the conversion of progesterone to androgens … · the conversion of progesterone to androgens by...

THE CONVERSION OF PROGESTERONE TO ANDROGENS BY TESTES*

BY WILLIAM S. LYNN, Jn.,t AND ROSE H. BROWN

(From the Department of Biochemistry and Medicine, Duke University School of Medicine, Durham, North Carolina)

(Received for publication, November 18, 1957)

Studies from this laboratory (l-3) and others (4, 5) have indicated that testicular extracts are capable of oxidizing progesterone to testosterone and acetic acid. Preliminary data from this laboratory indicated that the enzymatic activity was associated with the microsomes, that reduced triphosphopyridine nucleotide (TPNH’) and oxygen were essential for the reactions, and, further, that cyanide was impressively stimulatory. This report deals with some properties of the four enzymes thus far implicated; namely, (1) progesterone --) 17c+hydroxyprogesterone; (2) 17ar-hydroxyprogesterone + androstenedione + acetic acid; (3) androstenedione e testosterone; and (4) 17Lu-hydroxyprogesterone e A4-pregnene-17or, 2Op- diol-3-one (and some of their properties).

EXPERIMENTAL

Substrates-Progesterone-4-C14 and progesterone-21-Cl4 were obtained from commercial sources and were found to be chromatographically pure. A4-Pregnene-17a,20/3-diol-3-one, as well as the 2Ocr isomer, was obtained from Dr. T. Gallagher and Dr. I. Salamon of the Sloan-Kettering Insti- tute for Cancer Research. All other steroids were obtained from commercial sources. Melting points and chromatographic purity were checked for each of the substrates.

All of these water-insoluble substrates were dissolved in ethanol, and small volumes of the solutions were added directly to the incubation mix- tures. After incubation, proteins were precipitated by 2 volumes of ethanol, and the products were isolated from the filtrate.

Isolation and Identijication Procedures-In experiments in which progesterone-21-Cl4 was used, radioactive acetic acid was isolated directly from the incubation mixture by distillation. The acetic acid was further separated by two paper chromatographic systems, either as the free acid (6)

* Supported by the United States Public Health Service, grant No. RG4758. t Markle Scholar in Medical Sciences. 1 The following abbreviations are used in this paper: TPNH, reduced triphospho-

pyridine nucleotide; Tris, tris(hydroxymethyl)aminomethane; DPNH, reduced di- phosphopyridine nucleotide; TPN, oxidized triphosphopyridine nucleotide; PP, sodium pyrophosphate.

1015

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1016 PROGESTERONE CONVERSION TO ANDROGMNS

or as the ammonium salt (7). Final identification was accomplished by the addition of carrier acid and the preparation of two derivatives, the anilide and the p-toluide. These derivatives were recrystallized to constant specific activity.

To distinguish further between formic and acetic acids, a Duclaux constant was obtained (Table I). Slaunwhite and Samuels (4) reported that they found formic acid in a similar testicular extract; however, no formic acid was obtained in our incubations.

Radioactive Steroid Products--In experiments in which progesterone-4-Cl4 was the substrate, the steroids were extracted with ether and then separated by the paper chromatographic procedures of Zaffaroni and Burton (8) and Bush (9). After separation by these two different methods, the four enzymatic products, 17a-hydroxyprogesterone, testosterone, androstenedione, and A4-pregnene-17a!, 20P-diol-3-one, were eluted from the chromato- grams, carrier steroids were added, and crystallizations to constant specific activity were carried out (Table I). A4-Pregnene-17a!, 20/3-diol-3-one is easily separable from its 2Oa isomer in the chromatographic system of Zaffaroni (Table II). The dihydroxyketopregnene was further identified by the isolation of radioactive acetaldehyde as the product of the bismu- thate oxidation of the side chain of the radioactive dihydroxyketopregnene. The acetaldehyde was trapped as its 2,4-dinitrophenylhydrazone, and this product separated on the paper chromatographic system previously described (10). Carrier hydrazone was then added, and this was crystallized to constant specific activity. (Th e only two steroids of this group which are difficult to separate are testosterone and 17a-hydroxyprogesterone; however, these two steroids were separable in a modified Zaffaroni paper chromatographic system; i.e., the paper strips were dipped in a mixture of propylene glycol and methanol, ratio 1:3 rather than 1: 1) (Table I). In experiments with non-labeled substrates, the steroids were determined after separation and elution by measuring their absorption at 240 rnp. Since these testicular extracts contain no 3-keto reductases, quantitative recoveries could be obtained with the spectral assay. The 17-ketosteroids were also determined calorimetrically by the Zimmermann reaction (11).

