Partial purification and some properties of guinea pig kidney 17 β-hydroxy-C19 steroid dehydrogenase

701

~ARTIAL PURIFICATION AND SOME PROPERTIES OF GUINEA PIG KIDNEY 17~-HYDROXY-C~9 STEROID DEHYDROGENASE Dai Kee Liu and Charles D. Kochakian Laboratory of Experimental Endocrinology University of Alabama in Birmingham Birmingham, Alabama U.S.A. 35233 Received: 12/7/71 ABSTRACT Guinea pig kidney 17~-hydroxy-C~9-steroid dehydrogenase was partially purified by a combination of streptomycin sulfate and ammonium sulfate fractionation, Sephadex filtration, DEAE-ceilulose chromatography and a second Sephadex filtration. The specific activity of both TPN- and DPN-linked activities was increased fifty-fold and thirty two-fold for the respective recovered enzyme activities. The purest fraction was increased two hundred thirty-fold in specific activity. The partially purified enzyme sedimented as one symmetrical peak on ultracentrif~gation. The S~o,w was 3.0S and the D~o~w was 7.89xi0-Tcm2sec -l. On disc electrophoresis the TPN- and DPN-linked 178-0H C19-steroid dehydrogenase activities were revealed in five prominent bands~ three weakly stained protein bands showed no enzyme activity. The two cofactor linked activities were not separated throughout the purification steps and had a ratio~of i to 8 in favor of the TPN-linked activity. The molecular weight was 35,100, 31,600 and 31,200 by ultracentrifugation, Sephadex filtration, and disc electrophoresis respectively. No bound cofactor was detected in the partially purified enzyme fraction. Testosterone produced the highest activity among the steroids tested~ 17~-estradiol elicited only slight activity. A crude preparation of the enzyme in 0.25 M sucrose was'stable but readily lost activity after purification. Addition of 7mM ~-mercaptoethanol and 0.25 M sucrose or 20~ glycerol prevented the loss in activity. The enzyme activity was lost on making cytosol 4 M in urea or On dialysis against 8 M urea. The partially purified enzyme was labile to heat but was stable at 4 ° or -20 °. Thiol-blocking agents inhibited the enzyme activity. There were 2.8 moles-SH/mo!e protein. INTRODUCTION The presence and activity of 17~-hydroxy-C~9-steroid dehydrogenase (17~-hydroxy-Cle-steroid: reductase,

TPN (DPN) 17~-oxido-

E.D.i.I.I.K) vary not only among tissues but in the

702

ST ER O I D S

same tissue of different

19:5

species (1-3).

distribution of TPN- and DPN-linked

The subce!lular

enzyme activities also

varies among tissues and animal species (3). activities

The enzyme

in the liver and kidney were higher than in the

other tissues examined

(i, 2).

In the guinea pig the kidney

but not the liver 17~-OH-C19-steroid

dehydrogenase

gested as a regulator for maintenance of androgens

TPN-linked 178-OH-Cls-steroid

is located in the cytosol and the DPN-iinked

activity in the microsomai both coenzyme

of a homeostatic mixture

(4).

In the guinea pig liver, dehydrogenase

was sug-

fraction (3, 5, 6).

specific activities

However,

exist exclusively in the

cytosol of the guinea pig kidney (3) and in the microsomal fraction of the dog prostate

(7).

Whether the dual cofactor

activity in the kidney is due to one enzyme which utilizes both cofactors or to two separate entities is not known. Accordingly,

this study was undertaken to determine whether

the two activities could be separated. MATERIALS AND METHODS Materials: TPN and DPN (P-L Biochemicals, Inc.) were checked for purity by paper chromatography. The steroids (G. D. Searie and Co. and Syntex, S.A.) were recrystallized from methanol and water. The various grades of Sephadex were purchased from Pharmacia Fine Chemicals, Inc. Acrylamide, bisacrylamide, 3-dimethylaminoproprionitrile, ethylenediamine tetraacetic acid, 8-mercaptoethanol and DEAE-cellulose were the products of Eastman Organic Chemicals. Phenazine methosulfate, p-nitro blue tetrazolium, bovine serum albumin, horse heart cytochrome c, bovine RNase, yeast alcohol dehydrogenase, trypsin and p-chloromercuribenzoic acid were products of Sigma Chemical Company. 5, 5'-Dithiobis(2-nitrobenzoic

