first to suggest such an involvement with the desaturation of a fatty acid.

The use of SOD and deferoxamine and their lack of inhibition suggests that neither superoxide nor the hydroxide radical are involved in desaturation. How- ever, the strong inhibition with HQ would be consis- tent with the involvement of some form of free radical, but the data are not conclusive nor are they specific enough to make further comments.

Acknowledgements

This is a contribution of the Nevada Agricultural Experiment Station, and the research was also sup- ported in part by Grant No. DCB-8900088 from the NSF.

273

References

1 Brooks, G.T. (1976) in Insecticide Biochemistry and Physiology (Wilkinson, CF., ed.), pp. 3-6, Plenum Press, New York.

2 Heisler, C.R., Hodnick, W.F. and Ahmad, S. (1988) Camp. Biochem. Physiol. 91c, 469-472.

3 Negherbon, W.O. (1959) Handbook of Toxicology: Insects, Vol. III, Saunders, Philadelphia.

4 Long, K.Y. and Brattsten, L.B. (1982) Insect B&hem. 12, 367- 375.

5 Beesley, S., Compton, S.G. and Jones, D. (1985) J. Chem. Ecol. 11, 45-50.

6 Wang, D.L., Dillwith, J.W., Ryan, R.O., Blomquist, G.J. and Reitz, R.C. (1982) Insect B&hem. 12,545-551.

7 Vaz, A.H., Blomquist, G.J. and Reitz, R.C. (1988) Insect Biochem. 18, 177-184.

8 Bergstrom, J.D. and Reitz, R.C. (1980) Arch. B&hem. Biophys. 204, 71-79.

9 Goodman, D.S. (1957) Science 125, 1296-1297. 10 Bradford, M.M. (1976) Anal. Biochem. 71, 248-254. 11 Ahmad, S., Pritsos, C.A., Bowen, SM., Kirkland, K.E., Blomquist,

G.J. and Pardini, R.S. (1987) Arch. Insect Biochem. Physiol. 6, 85-96.

12 Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 911-917.

13 Metcalf, L.D., Schmitz, A.A. and Pelka, J.R. (1966) Anal. Chem. 38,514-515.

14 Joshi, V.C., Wilson, A.C. and Wakil, S.J. (1977) J. Lipid Res. 18, 32-36.

15 Berenbaum, M.R. (1986) in Molecular Aspects of Insect-Plant Associations (Brattsten, L.B. and Ahmad, S., eds.), pp. 257-212, Plenum Press, New York.

16 Hiwatashi, A., Ichikawa, Y. and Yamano, T. (1975) B&him. Biophys. Acta 388, 397-401.

17 Sugiyama, T., Miki, N. and Yamano, T. (1980) J. Biochem. 87, 1457-1467.

18 Miki, N., Sugiyama, T. and Yamano, T. (1980) J. B&hem. 88, 307-316.

19 Capdevila, J., Ahmad, N. and Argosin, M. (1975) J. Biol. Chem. 250, 1048-1060.

20 Shimakata, T., Mihara, K. and Sato, R. (1972) J. B&hem. (Tokyo) 72, 1163-1174.

21 Strittmatter, P., Spatz, L., Corcoran, D., Rogers, M.J., Setlow, B. and Redline, R. (1974) Proc. Natl. Acad. Sci. USA 71,4565-4569.

22 Trudell, J.R., Biisterling, B. and Trevor, A.J. (1981) Biochem. Biophys. Res. Commun. 102, 372-377.

23 Gillette, J.R., Mitchell, J.R. and Brodie, B.B. (1974) Annu. Rev. Pharmacol. 14, 271-288.

24 Chiang, J.Y.L. (1981) Arch. B&hem. Biophys. 211, 662-673. 25 Bdsterling, B., Trudell, J.R., Trevor, A.J. and Bendix, M. (1982)

J. Biol. Chem. 257, 4375-4380. 26 Bdsterling, B. and Trudell, J.R. (1982) J. Biol. Chem. 257, 4783-

4787. 27 Rettie, A.E., Rettenmeier, A.W., Howald, W.N. and Baillie, T.A.

(1987) Science 235, 890-893. 28 Nagata, K., Liberato, D.J., Gillette, J.R. and Sasame, H.A. (1986)

Drug Metab. Disp. 14,559-564.