Appropriate paper chromatographic blanks without substrate were run in all cases. To insure further that the 240 rnp absorption represented only steroid absorption, the spectra of ethanolic eluates between 210 and 300 rnp were obtained by a model 11 Cary recording spectrophotometer. In all experiments, the ultraviolet absorption in this region was charac- teristic of the A4,3-keto structure of the steroids.

Enzyme Preparation

Preparation of Microsomes-All four enzymes were found to be much more active in their particulate state. These particles were obtained by


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W. S. LYNN, JR., AND R. H. BROWN 1017

homogenizing (Potter-Elvehjem homogenizer) the decapsulated testes of guinea pigs in 0.02 M Tris-HCl buffer (pH 7.0). The large inactive parti-

TABLE I Radioactivity of Products and Derivatives Obtained from Radioactive Progesterone

-i-

Compound

Androstenedione

17a-Hydroxyprogesterone

A’-Pregnene-l’lor ,20@-diol-3- one

Acetaldehyde-2,4-dinitrophenylhydrazone

Testosterone

Anilide of acetic acid

p-Toluide of acetic acid

mat

172 174 174 173

221 223 223

195-202 195-200 196200

165 165 167

154 155 155

112 112 114

145 147 147

-

2. :Zystal- mtions

1 2 3 4

1 2 3

1 2 3

1 2 3

1 2 3

1 2 3 4

1 2 3

Solvent

tiethanol

Methanol

Methanol- water

Ethanol

Methanol

Methanol- water

Nethanol- water

Radioactivity*

Veight Total :ounts

w.

33 16 14.1 10.2

2000 930 798 552

45 5ooo 25 2560 9.7 1052

10.1 3500 5 1640 1.7 560

27 600 17 345 6.1 115

42 4500 21.2 2050 9.7 1025

400 6000 148 2010 22 340 6.1 101

100 95 26

4000 1760 460

-

- Duclaux constant of radioactive acetic acid (3800 counts)

1st 10 ml. Counts, 140 Found,$ 5.6 Theory,S 6.8 2nd 10 “ “ 166 “ 6.6 “ 7.1 3rd 10 “ “ 177 “ 7.6 “ 7.4

C.).rn. be* mg.

60 58 57 55

111 102 107

347 324 330

22 20 19

107 96

105

15 13 15 16

20 18.5 17.6

* Counts per minute per mg. represent the counts corrected to infinite thinness. t All melting points were performed on a Fisher-Johns electric apparatus. 1 Ratio.


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1018 PROGESTERONE CONVERSION TO ANDROGENS

cles were removed from this homogenate by centrifugation at 10,000 X g for 15 minutes, and the small active particles were obtained from this supernatant fluid by centrifugation at 100,000 X g for 20 minutes. The latter particles were then suspended by gentle homogenization in sufficient

TABLE II Localization of Testicular Enzymes

Enzyme

Supernatant fluid, 100,000 X g

Small particles, 10,00&100,000 X g

Large “ 2000-10,000 x g Supernatant fluid

O-50yo (NH4)zS04 fraction* Soluble 0-50yo (NHa) 804 fraction Insoluble 0-50yo (NHa) &Oh fraction

Small particles + supernatant fluid Washed small particles - supernatant fluid

“ I‘ “ + (‘ “

- 1 Radioactive products recovered

17a-Hydroxy- progesterone

)er cent

7

20

0

15 0

15 15 2

20

Acetate

pn cent

0

25

0

6 0

14 70 0

60

Enzyme, the amount of enzyme used per incubation corresponded to that amount of material obtained from one-half of a guinea pig testicle. Substrate, 0.3 rmole of progesterone-4-Cl4 (10,000 counts). Incubation, 0.02 M Tris-HCl, pH 7.0, at 30” for 1 hour. Gas phase, air. Additions, TPN 0.02 pmole, glucose-g-phosphate dehydrogenase 0.5 unit (see the footnote below). Glucose 6-phosphate 5.0 pmoles, and NaCN 1OV M. Washed small particles; particles washed twice with 10 ml. of 1 M NaCl.

* The supernatant fluid was precipitated with (NHJ804 (50 per cent saturated), and this precipitate, resuspended in Tris buffer and then recentrifuged at 100,000 X g for 15 minutes (see “Insoluble O-50 per cent (NH,) $04 fraction”), contained the enzymatic activity. The TPNH-generating system was equally active in all fractions.

Tris buffer to give an emulsion with an optical density of 1.0 at 280 ml.c. The constituents of these microsomes were previously reported (12).

