Adrenodoxin Biosynthesis by Bovine Adrenal Cells in Monolayer Culture

THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 24, Issue of December 25, pp. 14921-14925,1982 Printed in U.S.A.

Adrenodoxin Biosynthesis by Bovine Adrenal Cells in Monolayer Culture INDUCTION BY ADRENOCORTICOTROPIN* (Received for publication, May 3, 1982)

Robert E. Kramer#, Christen M. Andersonl, Julian A. Petersonl, Evan R. Simpson$l, and Michael R. Waterman111 From the YDivision of Molecular Biology, Department of Biochemistry and $Cecil H. and Ida Green Center for Reproductive Biolom Sciences and the Denartment of Obstetrics and Gynecology, The University of Texas Health Science Center at Dallay Dallas, Texas 75235-

drial proteins, a flavoprotein (adrenodoxin reductase) and an iron-sulfur protein (adrenodoxin) (6-8). The cytochromes P450 are integral components of the inner mitochondrial membrane. Adrenodoxin reductase and adrenodoxin, on the other hand, are matrix proteins which are loosely associated with the inner aspect of the inner membrane and function to transfer reducing equivalents from intramitochondrial NADPH to cytochromes P-450. It is generally accepted that adrenodoxin is the exclusive reductant of cytochromes P-450 in adrenocortical mitochondria (9, 10). Adrenodoxin and cytochrome P-450 are present in bovine adrenocortical mitochondria in equimolar concentrations (11, 12), and Miura et al. (13) demonstrated that the interaction between adrenodoxin and cytochrome P-450,,, in vitro was maximal whenthe ratio of the two proteins was approximately 1:l. These observations raise the possibility that the adrenocortical cell maintains an optimal steroidogenic capacity by coordinately regulating the levels of adrenodoxin and cytochrome P-450 within its mitochondria. This contention is supported by the observations of Purvis et al. (14) that adrenodoxin and cytochrome P-450 levels in rat adrenocortical mitochondria fall concomitantly after hypophysectomy and increase following the administration of ACTH to hypophysectomized animals. Similarly, Kowal et al. (15) noted that ACTH caused an increase in the concentration of both proteins in mouse Y-1 adrenocortical tumor cells. However, relatively little attention has been focused on the mechanisms by which ACTH maintains steroidogenic enzyme concentrations or on the relationships between the synthesis of the various components of the steroid hydroxylase systems. Asano Steroid hydroxylation reactions in the adrenal cortex are and Harding (16) reported that ACTH increased the rate of adrenodoxin biosynthesis in Y-1 cells, but did not examine the catalyzed bymixed function oxidases located in both the endoplasmic reticulum and the mitochondrion. Cholesterol synthesis of other steroidogenic enzymes. Studies recently side chain cleavage and 1lp-hydroxylation are reactions that carried out in this laboratory (17) have demonstrated that ACTH causes a time-dependent increase in the synthesis of occur within the mitochondria of adrenocortical cells (1-3) and are catalyzed by specific steroid hydroxylases termed cytochrome P-450,, in bovine adrenocortical cells as well as cytochrome P-450,,,’ and cytochrome P-45OI1, (4, 5), respec- an increase in the translational ability of mRNA sequences tively. These reactions also require two additional mitochon- specific for cytochrome P-450,,,. The present study was carried out to examine the effect of ACTH on adrenodoxin biosyn* This research was supported in part by United States Public Health Service Grants AM28350, HD11149, and GM19036 and Grant thesis in primary cultures of bovine adrenocortical cells and 1-624from The Robert A. Welch Foundation. The costs of publication to define the relationship between its synthesis and that of of this article were defrayed in part by the payment of page charges. cytochrome P-450,,,. The results indicate that ACTH induces This article must therefore be hereby marked “advertisement” in the synthesis of adrenodoxin, and together with those of accordance with 18 U.S.C. Section 1734 solely to indicate this fact. DuBois et al. (17), suggest that the synthesis of both adreno8 Postdoctoral Trainee supported by United States Public Health doxin and cytochrome P-450,- is controlled in a coordinate Service Training Grant T32-HD07190. fashion. 11 To whom correspondence should be sent.

