Studies of adrenal steroid hydroxylation

ARCHIVES

OF

BIOCHEMISTRY

AND

Studies

BIOPHYSICS

146, 194-198 (1971)

of Adrenal

I. Purification

Steroid

of the Microsomal

Hydroxylation

21-Hydroxylase

System’

BRUCE J4ACKLER,2 BYRON HAYNES, DIANA S. TATTONI, DORIS F. TIPPIT, AND VINCENT C. KELLEY Department

University of Washington, Seattle, Washington Received January 11, 1971; accepted April 2, 1971 of Pediatrics,

A steroid 21-hydroxylase has been isolated from steer adrenal microsomes and purified seven to ten fold. The microsomal enzyme initially catalyzes the 21-hydroxylation of A5-pregnenolone, progesterone, and 17.a-hydroxyprogesterone to a similar degree, but progressively loses the ability to hydroxylate A5-pregnenolone and progesterone at different stages of the purification procedure. The results suggest that a single enzyme syst.em catalyzes the hydroxylation of the three steroids, but that some of the mechanisms of the enzymic reaction differ for each of the three steroid substrates. Previously, other investigators have shown that steroid 21-hydroxylation is catalyzed by the microsomal fraction of the adrenal cortex. The enzymic system has been partially purified by solubilization with Triton (l), and catalyzes the 21-hydroxylation of A5-pregnenolone, progesterone and 17-ahydroxyprogesterone. The reaction has been shown to have the following stoichiometry :1

mole of TPNH oxidized per mole 02 utilized per mole of steroid hydroxylated (2). AS shown

by Ryan

and Engel

(3), the enzyme

system is inhibited by CO suggesting that the cytochrome P450present in the preparations

is active

in the reaction;

however,

in

other work, Matthijssen and Mandel (4) have reported the isolation of an adrenal 1 Supported in part by Grant Number HE 05457 and Training Grant in Pediatric Endocrinology Number AM0 5190 from the National Institutes of Health. 2 Performed during the tenure of Career Development Award 2K3-HD-1128 from the National Institutes of Health. 3 Postdoctoral trainee in endocrinology supported by Training Grant in Pediatric Endocrinology Number AM0 5190 from the National Institutes of Health.

steroid 21-hydroxylase which contains no cytochrome P.w . The present paper presents studies of further purification of the adrenal 21-hydroxylase system? and demonstrates that although the hydroxylation of the A5-pregnenolone, progesterone, and 17-ar-hydroxyprogesterone appears to be catalyzed by one particulate enzyme system there are differencesin the mechanismsof hydroxylation for the three steroids. METHODS Assays of enzymic activity were performed at 38” by two methods: first, by determination of the rate of 02 utilization wit.h an oxygen polarograph, after subtraction of any blank rate which was present (rate present before addition of steroid, but with enzyme and TPNH present) ; and second, by chemical determination of the hydroxylated product. The assay system was similar to that reported previously by Cooper et al. (I), except that Mg”+ was found to be unnecessary for maximal activity; the reaction mixture contained 1 ml of a solution of 2% dialyzed bovine albumin with 0.15 M NaCl (pH 7.4), 0.2 ml of 0.15 M glycyl glycine buffer of pH 7.4, 0.04 ml of 0.15 M KC1 solution, 0.05 ml of 3y0 TPNH solution, 0.02 ml of a solution in ethyl alcohol of the appropriate steroid (16 mg/ 194

ADRENAL

MICROSOMAL

ml), and 0.8 ml of a suspension of the enzyme preparation in 0.25 M sucrose (approximately 6-7 mg protein). In experiments where quantitative determination of steroids were performed, the assay system was similar to that described above except all constituent,s were increased sixfold. The conversion by 21-hydroxylation of three steroid subst,rates: 17.a-hydroxyprogesterone, progesterone, and A5-pregnenolone to 11-deoxycortisol, deoxycort.icosterone, and 21-hydroxy-A5-pregnenolone, respectively, was studied and steroid determinations were performed as follows. The enzymic reaction was stopped by immediate addition of excess chloroform. Steroids were extracted from the reaction mixture with chloroform, separat,ed and pllrified by chromatography, and quantitat’ed by an appropriate reaction for each st.eroid. In the experiments employing 17.a-hydroxyprogesterone as sltbstrate, the 17.a-hydroxyprogesterone and ll-deoxycortisol were separated and purified by florisil column chromatography (5), all 17-ahydroxyprogesterone being found in the 470 met,hanol eluate and all ll-deoxycortisol in the 25% methanol eluate. Aliquots of these elnates then were fractionated by paper chromatography employing Zaffaroni systems. ll-Deoxycortisol concent,ration was measured by the phenylhydrazine reaction (6) and 17.a-hydroxyprogesterone by a quantit,at,ive modification of the vanillin reaction (7). In the experiments employing A5-pregnenolone or progesterone an aliquot of the chloroform extract from the reaction mixture was applied directly to the paper chromatograms. A5-Pregnenolone was quantitated by the vanillin reaction (7) ; Zl-hydroxy-A5-pregnenolone by tetrazolillm blue (modified Mader-Buck (8) procedure); progesterone by t,he dinitrophenylhydrazine reaction (Gornall-Macdonald (9)) ; and Il-deoxycorticosterone by tetrazolium blue (8). Protein was determined by the method of Lowry et al. (10). Cytochrome P,ja was determined spectrophotometrically as described previously (1). TPNH, A5pregnenolone, progesterone and I’?-or-hydroxyprogesterone were obtained from the Sigma Chemical Company. RESULTS