Fractionation Procedures-Attempts by various procedures to dissolve these microsomes failed, but it was observed that the inactive soluble 100,000 X g supernatant fluid could be made active by precipitating the proteins either with salt or ethanol or by dialysis against buffer. How- ever, in each instance it was found that the generated activity resided in the insoluble precipitates produced by these procedures (Table II). These observations appeared to indicate that, although these enzymes were pres-


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ent in a soluble form, they were active only in the presence of particles. It was further observed that these active particles could be inactivated by washing them repeatedly with either 0.02 M Tris buffer or with 1 M NaCl and that their enzymatic activity could be regenerated by reexposure to more 100,000 X g supernatant fluid. From this observation it appeared possible to fractionate the enzymes soluble in the supernatant fluid and to assay them in the presence of the washed inactive particles. This proved to be the case. However, since these enzymes were usually largely inactivated by salt, ethanol, or isoelectric precipitations, the most useful method of separation thus far found was adsorption and gradient elution, with the hydroxyapatite columns of Tiselius et al. (13). Columns, 4 X 1 cm., containing 75 gm. of hydroxyapatite, were sufficient to adsorb and to separate over 90 per cent of the protein obtained in the supernatant fluid from six guinea pig testicles (about 20 ml. in volume). Elution and separation were effected by eluting these columns with four 10 ml. fractions of phosphate buffer, pH 6.8, of increasing ionic strength; Fraction 1, 0.001 M phosphate buffer; Fraction 2, 0.01 M; Fraction 3, 0.1 M; and Fraction 4, 1.0 M. The 1 M buffer removed most of the remaining protein, including hemoglobin (Table III). The last two fractions were then dialyzed against sufficient water to make the final phosphate concentration 0.05 M. As shown in Table III, the two reductases as well as the two oxidases could be easily separated; however, the androstenedione reductase contaminated both oxidase fractions. Since these fractions are still unstable (50 per cent of activity lost in 24 hours), no further purification has been obtained. Although the two reductases were initially present in the supernatant fluid (100,000 X g), the ratio of their activities could be greatly accelerated by the addition of the washed microsomes.

Incubations-These were routinely made in closed vessels at 30” with shaking at pH 7.0 in either 0.01 M phosphate or 0.02 M Tris buffer. Con- sumption of oxygen was measured by the conventional Warburg tech- nique.

Results

Guinea pig and rat testicles were used as the source of enzymes. Rat testicles were appreciably active only after injection of the animals daily for 2 weeks with 25 units of chorionic gonadotropin (Upjohn) per day. Guinea pig glands were equally active both before and after injections of chorionic gonadotropin. Since the rat glands contained only slight amounts of androstenedione reductase, the cleavage product from progesterone or 17a-hydroxyprogesterone was primarily androstenedione, whereas in the guinea pig testosterone was the cleavage product isolated. How- ever, the androstenedione reductase obtained from the guinea pig could be


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inhibited at pH 8.5, and, under these conditions (the cleavage enzyme is still appreciably active at pH 8.5), the product obtained with the guinea pig preparation was also androstenedione (Table VIII). Table IV indi-

TABLE III Enzyme Fractionation

Enzyme fraction Protein Enzymes present

Microsomes + supernatant fluid. . . . . . . .

Microsomes . . . . . . . . . . Washed microsomes* . . . . . .

“ “ + supernatant fluid.. . . . . .

Supernatant fluid only. . . . Washed microsomes + Frac-

tion O..................... Washed microsomes + Frac-

tion l..................... Washed microsomes + Frac-

tion 2..................... Washed microsomes + Frac-

tion 3.....................

Washed microsomes + Frac- tion 4.....................

*g. pn ml.

1.2

1.2 1.2

0.04

0.02

0.04

0.22

0.66

Activity

per Gent

All 4 ‘I 4

Traces

All 4

100 60

>l

86 >l

Traces >l

20&Reductase 70

“ 15

Androstenedione reductase, 60 17a-hydroxylase 90

Androstenedione reductase, 1701,20-keto cleavage enzyme

30 50

Enzyme, all incubations contain that portion of protein which was obtained from one-half of a guinea pig testicle by the fractionation procedures. The fractions represent eluates from hydroxyapatite columns (see the text). Incubations, Tris- HCl buffer, pH 7, for 30 minutes at 30” with air as the gas phase. Additions, TPN 0.02 pmole, glucose 6-phosphate 5.0 pmoles, glucose-g-phosphate dehydrogenase 0.5 unit. Substrate, 0.3 pmole of each substrate for each enzyme was added in 0.02 ml. of ethanol, and the products were assayed, after chromatographic separation, by their adsorption at 240 rnp. Per cent activity, amount of product produced in 30 minutes expressed as per cent of the activity of microsomes plus the supernatant fluid. Assays were for the 2@-reductase, 17a-hydroxylase, 17,20-cleavage enzyme and androstenedione reductase. To assay the 2O,%reductase, it was necessary to inhibit the 17,20 cleavage enzyme. This could be done under anaerobic conditions.