The long term effect of adrenocorticotropin (ACTH) on the synthesis of adrenodoxin in bovine adrenocortical cells was investigated. Primary, confluent monolayer culturesof adult bovine adrenocortical cells were incubated inthe presence or absence ofACTH M) for periods up to 72 h. The amount of adrenodoxin precursor synthesized in a cell-free translation system programmed with RNA isolated from ACTH-treated cells increased to approximately 3 times the control level by 36 h. Similarly, ACTH increased the rate of incorporation of [3sS]methionineinto mature adrenodoxin in radiolabeled adrenocortical cells, an effect that was maximal 36 h after initiation of ACTH treatment. At longer times (48-72 h), the stimulatory effect of ACTH was not maintained, and adrenodoxin synthesis in both radiolabeled cells and cell-free translation systems declined to control levels. The content of adrenodoxin in cells treated with ACTH for 36 h, as measured by electron paramagnetic resonance spectroscopy, was approximately twice that in control cells. The results indicate that ACTH induces the synthesis of adrenodoxin in bovine adrenocortical cells. Basedon the present results as well as those previously reported with respect to theinduction of cholesterol side chain cleavage cytochrome P-450 by ACTH (DuBois, R. N., Simpson, E. R., Kramer, R. E., and Waterman, M.R. (1981)J. Biol. Chem 256, 7000-7005),it is proposed that the synthesis of the mitochondrial components of the adrenocortical steroid hydroxylase system is controlled by ACTH in a coordinate fashion.

The abbreviations used are: P-450,, cholesterol side chain cleavage cytochrome P-450; P-45011p, Ilp-hydroxylase cytochrome P-450; ACTH, adrenocorticotropin; EPR, electron paramagnetic resonance; SDS, sodium dodecyl sulfate.

MATERIALS AND METHODS

Cell Culture-Primary cultures of bovine adrenocortical cells were established as previously described (17). Briefly, adrenocortical tissue

14921

Induction of Adrenodoxin Synthesis by ACTH

14922

fragments were subjected to collagenase (2 mg X rn") digestion, and isolatedcellswere prepared by mechanicaldispersion.Cellswere plated a t a napproximate density of 10' cells/lW-mrn dish and grown to confluence in the presence of fibroblast growth factor (FGF 50 ng X d-'; Collaborative Res., Inc., Waltham, MA).Confluence was generally achieved within 5-6 days. FGF was then removed from the culturemedium,andthe cell monolayerswere incubated for an additional 24-48 h. ACTH,_% (Cortrosyn;lo-" M; Organon Inc., West Orange, NJ) was added to the medium of half of the culture dishes, and cells were then incubated for periods of time up to 72 h. The incubation medium was replaced every 24 h. Cortisol was measured in culturemedia by direct radioimmunoassay using antisera obtained from Radioassay Systems Laboratories, Inc. (Carson, CA). The antiserum was directed against cortisol 3-carboxymethyl oxime/bovine serum albumin and had 2% cross-reactivity with corticosterone. ['HI Cortisol obtained from New England Nuclearwas employed as tracer in the assays. Radiolabeline of Cells and I n Vitro Translation of RNA-Cell proteins were radiolabeled at the indicated times by incubating cell monolayers for 2 h in methionine-free medium and for an additional 2 h in the presence of [35S]methionine ( 6 0 pCi X d-'; New England Nuclear) (17). Cells previously incubated in the presence of ACTH were maintained in the presence of ACTH during the radiolabeling procedure. Cells were harvested and stored frozen a t -70 "C until all samples were collected. Nonradiolabeled cells were also harvested, stored a t -70 "C, and used to isolate total cellular RNA (17, 18).The RNA was then used to program a cell-free, rabbit reticulocyte lysate system (NewEngland Nuclear). Newly synthesized, [%]methioninelabeled adrenodoxin was immunoisolated from both radiolabeled cells I 2 3 4 5 6 7 8 91011 1 2 and total cell-free translation products using the immunoglobulin FIG.1. Autoradiogram of adrenodoxin precursor spthefraction of an antiserum raised against purified bovine adrenocortical adrenodoxin (kindly provided by Dr. J. D. Lambeth, Emory Univer- sized in a cell-free translation system directed by adrenocorsity)andStaphylococcusaureus cell membranes as a source of tical cellular RNA. Monolayer cultures of bovine adrenocortical protein A. As has been previously shown, this antiadrenodoxin im- cells were incubated for up to 72 h in the absence or presence of M). At the times indicatedcellswere harvested, and total munoglobulin can be used to immunoisolate both the newly synthe- ACTH sized mature form from radiolabeled cells and the newly synthesized cellular RNA was isolated and used to program rabbit reticulocyte precursor form from cell-free translation products (19). Authentic, lysate translation systems. Th e volumes of the lysates were then purified adrenodoxin will compete with newly synthesized adreno- adjusted to contain the same amountof trichloroacetic acid-precipidoxin for the antibodyin such immunoisolations (19).Immunoisolates table radioactivity, and newly synthesized, [%]methionine-labeled were subjected to electrophoresis on SDS-15% polyacrylamide gels adrenodoxin was immunoisolated from total translation products and (20). Autoradiography and densitometry were carried out by using electrophoresed on an SDS-15% polyacrylamidegel. Lanes I-6, adretotal translation products standard techniques.Cytochrome P-450, wasimmunoisolated as nodoxin precursorimmunoisolatedfrom directed by RNA isolated from adrenocortical cellsmaintained in the previously described (17). E P R Spectroscopy of Adrenodoxin-Bovine adrenocortical cells absence of ACTH for 0, 12, 24, 36, 48, and 60 h, respectively. Lanes total translation products in confluent monolayer culture were harvested and resuspended in a 8-12, adrenodoxinimmunoisolatesfrom total volume of 0.4 ml of sucrose (0.25 M) 4-(2-hydroxyethyl)-l-piper- directed by RNA isolated from cells maintained in the presence of azineethanesulfonicacid(25 m) buffer, pH 7.4. A few grains of ACTH (lo-' M) for 12,24, 36,48, and 60 h respectively. Lane 7, sodium dithionite were added to the cell suspension which was then electrophoreticmobility of the precursor (19,OOO daltons, A ) and placed in a quartz EPR tube and frozen in liquid nitrogen. EP R mature (12,000 daltons, B ) forms of adrenodoxin. The molecular this gel using [I4C]methylatedmolecular spectra were obtained using a VarianAssociates (Palo Alto, CA) weights were calculated from model E4 X-band E P R spectrometer equipped witha liquid nitrogen weight standards and Coomassie stained mature adrenodoxin. cryostat. Spectra were collected and processed using a P D P l l minicomputer (Digital Equipment Corp.; Maynard, MS). An average of systems programmed with RNA isolated fromACTH-treated 32 scans were collected per sample, and the spectrawere normalized and control cells are shown in Fig. 2. A modest increasein the for protein concentration and variations in diameter between EPR tubes. The contentof adrenodoxin was quantitated by comparison of synthesis of adrenodoxin was observed a t 24 h in cell-free the areaof the integrated spectrumwith that of standard solutionsof translationsystems programmed with RNAisolated from purified putidaredoxin (21). control cells, but thereafter the ability of RNA from cells

