I72 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 55290

SITE SPECIFICITY OF BOVINE ADRENAL $&HYDROXYSTEROID

DEHYDROGENASE AND d5-3-KETOSTEROID ISOMERASE”

SARAH G. CHEATUM, ARTHUR W. DOUVILLE**, AND JAMES C. WARREN***

Departments of Obstetrics-Gynecology and of Bzochemistry, University of Kansas School of Medicine, Kansas City, Kansas (U.S.A.)

(Received June rjth, 1966) (Revised manuscript received September rgth, 1966)

SUMMARY

I. The $-hydroxysteroid dehydrogenase and As-3-ketosteroid isomerase activ- ities from the microsomal fraction of bovine adrenal cortex have been studied with emphasis on the site specificity for dehydrogenation and isomerization of the natural C,, and C,, steroid substrates.

2. The data indicate a distinct dehydrogenase site and a distinct isomerase site each of which is capable of utilizing both C,, and C,, substrates as shown by the following parameters : activity ratios during purification, pH curves, inactivation rates, and the kinetics of equimolar mixtures. These observations are similar to results with the same activities from bovine corpora lutea.

INTRODUCTION

The conversion of As-$-hydroxysteroids to A4-3-ketosteroids, first demonstrat- ed in mammalian tissues by SAMUELS et al.‘, is an important step in the biosynthesis of biologically-active steroid hormones. CHEATUM AND WARRENS have demonstrated that this conversion is mediated in bovine corpus luteum by a $I-hydroxysteroid dehydrogenase and a As-3-ketosteroid isomerase. The dehydrogenase is specific for the $-position and uses DPN+ as cofactor. Purification of these activities from the microsomal fraction and subsequent studies of stability and activity indicated one distinct dehydrogenation site and one distinct isomerization site, each utilizing both C,, and C,, natural substrates.

Because others.-5 have suggested the existence of two separate isomerases in bovine adrenal, we thought to evaluate that tissue using the methods employed with corpus luteum.

* The systematic names for steroids preceded by the trivial names used in this report : dehydroiso- androsterone, $-hydroxy-5-androsten-rT-one; pregnenolone, 3B-hydroxy-5-pregnen-so-one: and progesterone, 4-pregnen-j,zo-dione.

** Predoctoral trainee, National Institutes of Health. *** Career development awardee, National Institute of Child Health and Human Development.

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ADRENAL STEROID DEHYDROGENASEAND JSOMERASE I73

MATERIALS AND METHODS

The enzymes were purified essentially as described for ovary’. The medulla

and capsule were discarded and the cortex homogenized in 50 mM sodium phosphate buffer (pH 7.2), 0.25 M sucrose, 7 mM /?-mercaptoethanol I:I, w/v)(. Substrates, methods of homogenization, differential centrifugation, sonication, ammonium sulfate fractionation, dialysis and protein determination were identical to those described in the preceding report 2.

Assays Dehydrogenase activity was determined at 37” by monitoring formation of

DPNH at 340 rnp (As40 mp = 6220) in a Beckman DU spectrophotometer equipped with a Gilford attachment and a IO mV Honeywell Recorder. During inactivation studies, dehydrogenase activity was determined by fluorescence of the generated DPNH in an Aminco Bowman recording spectrophotofluorimeter using 340 rnp as the exciting wavelength and measuring fluorescence at 465 rnp. Each cuvette contained 50 mM sodium phosphate buffer (pH 7.2), 40 mM nicotinamide, 5 mM d&odium EDTA, 7 mM /?-mercaptoethanol, I mM KCN, the specified amounts of DPNf, and the design- ated amounts of dehydroisoandrosterone or pregnenolone in IOO pl of propylene glycol with a total volume of 3.0 ml. Corrections for endogenous DPNH-oxidation were made as required, but none was observed after the ammonium sulfate precipita- tion step of the purification scheme.