* Microsomes washed twice with 10 ml. of NaCl (1 M).

cates that TPNH and O2 are specific and are the only cofactors necessary for the oxidases, whereas TPNH is the only cofactor necessary for the reductases. Cyanide, 10e4 M, pyrophosphate, 0.002 M, and carbon monox- ide, 5 per cent, had stimulating effects on all of the enzymes. These effects were probably the result of the inhibition of cytochrome oxidase, which


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was present in these particles (as is TPNH cytochrome c reductase) (11). Although these particles contain only trace amounts of porphyrins (12), cytochrome oxidase, as measured by the oxidation of reduced cytochrome c,

TABLE IV Cofactor Requirements

Substrate additions

0 . . . . . . . . . . . . . . . . . . . . . . TPN 1.0 pmole.. . . . TPNH 1 .O pmole . . DPNH 1.0 “ . . . . . . Peroxide generators,? 3 pmoles. TPNH, 1 pmole + Nz. . .

“ 1 “ + CN 10- M. ‘I 1 “ + PP, 2 rmoles. ‘I 1 “ + CO 5 per

cent......................... TPNH, 1 pmole + malate, 5

rmoles....................... TPNH, 1 pmole -I- NaC1,0.02 M.

“ 1 pmole + Nz + NaCl, 0.02 M. . . , . .

TPNH, 1 pmole + catalase, 0.5 mg..........................

Progesterone-21-C”

lla-Hy- &OXY- progesterone

Qer cm1

4 10 15 4 4

10 4 8

-

A

_- Ql

cetate

CT cen1

0 0

35 0 0 4

90 85

15 70 10

20 75 Trace 15 60 10

20 0 45

21 37 7

-

- I 17a-Hydroxypro-

gesterone

- A

‘estos- terone

C” CC?kL Qcr cent per c.%t

0 0 2 0 7 15

30 10 80 0 3 11 0 0 6 4 29 75

80 7 100 80 10 100

50

50 55

5

15 100

17 100 20 100

50 100

Enzyme, microsomes obtained from one-half of a guinea pig testicle per incubation. Substrates, 0.3 rmole of each, added in 0.02 ml. of ethanol. Incubation, 1 hour at 30” in 0.02 M Tris-HCl at pH 7 in a final volume of 2 ml. Products, all steroids assayed after chromatographic separation by their absorption at 240 rnp. The radioactive acetate was counted. The numbers refer to the per cent of the added substrate.

* A4-Pregnene-l7cu,20/3-diol-3-one. t The peroxide generators used were glucose oxidase and xanthine oxidase (hy-

poxanthine used as a substrate for the xanthine oxidase).

J-K&o- megnene-

dial’

,ndrostene- dime

Testos- terone

is present in these particles, and also is inhibited by 10eb M NaCN. These particles are also much more active in the presence of salt (NakJOr, KzS04, NaHP04, KCl, NaCl, MgC12) in concentrations of 0.002 to 0.02 M. Ta- ble V shows that all four enzymes are more active in the presence of salt.

Stoichiometry-Table VI shows that each oxidative step, i.e. the hydroxylase and the cleavage enzyme, uses approximately 1 mole of 02 per


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1 mole of TPNH. The results are complicated by the fact that the enzymes could not be separated adequately, by the presence of non-specific TPNH oxidases in these microsomes, and by the presence of the 17cr-reductase which is also competing for the TPNH. However, the amount of TPNH used by the 17a-reductase, i.e. the amount of testosterone formed, is known, and, by subtracting this amount from the total, the amount of TPNH used in the oxidative steps can be obtained. The data indicate that the oxidation of 1 mole of progesterone to androstenedione

TABLE V

Stimulation of Microsomal Enzymes by Salts

dl

0.0005 0.002 0.02 0.002 0.02 0.002 0.02 0.02 0.02 0.02 0.02 0.002

-

Control NatSO

I‘ ‘I

NaHPOa “

PP ‘I

KC1 NaCl K&Oh MgSO4

‘I

per cent per c&d

14 10 16 10 92 70 85 50 90 60 60 15 95 100 25 20 60 30 60 30 90 40 40 30 32 35

Each incubation contained 0.3 pmole of substrate, 0.8 pmole of TPNH and NaCN, W6 M in 2 ml. volume. The enzymes were microsomes obtained from one-half of a guinea pig testicle per incubation in Tris-HCl buffer, pH 7. Gas phase, air, at 30”, for 1 hour. The products were assayed in the same manner as in Table IV.

