Cytochrome P450 and steroid 21-hydroxylation in microsomes from human adrenal cortex

Life Sciences Vol . 10, Part II, pp. 901-908, 1971. Printed in Great Britain

Pergamon Press

CYTOC,fi P450 AND STEROID 21-FiYDROICYLAT~ON IN MICR030I~S FROM HUMAN ADRENAL OORTEX Alvin M . Lewis 2 and George T . Bryan3 Division of Endocrinology, Department of Pediatrics, and Clinical Study Center, University of Tease Medical Branch, Galveaton, Texas 77550

(Received 12 Aprll 1971; in final form 30 June 1971) Sum mar Human adrenal microsomes, from patients without adrenal dysfunction, were examined for cytochrome P450 . These studies demonstrate cytochrome P450 with Type I binding spectra in the presence of progesterone, 17a.-hydroxyprogeaterone, pregnenolone end androstenedione, The spectral changes with progesterone and 17a-hydroxyprogsaterone were reversed by NADPH . Type II spectra were not observed . Although the human adrenal cortex contains cytochrome P450 associated with steroid 11-hydroxylation in mitochondria (1), no similar studies of the human adrenal cortical microsomal 21-hydroxylase system have been described,

In the present studies we demonstrate the presence of cytochrome

P450 in the microsomal fraction from human adrenal cortex sad describe the steroid induced spectral changes in the presence of certain steroidal substrates ae wall as the reversal of these spectral changea by NADPH . Materials and Methods Cedaveric human adrenals from patients without adrenal dysfunction ware chilled with ice water within LO-15 minutes after death .

The glands

were dissected free of adipose and connective tissue, weighed and bisected to expose the medulla, which was removed whenever possible .

Tha adrenals

(mainly cortex) were minced with a knife and homogenized 1n 4 volumes of 0,25 M sucrose with a glass-Teflon, motor driven homogenizer (10 passes) . 1 Supported by USPH3 DnEW Grants AM-07616 end RR-00073 . 2 Supported by USPHS DREW Postdoctoral Fellawahip AM-42420 . 3 Markle Scholar in Academic Medicine .

901

902

Suman Adrenal Cytochrome P450

Vol. 10, No. 18

The homogenate was centrifuged at 1000 x g for 15 minutes to remove cell debris and nuclei, at 25,000 x g for 30 minutes to sediment mitochondria, and at 105,000 x g for 1 hour to sediment the microsomea, cedure ores performed at 0-4 °C,

The entire pro-

In order to remove hemoglobin, the 105,000

x g pellet was crashed by resuspension in a volume of 150 mM RCL containing 3 mM MgCl2 equal to the original homogenate and recentrifuged at 105,000 x g for 30 minutes .

This pallet was auspnnded by brief homogenization in

1-1,5 ml of 0 .25 M sucrose and is called herein the "microsomal suspension ." Electron photomicrographs of thin material showed smooth endoplasmic reticulum with no intact mitochondria . All spectral studies were performed with a dual wavelength, double beam spectrophotometer (Parkin-Elmar Model 356) which had been modified by mounting a blower fan on the door to the Light source compartment .

The

air flaw from this fan maintained the temperature of the cell compartment chamber at 25 + 0 .2°C . during spectral scanning . pH 7 .40 containing 3 mM MgC12 . ethanol .

The buffer was 50 mM Tris-C1,

Steroids were dissolved in redistilled

Protein concentration was detnrmiaed by the Lowry method . Results

The presence of cgtochromn P450 is human adrenal cortex microsomea is indicated by the peak at 447 nm in the dithionite-reduced, carbon monoxide treated sample bhawn in Fig, l,

Spectral changes is the Soret region occurred

following the addition of substrate to the iaicrosomal suspension (2) . composite graph of several of these spectra is shown in Fig . 2 .

A

The presence

of progesterone induced a broad maximum around 385-390 and a minimum at 420 am ; L7o.-hydroxyprogeaterone showed a maximum at 385 and a minimum near 419 nm ; pregnanolone caused a maximum at 385 sad a minimum at 418 am .

These spectral

changes era similar to the Typo I shift (3) which has a maximum at 385 and a miai~ at 420 nm .

p4 -Androstenedione caused a broad maximum at 380-385 am

and a minimum at about 422 nm,

Vol. 10, No . 18

Suman Adrenal Cytochrome P450

903

HUMAN AC hSCROS011~S A.(NapS~I-(NaeS :OI~ 9. (Nos Sl04+CO)-(Na~O~ aoo

a2o

4ao

wAVEiE:NC~rH (mN

4so

aeo

FIG. 1

Carbon monoxide difference spectrum of human adrenal cortical microsomes . Each cuvette contained 0 .1 ml of the microsomal fraction suspended in 0 .25 H sucrose, and 2 .9 ml of 50 mM Tris-C1, pH 7 .4 containing 3 mM MgC12 . Curve A . Pinch of sodium dithionite added to both cuvettes . Curve B . Carbon monoxide then added to sample cuvette with resulting maximum at 447 nm . The total change in absorbents between 447-490 nm shown here is 0 .016 units, which is equivalent to 0 .176 GiM cytochrome P450, as calculated by the method of Estabrook, et al ., with an eztinction coefficient of 91 mM-l cm 1 . (5) Protein concentration is 0 .72 mg/ml .