May 1972

ST ER O ID S

70

acid) (DTNB) was from Aldrich Chemical Company. Sucrose and ammonium sulfate (both special enzyme grade), iodoacetic acid, and N-ethyl-maleimide were purchased from Mann Research Laboratories. Dithiothreitol was from Calbiochem. Methanol (Baker) was redistilled and the water was glass distilled. Animals: Young adult male guinea pigs, 550-900 gm. were purchased from stock dealers. They were fed commercial diet ad libitum and were fasted overnight before autopsy. Enzyme purification: All of the steps were performed at ~o. The kidneys were homogenized twice with four volumes of 0.25 M sucrose-i mM EDTA-7 mM mercaptoethanol in a Virtis45 homogenizer at ~30 speed for 30 seconds. The cytosol was separated by centrifugation (~), the soluble RNA was removed by precipitation with streptomycin sulfate (8) and the enzyme activity was precipitated at 40 to 80% ( ~ 4 ) ~ S 0 4 saturation. Sephadex G-150 filtration: About 75% of the original activ~t~ was recovered from a peak emerging after the main peak of protein (Fig. i) and was preceded by a small fraction of activity which was presumably either enzyme associated with non-enzyme protein or enzyme aggregates. Inclusion of 0.5 M KCI in the eluting buffer did not affect the appearance of this minor fraction of enzyme activity. Similar elution patterns were obtained on filtration through Sephadex G-100 and G-200. Figure i: Sephadex G-150 gel filtr~t~o~ ~ f SEPHADEX GI5~ the ~m~o~i--um s~l~aT@ =E 2.4 X ~27 cm t ~raction ( ~ 0 ~ 8 ~ s~t~rat ion). o co / 534 ~ prote~n--in T~y m~ ¢,,I of 0.02 M potassium phos,,,I.8 800- ~.-I o phate, pH 7.2 was added to z< a column (2.% x 127 cm) -< npreviously equilibrated O with the same buffer con4 0 0 (.d taining 7 mM mercapto3_ ethanol and 0.25 M sucrose. Elution was at 26 ml/hr. == A TPN- and O O DPN-linked activities] o 8£] - 160 24O 0.... 0 protein at 280 nm. FRACTION (:3 m l ) DEAE-cel!ulose chromatography: Both TPN- and DPN-linked enzym~ ~cTi~i~i~s were st~l~ ~l~t~d in a single p e a k (Fig. 2). Second Sephadex filtration: The protein and TPN- and D P N - i ~ m ~ e ~ a c ~ i ~ i T i ~ s were once more eluted at the major peak which had a preceding shoulder of low enzyme activity (Fig. 3). Addition of 0.i M or 0.5 M KCI to the buffer did not prevent the appearance of the small preceding shoulder and the disc

704

ST ER O I D S

19:5

gel electrophoresis pattern for 17~-0H-Clg-steroid dehydrogenase activity was similar to that of the main peak.

]

Figure 2: DEAE cellulose chromato2.4 x 3 0 cm / _~r_aphy_ o_f j2ost- Sepha~ / r------ "-i 1 ]1> dex G-_IS_0 fraction. 8 The major activity °'1.4 .2 200~ peak (Fig. i) was concentrated by treatoo2. ~ 1 z Pot.phos. ~ \ TPNJ~J ment with solid sucrose pH7.O I / /~/ and (NH4)2S04. The preo cipitate was dissolved in 0.002 M potassium phosphate, pH 7.2 containing 7 mM mercaptoethanol and 0.25 M 40 80 120 160 sucrose and dialysed FRACTION 5 m[ against the same solution. Ten ml (86 mg protein) was added to the column and eluted at a flow rate of 36 ml/hr. 0 0, protein at 280 ram; X X, phosphate (20); ~ _ _ ~, TPN a n d : DPN- linked activity.