Substrate: 17a-bydroxy- progesterone

Testosterone formed Acetate formed

requires approximately 2 moles each of O2 and TPNH, and that the oxidation of 17cr-hydroxyprogesterone to androstenedione requires 1 mole of each (3). Confirmation of this 1: 1 ratio of TPNH and 02 was also obtained by using washed microsomes plus the supernatant fractions of the hydroxyapatite columns. Since the stoichiometry implied that these enzymes were peroxidatic, other sources of enzymatic peroxide, xanthine oxidase and glucose oxidase, were used; however, these peroxide generators (Table IV) could not replace the need for TPNH. Moreover, catalase did not inhibit these oxidative enzymes. Therefore, if peroxide is the oxidant, it is not free peroxide, but peroxide generated in or on these particles by TPNH.


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Inhibitors-Table VII indicates that all of these enzymes, in the presence of microsomes, are inhibited by p-chloromercuribenzoate but not by iodoacetate. Interpretation of these data on inhibitors is difficult because of the fact that many of these inhibitors tended either to clump or to precipitate the microsomal emulsion; thus the results of such data may be interpreted simply by means of their “detergent” activity on the particles.

TABLE VI Stoichiometry of Enzymatic Reactions

Control (no substrate). ‘I ( “ TPNH)

Progesterone, 1 rmole. .

17cu-Hydroxyprogester- one, 1 rmole.. . . . .

Androstenedione, 1 pmole . . . . . . . .

Testosterone, 1 pmole. . .

-

(

-

-

h used

pmolc

0.11 0.08 0.76

pmoles

0.15

1.0

Jmwle

0.55

0.51 1.1 0.46

0.18 1.1 0.03 0.08 0.05

ubstrat mid&d

-

e s1 I I

-

ubstratl .educed

#mwlc

0.2

0.4

0.95

Product

p?wlc

0


Testosterone, 0.: 3-Ketopregnene-

dial,* 0.05

Testosterone, 0.4

I‘ 0.91 Androstenedione

0.05

.-

1

L

1 ,

-

TPNH used for

o$da-

O:%d

1.3

0.98

1.14

Enzyme, microsomes from 1 guinea pig testicle. Each flask contained 1 pmole of substrate (added in 0.02 ml. of ethanol), TPNH, 1.3 pmoles, NaCN, 10-4 M, Tris buffer 0.02 M, pH 7, to a final volume of 2 ml. at 30”. TPNH was measured by titra- tion of the incubation mixture with 2,6-dichlorophenol-indophenol at the end of the incubations (3). 02 was measured with standard Warburg flasks and substrates were measured, after separation by paper chromatography, by their adsorption at 240 rnp.

*A4-Pregnene-l7ru,20&diol-3-one.

However, since inhibitory phenomena are not present for all enzymes in the microsomes, the effects may be specific. The hydroxylase and the reductases are stable to most of the inhibitors, but the cleavage enzyme is inhibited by a number of reagents like Versene, and other metal-chelating agents such as diethyl dithiocarbamate, CY ,CY-dipyridyl, and o-phenanthroline, as well as heavy metals such as iron and copper. Although other hydroxylases are known to be ‘metal-containing, for example phenol oxidase (14), these data seem to imply that the cleavage enzyme rather than the steroid hydroxylase is a metal-containing enzyme.


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Effects of pH--Table VIII indicates the apparent pH optimum of the various enzymes in microsomes. The cleavage enzyme is most active at pH 7, whereas the hydroxylase is still quite active at pH 8.5. The 2Op reductase and androstenedione reductase are inhibited at alkaline pH values and are active at values as low as pH 6.

E$ects of Hydrolytic Enzymes-To ascertain the type of structure or membrane which maintained the insolubility and catalytic activity of these

TABLE VII Inhibitors of Steroid Enzymes

Inhibitor

Control .................................. 40 p-Chloromercuribenzoate, 10-a M .......... 0 VerSene, IF3 M ........................... 35 Diethyl dithiocarbamate, 10m3 M .......... 60 Cyanide, lO+ M ......................... 65 Pyrophosphate, lO+ M .................... 60 a,cu-Dipyridyl, 10m3 M ................... 45 o-Phenanthroline, 10m3 M. ................ 45 Iodoacetate, 10m3 M ....................... 0 Nitrogen ................................. 0 Fe++, 2 X lo-” M. ....................... 40 &I++,2 x lo-’ M ......................... 40 Catalase, 0.5 mg .......................... 35