Isomerase activity was measured spectrophotometrically at 37” by the forma-

tion of the d4-3-ketone at 248 rnp (A 248 mp 16800). Each cuvette contained 50 mM Tris-KC1 (pH 8.2), 5 mM disodium EDTA, 7 mM /?-mercaptoethanol and the design- ated amounts of 5-androsten-3,r7-dione or 5-pregnen-3,2o-dione in IOO pl of pro- pylene glycol with a total volume of 3.0 ml. Controls for spontaneous isomerization were run, and solutions of the &-g-ketone substrates in propyIene glycol were pre- pared fresh daily.

The reactions were initiated by addition of enzyme. In all cases control assays were run in the absence of steroid. In some cases, early in the purification scheme, precipitating particles caused changes in absorbance. Appropriate corrections, which never exceeded 10% of the observed optical change with steroid present, were made in these cases. Because 5-pregnen-3,2o-dione is the least soluble of the substrates used, its solubility in the actual assay solution was evaluated spectrophotometrically at 320 mp. With no enzyme, the steroid was soluble to the extent of 3 - 10-5 M, with enzyme to the extent of 5 - 10-5 M.

To conform to the recommendation of the Inte~atio~al Union of Biochem- istry8, one Unit of either dehydrogenase or isomerase activity is defined as the amount which generates 1.0 pmole of product per min at 37”.

RESULTS

During puri~cation, both activities were assayed using concentrations of the C,, and C,, substrates which approached but did not exceed steroid solubility limits in the reaction mixture. These concentrations were at least twice the Km value for the isomerase and more than ten times the Km value for the dehydrogenase. Table I lists the recovery during purification.

Biochim. Biophys. Ada, 137 (1967) 172-178

774 S. G. CHEATUM, A. W. DOUVILLE, J. C. WARREN

Sonication destroys much of the activity, but was used because it relocates activity into a 105 ooo xg supernatant. The “spun dialysate” of the o-407/0 (NH&SO, fractions were used routinely for assays because the great majority of both activities was found in this fraction.

Ratios of isomerase/dehydrogenase activities during purification vary ro-fold with C,, substrates and 5.4-fold with C,, substrates, while both dehydrogenase and isomerase C,,/C,, activity ratios vary only r.7-fold. These observations favor one distinct dehydrogenase site and one distinct isomerase site.

TABLE I

RECOVERY AND ACTIVITY RATIOS OF DEHYDROGENASE AND ISOMERASE IN BOVINE ADRENAL CORTEX

The fractions were assayed under conditions listed in the text using 3.0 pmoles DPN+ with either 1.0 ymolc

dehydroisoandrosterone or o. r5 pmole pregnenolone and, for the isomerase, either 0.6 pmole 5-androsten-3, r 7- dione or 0.3 pmole 5-pregnen-3,zo-dione. Activity is expressed as mUnits per g equivalent of original tissue, and specific activity in mUnits/mg protein. D, dehydrogenase; I, isomerase.

Fracion Sub- Dehydrogenase Isomerase Activity ratios strate Acti- Specific yO Re- Acti- Specific

vity activity covnvy vity % Re- CU’/U G//D Dc,,Ic,, Ic,,Ic,~

activity couevy -_ ~____._

Mitochondria C,, 47 12 ‘39 35 C 21 40 10 82 20 2.0 3.0 I.2 I.7

Microsomes C,, 166 2.5 100 750 III 100 C 21 ‘07 16 IO0 630 93 IO0 5.9 4.5 1.6 I.2

Supernatant C,, 683 24 1070 37 C 21 600 2.1 540 19 0.9 1.6 I.1 2.0

Sonicated C,, 21 I2 I.3 180 104 24 microsomes C,, II 6 IO 99 58 16 9.0 8.6 I.9 1.8

c-40 % C C::

19 21 IL 158 174 21 (NH,),SO, 14 16 13 83 92 I3 5.9 8.3 I.4 I.9

._~

In one preparation, the majority of the dehydrogenase and isomerase activities was found in the 40-60% (NH&SO, fraction. A 60-80~/~ pellet was collected, resus- pended and assayed for both activities. While 1.0 mg of this protein displayed 41.7 mUnits of isomerase activity with 5-androsten-3,17-dione and 26.7 mUnits of activity with 5-pregnen-3,2o-dione, no detectable dehydrogenase activity was present with either dehydroisoandrosterone or pregnenolone. Further, no endogenous oxidation of DPNH was noted.