- .

incubated in the absence of ACTH to direct adrenodoxin synthesis was relatively constant (Fig. 1, lunes 1-6 Fig. 2). A A representativeautoradiogram of immunoisolates ob- similar change in the synthesis of cytochrome P-450,, in tained with anti-adrenodoxinfrom total products synthesized untreated cells has been reported by this laboratory(17). The in a cell-free translation system programmed with RNA iso- reasonfor such changes incontrol cells is unclear at the lated from bovine adrenocortical cells incubated in the pres- present time. Treatment of adrenocortical cells with ACTH M) for up to 72 h is shown in markedlyincreased ence and absence of ACTH ( the ability of cellularRNA todirect Fig. 1. The immunoisolated protein had an apparent molecular adrenodoxin synthesis in cell-free translation systems (Fig. 1, weight (Mr= 19,OOO) similar to that previously reported for lunes 7-11; Fig. 2), an effect which was maximal a t 36 h. The the precursor form of adrenodoxin (19,22).The intensities of amount of adrenodoxin synthesized in cell-free systems dithe bands on the autoradiogram shown in Fig. 1, indicative of rected by RNA fromcells incubated in the presence of ACTH ["S]methionine incorporation into newly synthesized adre- for 36 h was more than 3 times greater than that by RNA nodoxin, were quantitated byscanning densitometry. The from cells incubated in the absence of ACTH. The ability of area under the absorbance peakcorresponding to the immu- RNA isolated from cells incubated in the presence of ACTH noisolatedadrenodoxinprecursorwas then calculated and for longer periods of time (48-60 h) to direct adrenodoxin used as an index of adrenodoxinsynthesis. The relative synthesis was similar to that of RNA from control cells. The amounts of adrenodoxin synthesized in cell-free translation translational activity and yield of RNA isolated from three RESULTS