POST-SEPHADEXGI50 DEAE-CELLULOSE

.6

.

.8

E_

°

.

.

.

POST-DEAE-CELLULOSE on SEPHADEX G200

A

300-

~TPN-

Z00-

~4

L

100-

280mp.

8-0 160 FRACTION ( 3 rnl)

240

Figure 3: Second Sephadex filtrat~o~ of post-DEAE-~e~l~lose fraction. T~e--f~a~tions containing the enzyme activities were pooled and concentrated. The concentrated solution, 8.7 ml (17.8 mg protein) was put on a column 2.4 x 127 cm which had been equilibrated with 0.02 M potassium phosphate pH 7.2 containing 7 mM mercaptoethanol and 0.25 M sucrose.

Enzyme assay: The final volume of the reaction (5) was 1.5 ml and contained 0.09 mmole of sodium pyrophosphate, pH 9.6; 2.15 ~moles of DPN or 0.625 ~mole of TPN; 0.14 pmole of testosterone in 0.04 ml methanol (7) and 0.02 to 0.i ml of the enzyme solution. The blank contained all of the ingredients except testosterone. The enzyme activity was measured from the linear inorease in absorption at 340 nm in a Cary Model-!4 spectrophotom e t e r at 37 ° . One unit of enzyme activity was defined as that amount w h i c h produced one nmole of DPNH or TPNH per minute. Specific activity was defined as unit of activity per mg of protein. The conversion of TPNH or DPNH absorbance at 340 nm to concentration was according to Kornberg (9). Protein was determined by the method of Lowry et al. (i0); the albumin standard and blank contained the same medium as the protein solution.

May 1972

ST ER O ID S

7 05

Concentration of dilute en.z~ne solutions: Dilute enzyme solut{6ns were routlnely d6ncentra{ed at ~o. If the volumes were large, the solutions were placed in a cellophane tube and embedded in finely ground solid sucrose. After reduction to one-third of the original volume, the sample was dialyzed against saturated ammonium sulfate solution which was maintained at pH 7.0 by the addition of ammonium hydroxide. The precipitated p r o t e i n was centrifuged at 30,000 rpm for I0 to 30 min in the No. 30 rotor of the Spinco ultracentrifuge at a chamber temperature of -5 ° . The precipitate was suspended in a minimum volume of appropriate buffer and dialyzed against the same buffer. Small volumes of dilute solutions were concentrated by u l t r a f i l t r a t i o n with either a sintered glass immersion filter (ii) or a collodion bag (Bulletin 41-5M, 1962, Schleicher and Schuell Company, Keene, New Hampshire). Disc gel electrophoresis: The gels were prepared according to Davis (2) with m i n o r modifications. The acrylamide was 6.75~ in the lower gel and the sample gel was omitted. Electrophoresis was at 4 °. The enzyme solution contained 0.25 M sucrose and was allowed to settle on the top of the upper gel u n d e r n e a t h the buffer. The gels were sliced by a Canalco gel slicer (#i803, Longitudinal). The p r o t e i n was stained with i~ Buffalo Black in 74 acetic acid and destained by i0~ acetic acid. The nitro blue tetrazolium method, with some modification, was used to stain the 17~-OHC~e-steroid dehydrogenase activity in the gel. In a total volume of Ii ml the mixture contained 7 ~moles testosterone in 0.4 ml methanol; 0.23 mmole of sodium pyrophosphate, pH 9.6; 8.5 mmoles of TPN or 30 bmoles of DPN; 0.06 mg of phenazine methosulfate and 2.6 mg of p-nitro blue tetrazolium. The b l a n k contained all the components except testosterone. The staining was carried out in the dark at 25 ° for 30 minutes for the TPN and 60 minutes for the DPN-linked enzyme activity. U!tracemtrifuge studies: Analyses by u i t r a c e n t r i f u g a t i o n were performed in a Beckman2Spinco Model E analytical ultracentrifuge equipped with a phase plate for schlieren optics. Sedimentation rates were calculated to standard conditions (S2o,W) according to Schachman (13). The partial specific volume of the protein was assumed to be 0.73 ml per gram. The concentration of the sedimenting protein was estimated from the area under the boundaries with appropriate correction for radial dilution. The method of Ehrenberg was used to determine the diffusion coefficient (14). The separate chambers of a capillary-type double sector and a synthetic b o u n d a r y cell were loaded separately with 0.14 ml of enzyme solution containing three to ten mg p r o t e i n per ml and 0.4 ml of buffer. A sharp boundary was formed during acceleration. The speed was maintained at 3,617 rpm. At appropriate intervals for three hours, the boundary spreading was photographed with