The original supernatant contained large amounts of activity. Electron micro- scopy of subcellular fractions of the ovarian preparations reported earlier2 revealed that there was incomplete separation of fractions. Those results combined with the fact that the specific activity of the microsomal fraction was greater than that of the supernatant caused us to wonder if the supernatant activity might be due to un- precipitated particulate fragments. Table II shows the result of recentrifugation of a supernatant fraction: 70% of the activity was precipitated by further 105000 xg

centrifugation, and an additional 27% was precipitated by 186000 xg centrifugation after diluting the sucrose from 0.25 M to 0.08 M. The activity, therefore, appears to be associated with particulate fragments, which are not precipitated in the initial 105 000 X g centrifugation.

Because EWALD, WERBIN AND CHAIKOFF~ have reported partial separation of

Biochim. Biophys. Acta, 137 (1967) 172-178

ADRENAL STEROID DEHYDROGENASE AND ISOMERASE ‘75

the C,, and C,, isomerase activities with their purification procedure while we had observed consistent ratios with ours, we repeated their purification scheme. Table III contains the results of this experiment. Whereas, in the previously published work a r5-fold difference in C,, 1 C,, ratios of isomerase was obtained, we noted only a r.6-fold difference.

T,4BLE II

RECOVERY OF DEHYDROGENASE AND ISOMERASE FROM RECENTRIFUGED SUPERNATANT OF BOVINE

ADRENAL CORTEX

The fractions were assayed under conditions listed in the text using 6.0 pmoles DPN+ and either 1.0 ,nmole dehydroisoandrosterone or 0.15 pmole pregnenolone and either 0.3 pumole 5-androsten- 3,17-dione or 0.3 pmole 5-pregnen-3,2o-dione. Activity is expressed as mUnits per g equivalent ot original tissue, specific activity in mUnits/mg protein. Each supernatant was pipetted off to prevent contamination by particles not firmly held in the pellet,

~_ Fraction Substrate Dehydrogenase Isomeruse

Activity Specific yO He- Activity Specific yO He- _ activity cover,y activity covery

- Supernatant C IS 66.3 7.5 100 710 80.0 100

C 21 65.0 7.3 100 260 29.4 100 Supernatant first recen-

trifugation 105 000 x g C,, 38.8 6.3 59 190 30.6 27 (0.25 M sucrose) C 21 51.3 8.3 79 IO1 16.3 39

Supernatant second recen- trifugation 186000 x g C,, 4.9 l.I 16 3.8 2

(0.08 M sucrose) C 21 5.5 1.3 8 1.8 3

TABLE III

COMPARISON OF THE SAME PREPARATION FROM TWO DIFFERENT LABORATORIES

Fractions designated and assays of previously reported ratios are those given in the purification scheme of EWALD, WERBIN AND CHAIKOFF~. Assays of the present study were done as in Table I.

Fraction

C D E

C,,/C,, isomeruse ratios

Previously reported Present study

I .85 1.30 3.27 0.92

0.22 1.46

The pH curve of dehydrogenase activity utilizing both C,, and C,, substrates exhibited a broad maximum between pH 7.o-10.0, whereas the pH curves of isomerase activity utilizing both C,, and C,, substrates had a sharp peak at 8.2.

Fig. I illustrates that rates of inactivation of dehydrogenase and isomerase differ while those of each activity with the C,, and C,, substrates are similar. These data suggest that two different proteins, or at least two different sites, are involved in dehydrogenation and isomerization.