- I

--

Per cent product formed in 30 minutes

lfa-Hydrox- ylase

-

_-

-

50 80 0 10 5 80 0 80

80 100 75 100 0 80

25 80 50 70 0 100 0 80 0 0

43 85

Lndrostenedione reductase

Enzyme, washed microsomes obtained from one-half of guinea pig testicle per flask, plus the appropriate purified enzyme from hydroxyapatite columns (see the text). Incubation was performed at pH 7 (Tris-HCl) in 2 ml. of final volume at 30” in an open flask. Each flask contained 0.3 pmole of the various substrates, 0.6 pmole of TPNH, and NaCN lo-’ M. The products were assayed after chromatographic separation by their absorption at 240 rnp.

particles, preliminary treatment of these microsomes with trypsin, ribonuclease, and pancreatic lipase was performed. Particles obtained from guinea pig testicles were incubated at 30” (pH 7, Tris buffer) with 1 mg. of each of the above enzymes and then again sedimented. The amount dissolved was assayed by measuring the increase of absorption at 260 and 280 rnp in the supernatant fluid. Enzyme activity was assayed in the usual way in the resuspended particles. Of the three hydrolytic enzymes used, only lipase caused any leaching of ultraviolet-absorbing material, and it was, likewise, the only enzyme which inactivated the steroid enzymes. If, however, particles which had been treated with lipase were


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then treated with trypsin and ribonuclease, a further marked increase in ultraviolet-absorbing material was obtained in the supernatant fluid (Ta- ble IX). This implies that the surface of these particles is largely fat and that this fat is necessary for enzymatic activity, and furthermore, that this fatty surface protects these particles from the hydrolytic activity of trypsin and ribonuclease.

Sequence of Enzymatic Events-With these impure enzymes, two ques- tions arose: was it the A4-pregnene-17a!, 20/Sdiol-3-one or the l’kr-hydroxyprogesterone which was the substrate that was cleaved; and was acetate

TABLE VIII

Effects of pH on Steroid Enzymes

I Substrate: Progesteronedl-C’“, 0.2 /~mole

PR I Products formed I

6.0 6.5 7.0 7.5 8.5

Acetate

gcr cent per cent per CMIL per cent

4 12 20 0 20 12 25 0 35 18 15 0 15 22 11 5 11 33 0 12t


-

Androstenedione

Enzyme, microsomes were obtained from one-half of a guinea pig testicle. Incu- bation was performed for 30 minutes at 30” in Tris-HCl, pH 7, volume 2 ml. Gas phase, air. Products were assayed in the same manner as in Table IV. Additions, 0.6 pmole of TPNH.

* A’-Pregnene-17a,2@-diol-3-one. t Because of the difficulty in separating progesterone from androstenedione, the

formation of androstenedione was noted, 17a-hydroxyprogesterone being used as the substrate, and the same results were obtained.

the actual cleavage product? The first question was answered when the 3-ketopregnenediol became available in quantity. The equilibrium of the 2Op reductase in the presence of excess TPNH was in favor (90 per cent) of the reduced product, and it could be shown under these conditions that very little cleavage could be effected, as compared to a similar incubation with 17a-hydroxyprogesterone as substrate (Table X). Moreover, in incubations performed at pH 8.5, cleavage of 17cY-hydroxyprogesterone could be obtained, but under these conditions microsomes could neither reduce 17a-hydroxyprogesterone to A4-pregnene-1701, 20&diol-3-one nor cleave the side chain of the latter compound or its 2ocU epimer. These observations made it likely that the actual substrate undergoing cleavage is 17cr-hy-


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TABLE IX

Effect of Lipase, Ribonuclease, and Trypsin

Substrate: 0.2 rmole of progesterone-21-C”

Control. . Ribonuclease-treated,t 1 mg Trypsin-treated,t 1 mg.. Lipase-treated,t 1 mg.. . Lipase + ribonuclease +

trypsin..................

Products formed

17eHydroxy- 3-Keto- progesterone pregnenediol’

fier cmt

10 10 12 30

5

Acetate

)er Gent per cent

0 80 20 55 15 59 31 5

10 0

Material released% 260 In@

0.05 0.03 0.350

0.95

Enzyme, microsomes obtained from one-half of a guinea pig testicle, preincubated with 1 mg. of each of the hydrolytic enzymes for 30 minutes at 30”, pH 7, and then resedimented at 109,000 X g for 20 minutes. The increase over controls in adsorption at 260 rnp was obtained on each supernatant fluid. Additions, 0.5 pmole of TPNH. Incubations, pH 7 (Tris 0.02 M), for 30 minutes at 30” in open flasks. Prod- ucts, assayed in same manner as in Table IV.

* A4-Pregnene-17a, 20&diol-3-one. t The hydrolytic enzymes were all commercially obtained and were enzymatically

active. 1 The material released had the typical broad adsorption peak at 260 rnp of nu-

cleotides.