HVwAAN AC MICR060NES STERO~ IrOUC.ED SPECTRA arroH PROC ( 9.PREGI~ENOl.01~E - C.PROGESTEROf~E~ ) D.e4-A (---)

0.006

Û

0.003 o000

a ~-OD03 -OAO6

340

400 420 WAVELENßTH (nN

440

460

FIG. 2

Human adrenal cortical mioroaomes showing steroid-induced spectral changes . A mixture of 0 .2 ml microsomal suspension and

904

Human Adrenal Cytochrome P460

Vol . 10, No. 18

5 .8 ml buffer vas divided between two 1 cm cuvettes . Steroids dissolves in ethanol warn added to sample cuvette in successive 5 ul increa~anta (with ethanol added to the reference) . The difference spectrum vas obtained after each addition . A composite of these spectra is shown here where the P450 concentration is constant (0 .28-0 .29 ~tl1), protein concentration is 0,52 mg/m1, and each steroid is at a concentration giving near maximal change in absorbents . A . 17a.-üydroxyprogesterone, 5 .0 ~.~M . B . Pregaenoloae 2 .4 ~M . C . Progesterone, 6 .4 ELM . D . p4 -Androstenedionn, 6 .4 ßùi . The effcct of increasing concentrations of these steroids on the absarbancy changes was determined by recording changes in apectrs following successive additions of the steroid to the sample cuvette . total plot of these data is ahovn in Fig . 3 .

A double-recip

The approximate spectral

FIG . 3 Humsn adrenal cortical microsomes . Double reciprocal plot of change is absorbaace (DA) vs steroid concentration . Experimental conditions are similar to those in Fig . 2 . The change in absorbanca wan calculated from the maximum (385 to 390) minus the minimum (418 to 422) absorbaace as depicted in Fig . 2 . The calculated valuna for Rs are : A . Pregnenolone, K a~0 .7 ;aM . 8 . Progesterone, Ks ~6,7 uM . C . 17a~Hydroxyprogesterone, K s ~ 1 .5 uèl. D . Androstnnadiona, K s~4 .0 ~M .

Vol. 10, No. 16

Human Adrenal Cytochrome P450

90 5

diaeociatioa constant, Ka , waa estimated for each steroid from the intercept oa the abscissa . 6 .7

4+~;

The relative Ks values obtained were :

progesterone,

p4 -androstenedione, 4 .0 WM ; 17a-hydroxyprogesterone,

pregnanoloue, 0,7

1,5

4ùi ;

,

and

4+M.

Tha spectral changes induced by progesterone and 17a-hydroxyprogesterona are stable unless a reducing agent is introduced .

In the presence of a

NADPH generating system the induced spectral changes disappesr with time (Fig . 4),

Limited quantities of the humsn microsomal fraction did not allow

extension of these kinetic studies to pregnenolone and p4 -androstenedione .

FIG . 4 Human adrenal cortical microsomes . Spectral kinetics . Reaction mixture contained 0 .2 ml microsomal suapeasion, 2 .7 ml Tria-MgCl2 buffer, and 0,145 ml of a NADPA generating system waaistlng of trisodium isocitrate (250 mM), 0,1 ml ; isocitric dehydrogenase in 50x glycerol, 0 .02 ml ; and NADPH, tetrasodium salt (40 mg/ml), 0 .025 ml .(3) Abaorbance changes were observed at 385 ctm (sample beam) and 420 nm (rafarance beam) using the dual wavelength mode, as a function of time . The steroids were added at sero time, in the following concentrations : A . Progesterone 0,96 4ùi . B . Progesterone 3,8 4ùi . C. 17a-hydroxyprogestarone 3 .8 4ùi. D. 17a-hydroxyprogesterone 12,0 uM .

908

Human Adrenal Cytochrome P450

Vol, 10, No. 18

Preliminary studies on the adrenal cortex mitochondrial fraction . from these human adrenals demonstrated the presence of cytochrome P450 sad Typ~ I spectral shifts with 11-deozycorticoaterona, 11-deoaycortisol, and metyrapone (2-methyl-l,2-bis (3 pyridyl) - propanone) . Discussion Tha presence of cytochrome P450 in human adrenal cortical microsomes is demonstrated by : (1) the absorbents peak at 447 nm with the carbon monoaida treated, dithionita reduced suepnnsion, (2) the induced spectral shifts with substrates such as progesterone and 17a-hydroayprogaeterone and (3) the reversal of these shifts in the presence of an electron donor (NADPH) . Cytochrome P450 in beef adrenal cortex microsomas has bean studied previously by others (4-6) as well as by us (7) .

Studies with mitochondria confirm

the observations of Wilson, et al (1), in which they demonstrated that human adrenal cortical mitochondria have an electron-transfer pathway similar to that which Omura, at al (4), have described for beef adrenals .