706

ST ER O I D S

19:5

the phase plate at 60 °. The photographic uated with a Nikon microcomparator.

plates were eval-

S e p h a d e x gel f i l t r a t i o n ( i S - 1 7 ) : The molecular weights of the reference proteins were assumed to be: bovine serum albumin, 67,000; yeast alcohol dehydrogenase, 150,000; cytochrome c, 13,000; RNase, 13,700; and trypsin, 23,800 (16, 17). The void volume, Vo, was determined with blue dextran 2,000. RESULTS Enzyme purification: ' The above p u r i f i c a t i o n procedures gave an increase of 50 fold in the TPN-iinked activity and of 30 fold in the DPN-linked of the total recovered activity.

specific

activity (Table I)

Furthermore,

the increase

in specific activity was 230 fold if only the fractions at the p e a k of the elution curve of the second Sephadex filtration were considered DPN-linked

(see Fig.

enzyme activities

in every step (see also Figs.

i0).

consistently

The TPN- and remained together

1-3.).

Homogeneity of the p a r t i a l l y purified enzYme preparation: The second post-Sephadex

fraction showed only one symmetri-

cal protein peak when it was refiltered E

(Fig.

4).

The TPN-

Figure % : Refiltration of the second post0 co Se_phadex fraction on a c~ Sephadex G-200 column. -0he ml (~.~ mg of protein) -0.4 40of the second postt.lJ (.~ z Sephadex fraction which Zi had been equilibrated with
O

REFILTRATION OF 2 ND POST-SEPHADEX FRACTION ON SEPHADEX G2OO

77.3 56.5

Sephadex G-200

178

i, 450

364.0

2~8.0

150. i

30. i

18.5

20,550

19,140

26,650

43,620

Not Determined

39,330

mg

2,120

Total unit s/rag units

....

TPN-

Protein

DEAE- cellulose

Sephadex G-100

Ammonium Sulfate 40-80~ Satn.

St reptomycin sulfate

Cytosol

Fraction

43.2

31.7

18.5

4.24

2.76

DPN. . . . . unit s/rag

2,440

2,450

3,300

5,400

5,850

Totai units

8

8

8

7

7

TPN DPN Ratio

17~-0H C-19-Steroid Dehydrogenase Activity

Purification of 17~-0H-C~9-Steroid Dehydrogenase activity of guinea pig kidney cytosol. The procedures were as described in the Text. The TPN- and DPN-enzyme activities in the i~omogenate were 7.3 and 1.4 units/mg protein.

TABLE I

O

O

708

ST ER O I D S

19:5

and DPN-linked enzyme activities correlated with the protein peak, but the specific activities increased to the height of the peak and then gradually decreased indicating the presence of non-enzyme protein.

Ultracentrifugation

also showed a symmetrical peak (Fig.

5).

The omission of

Figure 5: Sedimentation of I ~ - ~ P ~

U~7 ~t~rZ~ ~e~rZgenase. The enzyme Zo~u~ion, 15.5 mg protein/ml, was dialyzed against sodium phosphate, pH 7- 5, ionic strength 0.2. The schlieren patterns were photographed at S-minute intervals after a speed of 59,y80 rpm was reached.

i

~-mercaptoethanol and sucrose or glycerol during the purification procedures did not alter the sedimentation pattern. The protein of the second post-Sephadex fraction, however, was not homogenous on disc gel eiectrophoresis.