Pur$ication of the enzyme. All procedures were carried out at O-5”. Whole beef adrenals obt.ained fresh at the slaughterhouse were freed of fat and homogenized with an overhead high-speed blender in 0.25 M sucrose solution, and the microsomal fraction was isolated by repeated centrifugation as described by Cooper et al. (1) and stored at -20”. Upon thawing, the protein content

STEROID

21-HYDROXYLASE

195

was adjusted to about 10 mg/ml with 0.25 M sucrose, and 0.4 ml of a 10% solution of Triton x-100 (10% Triton x-100 in 0.25 M sucrose and 0.02 M phosphate solution of pH 7.4) was added per 3 ml of the microsomal suspension. The partially clarified suspension was centrifuged for 90 min at 35,000 rpm in the 40 rotor of a Spinco ultracentrifuge and the resulting residue was discarded. The supernatant was then centrifuged for 24 hr at 50,000 rpm in the 50 rotor of the Spinco ultracentrifuge, and a packed red residue (P2) was obtained. The residue (P2) was suspended in 0.25 M sucrose solution (the protein concentration adjusted to between 5 and 6 mg/ml) and the pH was adjusted to 7.4 with 0.15 M glycyl glycine buffer of pH 7.4. Solid KC1 was then added to a final concentration of 0.2 M (14.8 mg of KCl/ml of suspension) with continuous stirring, and the suspension was centrifuged in the 50 rotor of the Spinco ultracentrifuge for 30 min at 20,000 rpm. The supernatant solution was again centrifuged in the 50 rotor for 45 min at 50,000 rpm and the residue (P4) was suspended in 0.25 R/I sucrose solution (protein concentration adjusted to between 4 and 5 mg/ml). A solution of molar phosphate of pH 7.4 was then added to the suspension of P4 to a final concentration of 0.65 M, and the suspension was centrifuged for 40 min in the 50 rotor of the Spinco ultracentrifuge at 40,000 rpm. The supernatant solution was discarded and the final residue (P5) was suspended in 0.25 M sucrose solution. Table I shows the degree of purification and enzyme yield obtained during a typical purification procedure. As shown in the Table, the starting suspension of microsomee hydroxylated the three substrates (A5-pregnenolone, progesterone, and 17-a-hydroxyprogesterone) at approximately the same rate. Preparations of P2 showed an approximate twofold increase in the ability to hydroxylate progesterone and 17-a-hydroxyprogesterone, but had lost the ability to actively catalyze the 21-hydroxylation of A5-pregnenolone, Preparations of P4 and P5 showed an increasing 2l-hydroxylase activity with 17+hydroxyprogesterone as substrate, but progressively lost the ability to hydroxylate progesterone. Although specific act.ivities of the various

196

MACKLER

ET AL.

TABLE STEROID

81.HYDROXYLASE Microsomal

Substrate

ACTIVITIES

suspension

I

AT VARIOUS

STAGES

PZ

OF PURIFIC.~TION P4

PS

Specific activit+

Unitsb (Total)

Speciiic activity

Units

Specific activity

A5-Pregnenolone

2.2

1756

0.6

124

0.0

0

0.0

0

Progesterone

1.8

1436

4.3

886

4.6

166

3.3

68

17-a-Hydroxyprogesterone

2.0

1596

4.5

927

6.5

234

14.0

290

Units

Speciiic activity

Units

B Specific activity is defined as mbmoles of steroid hydroxylated per minute per mg of enzyme protein. b Units of activity are defined as total mrmoles of steroid hydroxylated per minute by the preparation.