Human

adrenal microsomea show spectral changes with progesterone and 17a-hydroxyprogesterone which are reversible in the presence of NADPH, and which are qualitatively similar to those found with beef adrenal cortical microsomas . Preliminary results in this laboratory suggest that p4 -androatenedione in concentrations large enough to induce nest-maximal spectral changes (i .e ., near-saturating amounts) vill inhibit, but not prevent, epactral changes following the subaaqueat addition of 17a-hydroxyprogesterone .

The

Type I shift produced by pregnenolone was unexpected, since with beef adrenal microsomea a Type II shift develops (min, at 390, max . a t 420), ae reported earlier (7) .

A Type I shift of this magnitude vithout 11-deoxycortieol

binding cannot be explained by mitothondrial contamination .

The 3~01-

dehydtogenase reattioa which occurs in mitrosomee could be the sour ca of the Typa I spectrum .

Vol . 10, No. 18

Human Adrenal Cytochrome P450

90?

Although the exact nature of the steroid-induced spectral change ie unknown, its appearance preceding hydroxylation, as well as the relative specificity for steroids resembling the natural substrates suggests a steroid-cytochrome P450 interaction and the formation of an enzyme-substrate complex (2) .

The spectral dissociation constants (Ks ) for progesterone

(6,7 uM) and 17¢-hydroxyprogesterone (1 .5 G.~M), shown in Fig . 3, are very similar to those K s values found in beef microsomes (progesterone 7 .1 ~aM and 17a-hydroxyprogesterone, 1 .0 i.iM) which ware reported earlier (7) . Despite these relative differences in the Ks values, the apparent Km values for progesterone and 17Q,-hydroxyprogesterone in beef ware similar,

7 .1 4~M .

At present there is insufficient kinetic data to estimate Km values for these substrates with human adrenal microsomes . The basis for aubetrete specificity in cytochrome P450-mediated hydroxylations remains unknown .

The protein moiety of the cytochrome P450

may be the determinant of substrate specificity, thus explaining the binding of substrates to adrenal mitochondrial end microsomsl P450 preferentially . Possibly pregnenolone and p4-androstenedione bind to their specific "sites" on the protein or to the 21-hydroxylation "site ."

Kinetic data and product

identification are insufficient at present to clarify the mechanisms responsible for these induced spectral changes .

The two types of substrate-induced

spectral changes (termed I ä II)(3) that have bean studied extensively in rabbit liver microeomes(and seen also 1n beef adrenal microeomes (7) a~ rat liver microsomea (3,8)) have been taken ae evidence for an interconversion of one form o~ cytochrome P450 to another form (9) .

There is no evidence

for a Type II spectral change in human adrenal mlcroeomes when titrated wlth progesterone, 17a-hydroxyprogesterone, pregnnnolone, 11-deoxycorticosterone, 11-deoxycortisol or p4 -androstenedione, all of which have elicited either a Type I shift or no spectral changes (deoxycorticoaterone sad deoxycortisol) . In beef adrenal microsomes, however, a Type II shift has been observed with pregnenolone (7) .

The present data show that beef and human adrenal cortical

908

Human Adrenal Cytochrome P480

Vol. 10, No. 18

microsomes differ in the type of spectral change induced by pregnenolone, indicating that pregnenolone may be bound in human adrenal microsomea by a form of P450 similar to that which binds progesterone, 17a-hydroxyprogeaterone, and p4-androatenedione . References 1.

L . D, WILSON, S . B . OLDHAM, and B . W . HARDING, J . Clin . Endocr, I~teb . 28, 1143 (1968) .

2.

D . Y . COOPER, S . NARASIMHULU, 0 . ROSENTHAL, and R . W, ESTABROOK in : Functi~na of the Adrenal Cortex, K . W . McKarne, editor, Vol . II, p . 897 . Appleton-Century-Ctofte, N . Y . (1968) .

3.

J . B, SCHENKMAN, H, REMMER, and R, W . ESTABROOK, Mol . Pharmacol, _3, 113 (1967),

4,

T . OMURA, R . SATO, D, Y . COOPER, 0 . R05ENTëfAL, and R . W, ESTABROOK, Fed . Proc . 24, 1181 (1965) .

5.

R . W . ESTABROOK, D . Y . COOPER, and 0 . ROSENTAAL, Biochem . 2 . 338, 141 (1963 ;

6.

D, Y, COOPER, R . W . ESTABROOR, and 0 . ROSENTHAL, J . Biol . Chem . 2~ , 1320 (1963),

7.

G, T . BRYAN, and A . M, LEWIS, Program of the 51st Meeting of the Endocrine Socie~, New York City, June 1969 . Abstract #93, p . 77 .

8.

F, MITANI, and S . HORIE, J . Hiochem . 65, 269 (1969) .

9.

A . G . HILDEBRANDT, and R . W . ESTABROOK in : Microsomea and Dru J . R . Gilette, et a1 ., editors, p . 331, Academic Press, N . Y .

Oxidations, 1969 ,