Protein

devoid of enzyme activity was shown in the fastest migrating band with Rf of 0.75 and two very faintly stained bands appeared as two small shoulders migrating in front of band 5 (Fig. 6).

In addition,

the enzyme activity was separated

into multiple bands which corresponded with the main protein bands.

A very weakly stained protein band which migrated

behind b and i also was stained very weakly for enzyme activity.

Although the bands were distinct,

some staining

was present between the bands apparently due to diffusion of

M a y 1972

S T E R O I D S

the dye prior to fixation.

The Rf values of protein bands

one through five were: 0.3, 0.36, respectively;

709

0.41, 0.~7,

and 0.56,

and the corresponding TPN- and DPN-linked

activity bands were: 0.30, 0.35, 0.40, 0.46 and 0.55. 0.6

Figure 6: Disc gel electrophoresis ~f--t~e---second p~s~-~ephad~x--

0.4

:::8 0 Z "-I

0.2

E

Tot

77

electrophoresis of the protein and the TPN-linked and DPN-linked enzyme activities were prepared at the same time. Each gel received 90 ~g of protein (33 units of TPN-linked and 4 units of DPN-linked activities). The electrophoresis and staining were carried out as described in the text. Testosterone was omitted in the staining reaction of the controls. The stained gels were scanned at 590 nm in a Gi!ford spectrophotom e t e r attached with a linear transport scanner. A= Protein~ B= TPN-linked a c t i v i t y ~ linked activity .... and control, .... .

0

tO

I

~ 04

w02 Z

o

0

m

°21 I

0

.

2

I

i

4 6 GEL LENTH cm

I

8

Determinati0n~ of molecular weight: and diffusion coefficients:

(a) Sedimentation

The second p o s t - S e p h a d e x

fraction sedimented as a single b o u n d a r y

(Fig.

5).

The

S~o,W was 3.0S and The D$o,W was 7.89 x iO-Tcm2sec -! (Fig. 7).

The insertion of the above constants

into the

Svedberg equation gave a m o l e c u l a r weight of 35,100.

If the

D2o,W and S2o,W values of an individual protein concentration were used,

the molecular weight was 33,000.

dex filtration.

At least three determinations

(b) Sepha-

were made

ST ER O I D S

710

19:5

2.6

i 4

B

J2

l

%JT#-OH SDH

Ve_t

~(Mw=

31,600)

B

'o s.o

%

~0 70 x

o

60

ADH .4 ,

,

,

~

PROTEIN CONCENTRATION

............. ,~,~.................. mg/ml |

i

42 Fig. 7

[

log

4.6 Mw

Eig.

8

*

!

5.0

Figure 7A: Concentration dependence of the sedimentation coefficienT.-- ~ h ~ a ~ a l y s i s was carried o u t - - i ~ o ~ i u m phosphate buffer, pH 7.5, ionic strength 0.2. B. Concentration dependence of the diffusion coefficient. The--s-61~t~o~ co~tai-h-ed 3.38 To-l~.~2 mg protein per ml i~ the same buffer as in A. The lines in A and B were obtained by the least squares method. Figure 8: Molecular weight determination by the Sephadex method.-- ~h~ ~e~h~d-$S G - 2 ~ colum~ ~l--x--l~S--cm) was eq~i~ibra-~e~ with 0.02 M potassium phosphate buffer containing 0.5 M KCi, pH 7.0. The protein (I to 2 mg) and dextran blue 2000 were dissolved in I mi of the buffer. The flow rate was about 5 ml per hour. for each protein (Fig.

8).

of the 17~-OH-Czg-steroid gel electrophoresis: with the enzyme bands,

The indicated molecular weight dehydrogenase

was 31,600.

(c) Disc

Since the protein bands coincided only enzyme activities were determined.