TABLE X

Substrate for Cleavage Enzyme

pe7 cent per cent )er cent

17c+Hydroxyprogesterone, pH 7. ................. 66 15 “ “ 8.5 ................. 22 0

A4-Pregnene-17a,20j3,diol-3-one, pH 7 ............. 10 11 “ “ 8.5 ............ 0 0

A4-Pregnene-l7or,2&,diol-3-one, pH 7 .............. 0 0 0

Enzyme, microsomes obtained from one-half of a guinea pig testicle. Incubation, in same manner as in Table IV. Addition, TPN 0.2 pmole, glucose 6-phosphate 5 pmoles, glucose-g-phosphate dehydrogenase 0.2 unit, NaCN 10-s M. Products, assayed in same manner as in Table IV. All substrates were added in 0.2 pmole amounts.

* The cleavage product was testosterone at pH 7, but 90 per cent androstenedione at pH 8.5.


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droxyprogesterone. Also, the fact that the 17-hydroxyl function is (Y in the substrate and p in the product strongly implies that the 17-ketone had to be the intermediate.

The answer to the second question is less clear. In repeated experiments, with progesterone-21-Cl4 and the partially purified enzymes, the only radioactive non-steroidal material present at the end of the incubation was acetic acid. Many attempts to isolate radioactive acetaldehyde or ethanol, by use of trapping agents or carrier acetaldehyde, have failed. Also, the stoichiometric measurements, which indicate that the oxidation equivalent of 1 mole of Hz02 is needed per mole of substrate cleaved, imply a peroxidatic type oxidation, thus:

CHa

c=O + HaOa +

I -C-OH

I

If the above mechanism is correct, both fragments, certainly the acetic acid and possibly the androstenedione, should contain oxygen derived from the atmosphere. Ols measurements to test this hypothesis have been made, but, because of the low activity of the enzymes and insufficient amounts of the products, the 018 measurements are still equivocal.

DISCUSSION

These data indicate that there are at least four enzymes, androstenedione reductase, 20-keto reductase, 17a-hydroxy-20-keto cleavage enzyme, and 17a-hydroxylase, involved in the production of testosterone from progesterone in testes. All these require TPNH (DPNH was inactive) and two require molecular oxygen. These two which require oxygen, l’kr-hydroxylase and the 17a-hydroxy-20-keto cleavage enzyme, are found in microsomes and were appreciably active only in the presence of particles. However, the two reductases, androstenedione reductase and the 17a-hydroxy-20- keto reductase, are active in the soluble state, but their activity was impressively stimulated by the presence of washed inactive particles. It has also been demonstrated that these particles are non-specific in that they can be generated from the soluble supernatant fluid by such procedures as dialysis and salt precipitation. It has been shown previously that these particles have a great affinity for water-insoluble substances (12) such as steroids and Amphenone B, and can quantitatively remove these substances from aqueous solution. Therefore, the ability of these particles to stimulate these steroid enzymes could be due to their surface activ-


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ity; i.e., their ability to adsorb the steroids as a monomolecular film, thus effecting emulsification of the globules of steroids which were added as substrate. By using these particles as emulsifying agents, partial purification of the four enzymes was accomplished by chromatography on columns of hydroxyapatite. Many other procedures for fractionating proteins proved to be ineffective.

Since two of the enzymes involved, the hydroxylase and the cleavage enzyme, require molecular oxygen, an understanding of the mechanism of this type of oxidation was sought. The TPNH-O2 stoichiometry of each enzyme indicates that the equivalent oxidative power of hydrogen peroxide is necessary. A great number of other similar enzymes have now been described and have been shown to add molecular oxygen to various substrates such as phenol oxidases, steroid oxidases (15), tryptophan oxidase (16), and lipoxidase (17), but no clear understanding of the mechanism of any of these has evolved. Some are stimulated by catalase; some are inhibited. Some can use any source of enzyme-generated peroxide and some are quite specific. Copper is involved in the phenol oxidases, iron in tryptophan oxidase, but neither metals nor sulfhydryl groups are involved in the enzymatic action of lipoxidase. The studies reported here on two steroid oxidases indicate that two different types of enzymatic mechanisms are present, although both require molecular oxygen as well as a specific source of reductive power, i.e. TPNH. Both enzymes, by stoichiometric measurements, require either hydroxyl radicals or hydrogen peroxide, but they most likely require the free radicals since neither catalase nor enzymatically generated peroxide has any effect. However, it was previously shown that these particulate preparations contain large quantities of catalase activity (12), and the addition of more catalase would be expected to be without effect (Table IV). Metals do not seem to be involved in the action of the 17cu-hydroxylase because metal-binding agents are without effect, and it is not likely that porphyrins are involved, for there is no measurable (photometrically) amount of porphyrin present (12). These particles do, however, contain sizable quantities of uncharacterized flavins (12). Perhaps these flavins are the coenzymes involved in the reduction of the oxygen; however, addition of flavin mono- nucleotide, flavin adenine dinucleotide, or riboflavin was without effect on either enzyme.