Kinetic studies using equimolar mixtures7-9 of C,, and C,, substrates plotted by the double reciprocal method of LINEWEAVER AND BURK~O show the rate with the equimolar mixture at maximal velocity (vmaX .) to be intermediate between those with the C,, and C,, substrates alone, rather than additive. In mUunits/min (Fig. z), urnax. dehydroisoandrosterone is 5.4; vmaX. pregnenolone, 8.0; and vmaX. for the equimolar mixture, 6.2. In’ Fig. 3, vmax. 5-androsten-3,r7-dione is 11.8; vUmax. 5-pregnen-3,zo- dione, 15.4; and vmax. for the equimolar mixture, 13.5.

Biochim. Biophys. Acta, 137 (1967) 172-178

176 S. G. CHEATUM, A. W. DOUVILLE, J. C. WARREN

01 I 1 I I I

0 2 4 6 8 Time at 257 pH 7.2 (h) l/[Sl x 10-4

Fig. I. Inactivation of dehydrogenase and isomerase activities at pH 7.2, 25’. Per cent activity is in reference to the activity of a spun dialysate fraction at zero time. The activity used was urna%. (calculated from averages of duplicate assays at four substrate concentrations between I ,uM and IO PM for the dehydrogenase and duplicate assays at three substrate concentrations between 12.5 yM and 50 PM substrate tar the isomerase). This range insures that substrate activation or inhi- bition does not give a misleading value. Aliquots were removed from the spun dialysate fraction at the times designated, taken to 5” and assayed immediately. The dehydrogenase activity was measured at 37O on a recording Aminco Bowman spectrophotofluorimeter using 50 /Lg protein and 6.0 /Imoles DPN+. The isomerase activity was measured at 37” with a DU spectrophotometer equipped with a Giltord attachment. The residual activity of the dehydrogenase with dehydro- isoandrosterone (O-O) and pregnenolone (o-o) as substrates follows the same pattern, and the changes in isomerization of 5-androsten-3,17-dione (o-n) and 5-pregnen-3,zo-dione (m-m) appear to be similar, although different from the dehydrogenase.

Fig. 2. Equimolar substrates of dehydrogenase. z, is velocity in mUnits/min; S, substrate con- centration in moles/l of dehydroisoandrosterone, O-O; of pregnenolone, 0-e; and of each substrate in the equimolar mixture, A-A. The assays were performed with the Gilford attachment using 1.0 pmole DPNf and were initiated with 0.36 mg protein from a spun dialysate fraction.

Fig. 3. Equimolar substrates of isomerase. ZI is velocity in mUnits/min; S, substrate concentration in moles/l of 5-androsten-3,r7-dione, O-O ; of 5-pregnen-3,2o-dione, )-B; and of each sub- strate in the equimolar mixture, A-A. The reactions were initiated by the addition of 0.12 mg protein from a spun dialysate fraction.

Biochim. Biophys. Acta, 137 (1967) 172-178

ADRENAL STEROID DEHYDROGENASE AND ISOMERASE I77

Thus, dehydrogenation of both dehydroisoandrosterone and pregnenolone appears to be effected by a single dehydrogenase site, and isomerization of the result- ing C,, and C,, As-3-ketosteroids appears to be effected by a single isomerase site.

DISCUSSION

Previously studies with bovine ovary suggested one distinct dehydrogenase site and one distinct isomerase site each utilizing both C,, and C,, substrates2. Current opinion relating to protein synthesis would suggest that this might be the case in adrenal if the two tissues possess enzymes with identical amino acid sequence, al- though different amino acid sequence or the presence of isozymes with different predominance in the two tissues cannot be ruled out.

The ideal approach to such problems of identity and specificity is absolute solubilization and subsequent extensive purification during which complete separa- tion of activities is or is is not accomplished. We have effected separation of a fraction that has isomerase activity, but lacks dehydrogenase activity. This separation coin- cides with other indirect evidence for the existence of distinct isomerase and dehydro- genase sites 2,a.