The plot of the logarithm of their relative mobilities versus gel concentration gave parallel lines.

The average value of

the slopes gave a molecular weight of 31,200

(Fig.9).

May 1972

ST ER O ID S

16

BSAoe ~ f ii ADH

n 0.J 03 I

BS/~e ~ ~

rot

LDH

liver

8

CytcD ;YtC~I < ~.-ilT~'OHSDH(Mw=51,200)

711

Figure 9: M o l e c u l a r weight determina~i~n--b~-~ i~ c--g~ i--e~ e~ t~o~h~ r~ s i s. The procedure was as described in the text. M, D, and T-subscripts indicate mono-, di- and trimer. The curve was obtained by the least squares method.

RNose

~"

t~ Mw

xld i6

pH optimum:

:~0

The TPN-iinked activity had a pH optimum

at approximately 9.9 (Fig.

i0).

The curve was flat at the

v i c i n i t y of the optimal pH, between 9.6 and 10.2.

The DPN-

linked enzyme activity also had a broad peak but at a slightly lower pH, between 9.4 and 9.7.

L24

8

9

pH

I0

I

Figure i0: The effect of pH on enzyme activity. ~h~ reacTi~n--m~x~u~e-was as described in the text. The enzyme protein for each determination was 14 DgThe pH of the reaction mixture was measured at the end of the assay at 25 °. The h i g h specific activity of this enzyme preparation was due to selection of only the fractions at the top of the peak of elution in the second Sephadex filtration.

II

Absorption s p e c t r u m of the partially purifie d enzyme! The second post-Sephadex p e a k at 280 nm.

fraction gave a discrete absorption

Addition of sodium hydros~!fite to 0.042 M

did not alter the spectrum indicating that the enzyme protein

712

19:5

ST ER O I D S

contained

no endogeneous!y

Stability: cytosol

were

slightly total

affected

and several more,

Both cofactor

stable

activity

at 4 ° .

remained

incidences

freshly prepared

activity

the addition

of 7 mM

or 20% glycerol. the activity

against

water

sample

against

restore

The enzyme

of the

the DEAE-

was prevented

by

and 0.25 M sucrose

extent. lost after

Redialysis

the enzyme

and almost

did not

was completely

activity was sensitive

for I0 minutes,

pH 7.2,

when the cytosol was

activity

on heating

dialysis

of the urea treated

buffer,

Furthermore,

remained

to heat.

the

a total loss

lost.

Only 26%

solution

at 45 °

in activity was

after 60 ° for i0 minutes.

Sulfhydryl enzyme

Further-

was less

following

was totally

of the activity

observed

at -20 °

alone up to 0.5 M stabilized

0.02 M phosphate

made to 4 M urea,

the enzyme

in activity

or 8 M urea.

the activity.

of storage

with that

~-mercaptoethanol

activity

in the

only

and thawing.

coincided

only to a slight

The enzyme

and thawing

specially

Sucrose

activities

six months

However,

The loss

(21).

more than 75% of the

of freezing

enzyme.

step.

Freezing

after

after purification,

cellulose

cofactor

specific

the stability;

the specific

stable

bound

group

in 10 -4 PCMB,

iodoacetic

acid

requirement:

Preincubation

i0 -~ M N-ethylmaleimide

(Table

Ii)

completely

of the

or IO-2M

inhibited

the enzyme

May 1972

ST ER O ID S

activity.

Addition

inhibited

the enzyme

was preincubated the enzyme ever,

of 0.01 M dithiothreitol activity.

was preserved.

could not be restored

the enzyme had been

slightly

When the dithiothreitol

with the enzyme p r e p a r a t i o n

activity

.9

713

and PC~B,

The activity,

by the dithiothreitol

inactivated

howafter

by PCMB for i0 minutes.