Induction of Adrenodoxin Synthesis by ACTH 100-mm dishes of cells maintained in the presence or absence of ACTH is shown in Table I. The reduction in adrenodoxin synthesis at longer times is not due to loss of translational activity. Newly synthesized, mature adrenodoxin (12,000 daltons) was immunoisolated from total cellular proteins radiolabeled by incubating adrenocortical cells with [35S]methionine.Immunoisolates were subjected to SDS-polyacrylamidegel electrophoresis and autoradiography, and the intensities of the autoradiograms were quantitated by densitometry. The relationship between the rate of adrenodoxin synthesis in [35S] methionine-pulsed adrenocortical cells and the duration of ACTH treatment was examinedin three separateexperiments with similar results. The results of one of these experiments are shown in Fig. 3. The ability of cells maintained in the absence of ACTH to synthesize adrenodoxin increased slightly over the first 24 h and then was relatively constant throughout the remainder of the experiment. Although unaffected at 12 h, the rate of adrenodoxin synthesis was markedly increased in adrenocortical cells incubated in the presence of ACTH ( M) for 24 h and was maximal in cells treated with ACTH for 36 h. Treatment of adrenocortical cells with ACTH for 36 h resulted in a 2-3-fold increase in the rate of adrenodoxin synthesis as compared to that in cells maintained in the absence of ACTH. Thereafter, the rate of adrenodoxin synthesis by cells incubated in the presence of ACTH decreased to control levels. Newly synthesized cytochrome P-450,, wasimmunoisolated from aliquots of the same radiolabeled cell proteins and translation products that were used for the immunoisolation of newly synthesized adrenodoxin. The effects of ACTH on the synthesis of cytochrome P-450,, observed in the present study (Fig. 3) were similar to those already reported (17) and closely parallel the changes observed in adrenodoxin biosynthesis. Cortisol production by bovineadrenocortical cells in monolayer culture is shown in Table 11. It is seen that the cortisol output is highest at 12 h and decreases to control levels by 72 h of continued ACTH treatment. At 36 h, the time of maximal adrenodoxin synthesis, the desensitization of cortisol production is clearly apparent. EPR spectroscopy was used to determine if the effect of ACTH on the rate of adrenodoxin synthesis was manifested as a change in adrenodoxin concentration in the adrenocortical 200

.-. 0-0

6

ACTH Control

14923

TABLEI Translational activity of total RNA isolated from cultured bovine adrenocortical cells maintained in the presence or absence of ACTH Yieid of RNA from three 100-mmdishes

Time of RNA isolation

Activity

h

cpm/pl translation/pg RNA

w

0

24,275 22,591 12,695 18,068 23,139 17,756 13,738 16,414 17,925 21,997 17,409 19,722 15,991

260 264 356 288 274 314 368 324 296 352 338 304 320

12 (-ACTH) 12 (+ACTH) 24 (-ACTH) 24 (+ACTH) 36 (-ACTH) 36 (+ACTH) 48 (-ACTH) 48 (+ACTH) 60 (-ACTH) 60 (+ACTH) 72 (-ACTH) 72 (+ACTH)

ADRENODOXIN

o--.oAClH

0

12 24 36 48 60 72 ation ion of A C T H Beotrnenl (hours1

FIG. 3. Effect of ACTH on the synthesis of adrenodoxin and cytochrome P-450,,,in bovine adrenocortical cells. Cells were M) for periods incubated in the presence or absence of ACTH up to 72 h. At the times indicated, cells were incubated with ["SI methionine for 2 h, and mature adrenodoxin and cytochrome P-450,,, were then immunoisolatedfrom the same amount of trichloroacetic acid-precipitableradioactivity.Immunoisolates were subjected to SDS-polyacrylamidegelelectrophoresis, andautoradiogramswere quantitatedby scanningdensitometry.The area underthe absorbance peak corresponding to mature adrenodoxin or cytochrome P-450,,, was usedas an index of synthesis.

TABLEI1 Cortisolproduction by bovine adrenocortical cells inmonolayer culture

1

Time ACTH h

3.1 1.5 1.2 0 12 24 36 48 60 DuratlonOf ACTH Treatment (hours1

FIG. 2. Effect of ACTH on the synthesis of adrenodoxin in cell-free translation systems programmed with RNA isolated from bovineadrenocortical cells. The lanes of the autoradiogram shown in Fig, 1 were quantitated by scanning densitometry. Each data point is expressed as the areaunder the absorbancepeak corresponding to the adrenodoxinprecursorandrepresents the amount of adrenodoxin synthesized.