fractions varied from one purification experiment to another, the relative activities of fractions within an experiment always followed the samepabtern described in Table I. Preparations of Pci of the highest purity had specific activities of about, 20 mpmolesof steroid hydroxylated per minute per mg of enzyme protein with 17-cr-hydroxyprogesterone as sub&rate, and hydroxylated progesterone and A5-pregnenolone at much lower rates. In addition, the ability to actively hydroxylate A5-pregnenolone always was greatly reduced at the P2 stage of purification. Studies of the microsomal-Triton x-100 suspension demonstrated that the loss of ability to hydroxylate A&pregnenolone was progressive with time at 0” and was SO-90 % complete in 24 hr. When the microsomalTrit)on x-100 suspensions were dialyzed against 0.02 M phosphate buffer of pH 7.5 to remove t,he Triton, the 21-hydroxylase act,ivit.y was markedly reduced with all three substrates. Treatment of the microsomes with Triton K-101 instead of Triton x-100 led to loss of A5-pregnenolone hydroxylase activity within 10 min and as in the case of T&on x-100 had no inhibitory effect on the hydroxylase activity with progest,erone and 17-a-hydroxyprogesterone as substrates. When deoxycholate was used inst,ead of Triton x-100, the 21-hydroxylase activity with all substrates was markedly reduced. Concentrations of cytochrome POX were determined in the preparat,ions of enzyme at various stagesof purificaGon and were found to be 0.47, 0.42, and 0.30 mkmoles/mg of enzyme protein for the microsomal, P2 and

P5 preparations, respectively. (All results are the averages of determinations on four or more preparat’ions.) Stoichiometry of the reactions. Preparations of P2 were studied to determine whether the oxygen utilized during the reactions (as measured by the oxygen polarograph) could be accounted for by the specific 21-hydroxylase reactions. Table II shows the results of experiments with either progesterone or 17-a-hydroxyprogesterone as substrate where both oxygen utilization and specific hydroxylat,ed product were measured simultaneously. As shown in the Table, ll-deoxycortisol and 11-deoxycorticosterone were formed during the enzymic reaction with 17-cr-hydroxyprogesterone and progesterone, respectively, as substrates, and the ratio of oxygen utilization during t#heexperiments to 21-hydroxylated steroid formed was approximately 1: 1 for both steroid substrates. In each instance only one product, namely, lldeoxycortisol or 11-deoxycorticost,erone, was found to be formed during the reaction, suggesting that other enzymes such as the 17-ahydroxylase were not present in the preparations. In control experiments with ll-deoxycorticosterone added instead of progesterone to the enzyme-reaction mixture, no oxygen was utilized and a recovery of only 75% of the added steroid was obtained. When the result,s for ll-deoxycort’icost’erone formation shown in Table II are corrected for this loss, an oxygen to 21-hydroxylated steroid ratio of 1: 1 results. The finding that’ 1 mole of oxygen was utilized per mole of steroid hydroxylated is in

ADRE?U’AL

MICILOSOMAL

STEROID

accord with the reaction mechanism of a mixed function oxidase and with the previous findings of Cooper et al. (2). Quantitat,ive studies of the formation of 21-OH, A5-pregnenolone by preparations of impure microsomes were not performed, but qualitative test,s performed on the assays in which A5 pregnenolone was used as substrate demonstrated that 21-OH, A5-pregnenolone was formed during the reaction. Mechanisms of the reaction. Studies were performed to determine whether the hydroxylation of t)he three steroid substrates was catalyzed by one enzyme system or by several. Virst, all fractions separated during the TBBLE RELATIONSHIP BND

BDTWEEN

‘%HYDROXYLATIO~

UTILIZ.~TION

.M

PREP~RITIONS

_ .--

purification procedure were assayed for %lhydroxylase activity with the three substrates. Only traces of activity jvere present in the discard fract,ions suggesting that only one dehydroxylase was present, but that the system was modified during the isolat’ion procedures so that it progressively lost, the ability t,o hydroxylate A5-pregnenolone and prog&erone. Second, experiments were performed in which the activity of the enzyme preparations was determined in the presence of one steroid and then again after addition of a secondsteroid sample. The results of t#he experimems are shown in Table III, and demonstrate that. there is no increase in maximal hydroxylase act,ivity of the preparations (rate of oxygen utilization) after addition of a second steroid substrate. This finding strongly suggests that a common rate-limiting step is present in the 21-hydroxylat,ion react.ions for the three steroids, and, therefore, t’hat one enzyme system catalyzes the three 21-hydroxylations.