Figure Ii: Det e r m i n a t i o n of the mole SH Y h ~ o ~ groups. T~e-.8 react i~n--mixture -and the procedure were .7 as described in Enz + Guo-HCI Methods. 0.43 mg i i i Z~412 .3 o 40 80 120~ protein of the second MIN post-Sephadex fraction .2 was used in each reEnz action. The enzyme .I was treated at a final concentration I I I of 4 M guanidine 4-0 80 120 hydrochloride for i MIN hour at 25 ° before addition of the DTNB reagent. 0.i pmole of glutathione per reaction mixture was used as a reference standard. The m o l a r extinction coefficient of glutathione, 12,900 , was used in the calculation.

~

GSH

,

The presence

\

,

faster reaction

of DTNB with the enzyme

of 4 M guanidine

the -SH groups (Fig.

nz + Gua.HCI

ii).

in the molecule

The number

of guanidine

hydrochloride

protein.

Therefore,

of -SH,

which was well

had become

of thioi

hydrochloride

suggested

groups

in the that

fully exposed

in the presence

was about 2.8 moles per mole

it appeared embedded

that there was one mole in the enzyme molecule.

of

714

ST ER O I D S TABLE

19:5

Ii

I n h i b i t i o n of 1 7 ~ - O H - C l g - s t e r o i d d e h y d r o g e n a s e a c t i v i t y b y s u l f h y d r y l b l o c k i n g agents. The i n c u b a t i o n m i x t u r e was as d e s c r i b e d in the text. The e n z y m e s o l u t i o n was t h o r o u g h l y d i a l y z e d a g a i n s t 0 . i M p o t a s s i u m p h o s p h a t e , p H 7.2, a n d 0 . 2 5 M s u c r o s e to r e m o v e the ~ - m e r c a p t o e t h a n o l . The e n z y m e solution, 0 . 0 2 m l c o n t a i n i n g 13.6 Bg p r o t e i n , was p r e i n c u b a t e d for i0 m i n u t e s at 37 ° w i t h b u f f e r a n d the i n h i b i t o r or d i t h i o t h r e i t o l . The r e a c t i o n was i n i t i a t e d b y the a d d i t i o n of the s u b s t r a t e and cofactor.

Additive

C o n c e n t rat ion

Activity TPN-

,DPN-

100

i00

98 9~ 13

98 54 2

10 -4

0

0

N- et h y l m a l e imide

i x 10 -5 i x 10 -4 i x i0 -s

80 33 0

64 25 0

lodoacetic

i x 10 -4 i x i0 -s i x 10 -2

90 88 0

75 75 0

i x

95 81

95 ~ 81

i x 10 -8 i x i0 - 5

92

98

2 x i0 -s ix 10 --5

93

96

i x 10 -4 i x I0 -s

0

0

ixl0 -4 2x!0-s

0

0

M None i x 10 -7 i x 10 -6 i x i0 -s

PCMB

i x

acid

Dithiothreitol

! 0 -3

2 x i0 -s Dithiothreitol~ PCMB*

PCMB** Dithiothreitol**

* D i t h i o t h r e i t o i and P C M B w e r e p r e i n c u b a t e d s i m u l t a n e o u s l y w i t h the enzyme. * * D i t h i o t h r e i t o l was a d d e d i0 m i n u t e s a f t e r the P C M B p r e i n c u b a t i o n w i t h the enzyme.

May 1972

ST ER O ID S

Substrate:

Testosterone

among the suIbstrates tested

71 5

gave the highest activity

(Table iV).

The specific

activity was relatively high with 17B-hydroxy, 5~-anidrostan3-one as substrate in spite of this steroid's lower concentration in the assay mixture.

Only a trace of enzyme

activity was observed when 17~-estradiol

was the substrate.