Control nmol cortisol/l2 h/mg

12 24 36 48 60

1.9

73

n.6

1.0

M)

protein

20.8 17.0 12.3 5.0 5.1 1.5

cell. The EPR spectra of cells incubated in the absence and presence of ACTH M) for 36 h are shown in Fig. 4. The adrenodoxin concentration in adrenocortical cells maintained in the absence of ACTH was 0.12 nmol X mg-'of protein, while that in cells incubated in the presence of ACTH was 0.22 nmol X mg"of protein. These values compare to an

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Induction of Adrenodoxin Synthesis by ACTH

and Harding (16) also noted that the rate of adrendoxin biosynthesis by Y- 1cells maintained inthe presence of ACTH gradually returned to control levels. The fall in the ACTHinduced rate of adrenodoxin biosynthesis in adrenocortical cells was also reflected in a decrease in the ability to cellular RNA to direct adrenodoxin synthesis in acell-free translation system. Although the mechanism(s) involved in the decrease in adrenodoxin biosynthesis in ACTH-stimulated adrenocortical cells is presently unknown, it appears to reflect an ACTHdependent refractoriness of the adrenocortical cell which is also manifested in decreased steroid output as shown in Table I1 (25, 26). It does not appear toreflect a loss of cell viability. Trypan blue exclusion studies, examination of morphology by election microscopy: incorporation of [35S]methionine into total cellular protein (17), and the datain Table I all indicate that cell viability is not the source of the desensitization I 1 I I I 3275 3375 3475 phenomenon. MAGNETIC FIELD (gauss) The time-dependent, ACTH-induced changes in the rateof FIG. 4. EPR signal of adrenodoxin in bovine adrenocortical adrenodoxin biosynthesis in radiolabeled bovine adrenocorticells maintained in the absence ( A ) and presence ( B )of ACTH cal cells as well as in thecapacity of adrenocortical cell RNA for 36 h. The EPR signal was recorded at 103 K with the following to direct adrenodoxin synthesis in a cell-free translation sysinstrumentparameters: ( a ) modulation amplitude, 10 gauss; ( b ) tem correlate closely with changes in the synthesis of cytopower, 50 milliwatts; ( c ) microwave frequency, 9.15GHz;and ( d ) chrome P-450,, in both this and a previous study (17). In gain, 6,200. Spectra were corrected for the baseline and EPR tube geometry. Each spectrum is an average of32 scans, and the magnitude addition, preliminary results3 indicate that the synthesis of cytochrome P-45OIlpin ACTH-treated bovine adrenocortical of each signal was normalizedto 10 mg of protein/ml. cellsfollows a similar pattern. The synthesis of all three enzymes is increased by ACTH and reaches a maximum 36 h adrenodoxin content of 0.41 nmol X mg" of protein infreshly after the initiation of ACTH treatment. Thus, itappears that dispersed adrenocortical cells (data not shown). the synthesis of the various components of the mitochondrial steroid hydroxylase systems in the bovine adrenal cortex is DISCUSSION induced by ACTH in a coordinate fashion. The coordinate The presentstudies were carried out to investigate the regulation of cytochrome P-450,,, cytochrome P-45OI1p, adreeffect of ACTH on the synthesis of adrenodoxin by bovine nodoxin, and perhaps also adrenodoxin reductase synthesis adrenocortical cells maintained inprimary monolayer culture. may provide the adrenocortical cell with a meansof maintainLong term (12 h to 36 h) treatment of bovine adrenocortical ing appropriate molar ratios of these proteins (11, 12), and M) resulted in a 2-3-fold increasein cells with ACTH thus optimizing the efficiency of mitochondrial steroidogenethe rate of adrenodoxin biosynthesis. The effect of ACTH to sis. increase the synthesis of adrenodoxin was demonstrable in both cell-free translation systems programmed with bovine Acknowledgments-We gratefully acknowledge the expert techniadrenocortical RNA and [35S]methionine-labeledadrenocor- cal assistance of Grace Carlton andJanette Tuckey. tical cells. 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N. the magnitude of the adrenodoxin EPR signal in Y-1 adrenoY.Acad. Sci. 212, 76-88 cortical cells that had been treated with ACTH M) for 11. Kimura, T., Parcells, J . H., and Wang, H. P. (1978) Methods Enzymol. 52, 132-142 72 h. Similarly, Purvis et al. (14) have shown that ACTH increases levels of adrenodoxin in adrenals of hypophysecto- 12. Wang, H.-P.,Pfeiffer, D. R., Kimura, T., and Tchen, T. T. (1974) mized rats. Interestingly, ACTH failed to maintain an elevated rate of adrenodoxin biosynthesis. Thus, bovine adrenocortical cells J. M. Snyder, R. E. Kramer, E. R. Simpson, and M. R. Waterman, that had been incubated in the presence of ACTH for 48-60 unpublished observations. h svnthesized adrenodoxin at rates similar to those of cells R. E. Kramer, E. R. Simpson, and M. R. Waterman, unpublished that had been maintainedin the absence of ACTH. Asano observations.

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