The cleavage enzyme differs from the hydroxylase in that it is inhibited by metal-binding agents, i.e. Versene, diethyl dithiocarbamate, and (Y,(Y- dipyridyl, but not by cyanide or pyrophosphate. The fact that both enzymes are not inhibited but actually stimulated by cyanide as well as pyrophosphate makes it uncertain that metals are involved. All these interpretations are, of course, open to question because of the particulate nature of these enzyme preparations.


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Finally, the fact that these steroid as well as other enzymes (previously demonstrated) (12) can be activated by surface-active agents such as intracellular particles, and the fact that agents which are adsorbed by these particles, such as Amphenone B, digitonin, and other detergents (3), can also affect the rate of enzymatic activity, plus the fact that the ionic strength of the medium exerts an appreciable effect on these phenomena, make very attractive the idea that these microsomal particles may exert considerable control over intracellular phenomena. Similar surface phenomena at cell membranes must also undoubtedly occur. Such phenomena have been hypothesized for the action of the fat-soluble vitamin D on the gut and bone (18). Data reported here seem both to indicate that the cell membrane is not the only surface of the cell which has regulatory functions and to imply that surface phenomena within the cell are equally important. Fraser and Kaplan (19) from their studies on the activation of catalase in yeast, and Robert and Polonovski (20) from their work on the activation of the lipoprotein particles of xanthine oxidase in milk, have drawn similar conclusions.

SUMMARY

Four enzymes from testes concerned with the oxidation of progesterone to testosterone and acetic acid have been partially purified. All of them require reduced triphosphopyridine nucleotide and two require oxygen. Each is markedly catalyzed by the presence of cytoplasmic particles. A4-pregnene-17ol, 20p-diol-3-one has been identified as one of the enzymatic products.

BIBLIOGRAPHY

1. Lynn, W. S., Jr., Federation Proc., 16, 305 (1956). 2. Lynn, W. S., Jr., and Brown, R., Biochim. el biophys. ucta, 21, 403 (1956). 3. Lynn, W. S., Jr., and Brown, R. H., J. Biol. Chem., 232, 1005 (1958). 4. Slaunwhite, W. R., Jr., and Samuels, L. T., J. Biol. Chem., 220, 341 (1956). 5. Viscelli, T. A., Lombardo, M. E., and Hudson, P. B., Federation Proc., 16, 265

(1957). 6. Jones, A. R., Dowling, E. J., and Skraba, W. J., Anal. Chem., 26, 394 (1953). 7. Brown, F., Biochem. J., 47, 593 (1950). 8. Zaffaroni, A., and Burton, R. B., J. Biol. Chem., 193, 749 (1951). 9. Bush, I. E., Biochem. J., 60, 370 (1951-52).

10. Lynn, W. S., Jr., Steel, L. A., and Staple, E., Anal. Chem., 26, 132 (1956). 11. Zimmermann, F., Vitamins and Hormones, 6, 1 (1947). 12. Lynn, W. S., Jr., Brown, R. H., and Mullins, J., J. BioZ. Chem., 232, 995 (1958). 13. Tiselius, A., Hjerten, S., and Levin, c)., Arch. Biochem. and Biophys., 66, 132 (1956). 14. Dawson, C. R., and Magee, R. J., in Colowick, S. P., and Kaplan, N. O., Methods

in enzymology, New York, 2, 17 (1955). 15. Hayano, M., Lindberg, M. C., Dorfman, R. I., Hancock, J. E. H., and Doering,

W. von E., Arch. Biochem. and Biophys., 69, 529 (1965).


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16. Knox, W. E., and Mehler, A. H., J. Biol. Chem., 187, 419 (1950). 17. Bergstr6m, S., and Holman, R. T., Advances in Enzymol., 3, 447 (1943). 18. Kodicek, E., Bone structure and metabolism, Ciba Foundation symposium,

London, 13 (1955). 19. Fraser, M. J., and Kaplan, J. G., J. Gen. Physiol., 38, 515 (1955). 20. Robert, L., and Polonovski, J., Discussions Faraday Sot., al,54 (1956).


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William S. Lynn, Jr. and Rose H. BrownTESTES

PROGESTERONE TO ANDROGENS BY THE CONVERSION OF

1958, 232:1015-1030.J. Biol. Chem.

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