The TPN-linked activity was always higher than that of the DPN-linked activity. TABLE III Substrate specificity of 17B-OH-Clg-steroid dehydrogenase. ~h~ ~ e ~ c T i ~ n - m ~ x T u ~ @ Wad t~@ sam~ ~s d~s~r~b~d--i~ -~ h ~ T e x t except that the steroids were dissolved in water instead of in methanol and 17B-estradiol was in O.O03N NaOH. The reaction mixture contained 0.2 ~mole of steroid, except 17B-hydroxy, 5~-androstan-3-one was 0.037 ~mole because of its low solubility. The final pH of the reaction mixture was 9.6 except for estradiol which was 9.85. Sub st rat e

Act ivity TPNDPNunit s/rag,p rot e in

Testosterone

140

16

17~-Hydroxy~-Androstan-3-one

74

9

17B-Hydroxy-5B-Androstan-3-one

21

17~-Estradiol

4

0

DISCUSSION The 17~-OH-C~9-steroid

dehydrogenase

activity of the

guinea pig kidney appears to be contained in a single protein which preferentially utilizes TPN than DPN as a coenzyme.

716

ST E R O I D S

19:5

The various purification, steps did not separate the activities of the two coenzymes and the ratio of their relative effectiveness remained constant. The enzyme activity was purified 230-foid but nonenzymic proteins with molecular weights very similar to that of the enzyme were present.

The impurities were

revealed by disc electrophoresis and comparison of the intensity of the stained bands suggested that they comprised only a very small portion of the purified fraction.

Further-

more, uitracentrifugation gave a single uniform peak and refi!tration of the purified preparation on Sephadex G-200 yielded uniform protein and enzyme activity peaks but the specific activity of the different fractions increased to the peak of the curves and then decreased. Disc electrophoresis revealed 5 distinct bands with enzyme activity.

A more detailed consideration of the

multiplicity of the enzyme is presented in the following report. There are a few reports (5, 6, 22-25)

on the partial

purification of testosterone specific dehydrogenase from animal tissues.

Dah~ and Breuer concentrated the rat

adrenal 17~-OH-C19-steroid dehydrogenase 70-fold (22) and that of rat kidney 12-foid (23).

Endahl and Kochakian (24)

concentrated the TPN-linked guinea pig liver enzyme activity five-fold in three steps with a 65~ recovery and Villee and Spencer (6) increased the purification to 16-fold with a 32~ recovery in 14 steps.

Joshi et al (25) obtained a

May 1972

ST ER O I D S

200-230

717

fold purification which is comparable to our purest

guinea pig kidney preparation. Unlike the 17~-OH-Cls-steroid

dehydrogenase

of the

guinea pig liver in which the TPN- and DPN-linked activities were readily separated into the cytosol and microsoma!

fraction respectively

DPN-linked activities

(3-6), both the TPN and

of the guinea pig kidney were in

the cytosol (3) and those of the dog prostate microsomal

fraction (7).

We have failed to separate the

two cofactor specific activities

of the kidney.

cofactor linked activities of the rat adrenal rat kidney (22) dehydrogenases Sweat e t a ! .

The two

(23) and

also were not separated.

(31) reported that the maximum molecular

weight of 17~-0H-Cls-steroid liver was 43,00G.

in the

dehydrogenase

from steer

The guinea pig kidney 17~-0H-C~9-

steroid dehydrogenase had a molecular weight of 35,000 calculated

from the Svedberg equation; 31,600 by Sephadex

filtration and 31,200 by electrophoresis. (unpublished)

A similar value,

31,400 was found for the TPN specific enzyme

in the guinea pig and mouse liver cytosols

(3).

The guinea pig kidney 17~-OH-C~s-steroid

dehydrogenase

was inhibited by -SH blocking agents as was the enzyme of other tissues (22~24).

Both TPN- and DPN-linked

activities were inhibited to the same extent. ACKNOWLEDGMENTS The authors are indebted to Dr. R. E. Schrohenloher and Mrs. K. Actin for invaluable discussion and desig~ and execution of the analytical ultracentrifuge experiments.

718

ST ER O I D S

19:5

This investigation was supported by National Institute of Health General Research Support Grants FR-05300 and FR-05349 and Grant AM-If060 from the National Institute of Arthritis and Metabolic Diseases, U.S.P.H.S. An abstract of part of this work has appeared, Fed. Proc., 27, 692, (1968). REFERENCES i.

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