II OXYGEN

CAT.4LYZF.D

BY

P2

OF

21-Hydroxylated Oxygen Substrate

steroid formed (Jmmles)

utilized (~moles)

ll-deoxy-

ll-deoxy! cortisol

17-WHYdroxyproges-

0.82 0.70

02 Utilized Steroid hydroxylated

DISCUSSION

corticosterone

1 0.77 ; 0.90

’

~ -

The experiments presented earlier in t,he paper describe the isolat,ion and purification of a steroid 21-hydroxylase syst,rm from bovine adrenal glands. Initial suspensionsof microsomesshow an equal abilit,y to hydroxylate 17-a-hydroxyprogesterone, progesterone, and A5-pregnenolone, but the abilities to hydroxylat,e A5-pregnenolone and proges-

1.1 0.78 = 0.94

Av

terone Progesterone

0.90

-

0.68

1.31

I

TABLE EFFI~XTS

OF ADDITION

OF -1 SECOND

STEROID

Specific Preparation As-Pregnenolone

Microsomal preparation

P?

2.4 -

activityb

with

AND single

Progesterone

ON

PREPIRATIONS

initial

steroid

I7-a-Hydroxyprogesterone

-

5.4

THE

R.&TE

OF 21-HYDROxYL.GE

ACTIVITYQ

OF P? Specific

activity

AS-Pregnenolonec

after

addition

ProgesteroneC

steroid

17-a-Hydroxy progesteroneC

2.5

2.0

3.2 1.G 2.5

5.1

1.0 4.5 4.7

5.5 4.8 3.6

4.3 4.6 4.3

a All results are the averages of 3 or more experiments. * Specific activity is defined as mrmoles of oxygen utilized c The activities in these columns are the activities present the reaction as a second steroid, the first. steroid added being

2.6

of second

1.9 2.5 2.2

2.4

0.9 -

III

SUBSTRSTE

OF MKROSOMES

IW

PI-HYDROXYLASE

per minute per mg of enzyme protein. after addition of the indicated steroid indicated in the first half of the Table.

to

198

MACKLER

terone are progressively lost during further purification although there has been a concommitant seven to tenfold increase in the rate of hydroxylation of 17-a-hydroxyprogesterone. This finding suggests either that more than one 21-hydroxylase is present in the microsomal preparations or that one enzyme system catalyzes the three 21-hydroxylations but that the mechanismsof hydroxylation differ in some respects for each of the substrates. The results of studies of the activity of the enzyme system with addition of more than one steroid substrate during the enzymic reaction demonstrates that a common rate-limiting reaction is operative in the hydroxylation mechanisms for the three steroid substrates, and suggestsstrongly that one enzyme system catalyzes the three 21hydroxylations. However, the finding that the hydroxylase activities of the preparations for the three steroid substrates vary at different stages of t’he purification procedure with the loss of the ability to hydroxylate first A5-pregnenolone and then progesterone demonstrabesclearly that there are differences in the mechanismsinvolved in the 21-hydroxylation of the three steroids. Such differences may involve separate sites of binding of the steroids to the enzyme or partially divergent pathways with separate cofactors for the individual hydroxylations, and may be the basis for the observed alterations in the

ET AL.

21-hydroxylation of progesterone and 17-ahydroprogesterone in congenital adrenal hyperplasia where the ability to carry out 21-hydroxylation may be interfered with (a) for both substrates or (b) only for 17-ahydroxyprogesterone. REFERENCES 1.

D. Y.,

COOPER, THAL,

NARASIMHULU,

S.,

ROSEN-

O., AND ESTABROOK, R. W., in “Functions of the Adrenal Cortex” (K. W. McKerns, ed.), Vol. II pp. 897-942.Appleton-century-crofts, New York (1968). D.

2. COOPER,

Y.,

(1963). K. J., AND 103 (1957).

3. RYAN, 226, 4.

Biophys.

5. WAXMAN, J. Clin. 6. PORTER,

Endocrinol.

S. H.,

AND

C. C., SILBER,

Biuphys.

W. J.,

8. MADER,

Chem.

MANDEL,

146,613

.IND

238,

L. L., J. Biol.

Acta

TIPPIT,

166, 201 (1950). 7. MCALEER, W. J.,

Biol.

ENGEL,

Biochim.

them.

J.

C.,

MATTHIJSSEN,

R. W.,

ESTABROOK,

O.,

ROSENTHAL,

1320 Chem.

J. (1967).

E.,

D. F., KELLEY, V. C., Metab. 21,943 (1961). R. H., J. Biol. Chem.

KOZLOWSKI,

62,196 BUCK,

M.,

Arch.

(1956). R. R., Anal.

Chem.

Bio24,

666 (1952).

A. G.,

9. GORNALL, Chem. 10.

0.

LOWRY,

H.,

L.,

AND

265

(1951).

MACDONALD,

M. P., J. Biol.

(1953).

201,279

ROSEBROUGH,

RANDALL,

N. J.,

R. J., J. Biol.

FARR,

Chem.

A. 193,