Specificity of these sites in terms of C,, and C,,substrates is yet another matter. Other investigators have generally suggested a single dehydrogenase site in bovine corpora lutea and in bovine and rat adrenals. However, these suggestions usually11~12 but not invariablyI are based on activity ratios of complete conversion of AS-~,!?- hydroxysteroids to A4-3-ketones. EWALD, WERBIN AND CHAIKOFF~.~ using a differ- ential centrifugation scheme reported marked differences (but no complete separation) in activity ratios with C,, and C,, isomerase substrates. Their data would support existence of two isomerases in adrenal, but no clear-cut proof of solubilization was given. On repeating this scheme, little difference in activity ratios was observed, again supporting a single isomerase site.

Further, it has been shown that various treatments and even variation in salt concentration can alter relative rates at which a single site converts different sub- strates 14,16.

Methods applicable to determine identity or non-identity of sites when solubili- zation and extensive purification cannot be effected require strict attention to several details. Evaluation of activity demands use of initial linear velocities or rate constants. Velocities should be determined at several substrate concentrations and handled as double-reciprocal plots, especially with a steroid substrate, to rule out inhibition by steroids at high concentrations or the exceeding of substrate solubility limits. Correc- tions must be made for spontaneous or non-enzymatic generation and possible destruc- tion of the measured product. Enzymes under study should ideally be solubilized to avoid limitation of access of substrate. Identical enzyme molecules residing in par- ticles of differing structure could demonstrate altered activity ratios with two sub- strates as a result of differing access of those substrates to an identical active-site.

In this study we have adhered rigidly to all the above requirements except the one of solubilization. We have not’been-able-to prepare-a-fraction with activity that was not sedimented by centrifugation at rag ooo xg for 3 h. EWALD, WERBIN AND

CHAIKOFF’~ have also reported difficulty in solubilizing the isomerase activity from

bovine adrenals. Nevertheless, the following data are consistent with the existence

Biochim. Biophys. Acta, 137 (1967) 172-178

178 S. G. CHEATUM, A. W. DOUVILLE, J. C. WARREN

in adrenal of a single dehydrogenase site effecting conversion of both natural C,, and C,, AS-3B-hydroxysteroids to their respective As-3-ketones and a separate single iso- merase site converting both A6-3-ketones to A4-3-ketones: (I) Ratios of isomerase/ dehydrogenase activities routinely change 5- to IO-fold during purification while C,,/C,, ratios of both activities change only r.7-fold. (2) A 60-80% ammonium sulfate fraction displays considerable isomerase activity and lacks dehydrogenase activity. (3) The pH dependency of dehydrogenase activity is similar using both C,, and C,, substrates. The pH dependency of isomerase activity is similar with both C,, and C,, carbon substrates and differs from that of dehydrogenase. (4) The inactivation rate of isomerase is similar whether determined with C,, or C,, substrates but differs from that of dehydrogenase which is also similar whether determined by C,, or C,, sub- strates. (5) For both isomerase and dehydrogenase activities, equimolar mixtures of C,, and C,, substrates resulted in maximal velocities that were not additive, but rather intermediate between those of the separate substrates.

In all these respects, activities from bovine adrenal are similar to those from bovine ovary. The existence of single isomerase and dehydrogenase sites capable of converting both C,, and C,, steroids is not surprising considering that a single active site of estradiol-r7/3 dehydrogenase from human placenta can dehydrogenate estra- diol, testosterone and estradiol-3-sulfate” as well as the zod-hydroxy group I8 and

that adrenal IQ-hydroxylase can utilize both C,, and C,, steroid substrates 19. These observations would also imply that the C,, substrate, dehydroisoandrosterone, could interfere with conversion of C,,-A5-3/3-hydroxysteroids to A4-3-ketones in viva, while formation of dehydroisoandrosterone sulfate, the major form in which this steroid is stored and released in adrenal might abolish this inhibition.

ACKNOWLEDGEMENT

This study was supported by a grant from the National Institute of Arthritis

and Metabolic Diseases, United States Public Health Service (AM-05546).

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Biochim. Biophys. Acta, 137 (1967) 172-178