Some characteristics of 17β-estradiol dehydrogenase from bovine placenta

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SOME CHARACTERISTICSOF 17S-ESTRADIOLDEHYDROGENASE FROM BOVINE PLACENTA Kamal G. Bitar, Michael Cochran and James C. Warren Departments of Obstetrics and Gynecology, Anatomy and Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110 (U.S.A.) Received S-24-79 ABSTRACT The activity of 17S-estradioldehydrogenase (E.C. 1.1.1.62) was measured, and its distribution in the subcellular fractions of bovine placenta was compared. Assay of activity was based on the formation of radioactive estrone from 176[4-'"Cl-estradiol. Either NAD+ or NADP+ can serve as cofactor for the enzyme. The nuclear and microsomal fractions of the placental homogenate exhibited the highest specific enzymatic activities before and after treatment with Triton X-100. Electron micrographs of these two fractions prior to treatment with Triton X-100 showed satisfactory purity. 17$-estradioldehydrogenase from bovine placenta exhibits a pH optimum of about 9.5-10.5, and is activated by 5 x 10q6M ZnC12; comparable concentrationsof CaC12 and MgCl2 inactivate the enzyme. The apparent Michaelis constants, Km, for 17$-estradiol and NAD+ are 1.4 x 10s6M and 5.5 x 10a5M respectively. No 17cr-estradiol dehydrogenase activity was demonstrable Ghen using 17a-estradiol as substrate. INTRODUCTION Reports appearing in the literature over the past decade have characterized the NAD+-dependent 17S-estradioldehydrogenase from several species. Although the general belief is that the enzyme is associated with the cytosol, several membrane-bound forms of 17Bestradiol dehydrogenase have been found in liver, kidney, skin, placental, testicular and endometrial tissue (l-6). In order to compare structure of proteins which effect the enzyme catalyzed dehydrogenation of the epimeric 17-hydrogens in the estradiol molecule, we have begun a systematic search for a placental tissue that exhibits both 17S- and 17o-estradiol dehydrogenase activities. Bovine placenta was chosen as a starting point due to its abundance and availability. Unfortunately, only 17B-estradiol dehydrogenase activity was

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demonstrable. Further it differs from that of human placenta in that it is associated predominantly with the particulate fractions of the cell rather than the cytosol. The partial characterizationof 17$estradiol dehydrogenase activity from bovine placenta is reported in this communication for the first time. MATERIALS AND METHODS Reagents- Organic, inorganic chemicals and steroids were of analytical grade purchased from Sigma Chemical Co. or Fisher Scientific co. - [4-'"C‘jestradiol (52 mCi/mmolj and 17a[2,4(n)-3H]estradiol (58 Ci/mmol) were purchased from Amersham-Searle. Collection of bovine placenta- Fresh bovine placentas were obtained shortly after kili%ngals slaughtered in late pregnancy at a slaughterhouse in St. Louis. The villous tissue was dissected free of connective tissue and fatty material. Preparation of placental homogenate- Fifty grams of fresh placental tissue Icontainingxoth foetal and maternal contributions)was homogenized-in 200 ml-of extraction buffer (10 mM potassium phosphate buffer, 10 mM 2-mercaptoethanoland 0.25 M sucrose at a final pH of 7.2) for 30 set at full speed in a l-liter War&g blendor container. The homogenate obtained was stirred for 10 min at 4OC and then filtered through four layers of cheese cloth. Subcellular localization of 178-estradiol dehydrogenase- Nuclei, mitochondria, microsomes and thFl05,OOO x g supernatant were prepared according to published procedures (2,7). The nuclear fraction was further purified by suspending in about 10 ml of 0.25 E sucrose and layering over a discontineous sucrose gradient of 6 ml of 2.0 g and 15 ml of 2.4 g sucrose, both containing 0.5 mM CaC12. Centrifugation was performed for 30 min at 60,000 x g. Both the mitochondrial and microsomal fractions were washed at least one time with the extraction buffer prior to use. Assay of 17$-estradiol dehydrogenase activity--An assay based on the use ofxoztsnbstrate was routinelv followed. Incubation mixtures contained 0.15 umole 17B-estradiol (50 ,lj, [4-'4C]-estradiol(5 1.11; 13000 counts/min), one nmole of NAD+(l.O ml) and 0.2 M Na-pyrophosphate buffer (2.0 ml), pH 9.5. To this was added 2 ml of c&de enzyme (S-15 mg total protein) or partially purified enzyme (ammonium sulfate precipitate) in extraction buffer, resulting in a total volume of 5.1 ml. The contents in 15 ml glass-stoppered tubes were incubated at 37OC for one hour, and then immersed in a beaker containing ice water. Radioactive estrone and estradiol were extracted with two portions of anhydrous ether (each 10 ml). The ether extracts were evaporated under a stream of nitrogen and residues dissolved in 0.2 ml of chloroform. Ten microliters were spotted on a t.1.c. plate coated with silica gel (from Eastman Kodak Inc.) and the plate developed with chloroform. Spots corresponding to estradiol and estrone we visualized by spraying the plates with a dilute solution of Folin-Ciocalteu reagent (8) followed by exposure to ammonia vapors. The amount of radioactivity in each spot was quantitated in a model 3320 Packard Tricarb liquid scintillation spectrometer. No correction for quench was necessary.

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Activity is defined as pmol estrone formed per hour at 37OC. Specific activity is activity per mg protein. Electronmicroscopy-For morphometric analyses, wedge-shaped sectors of each of the subcellular fractions (i,e. nuclear, mitochondrial and microsomal) were fixed in 2% glutaraldehyde-0.1M phosphate buffer, pH 7.2, overnight, followed by fixation with 2% osm&m tetroxide. The samples were dehydrated through an alcohol series and then embedded in Spurr's resin (9). Thin sections were stained with uranyl acetate and leid citrate prior to examination with a Philips EM/300. Partial characterizationof 17&estradiol dehydrogenase activityThe nuclear fraction of the bovze placental homogenate was sonicated for 20 min with a 300/w sonicator. The suspension was then centrifuged at 10,000 x g for 30 min and solid ammonium sulfate was added to the supernatant to achieve 50% saturation. The precipitate collected by centrifugation was dissolved in the minimum of extraction buffer and dialyzed against the same overnight. This material was used as a source of enzyme in most of the experiments described in this communication. RESULTS Because of the very low concentration of the enzyme, conventional spectrophotometricassay of NAD+-dependent dehydrogenasesbased on the measurement of the increase in absorbance at 340 nm was found unsatisfactory for the assay of 17B-estradiol dehydrogenase from bovine placenta. Therefore, an assay system was adapted that utilized [4-1'Cl-estradiolas the substrate. The amount of radioactive estrone formed per hour was taken as measure of 17B-estradiol dehydrogenase activity. When 17a[2,4(n)-3H]estradiol was used as the substrate, no 17a-estradioldehydrogenase activity was evident, either in the whole placental homogenate or in the subcellular fractions isolated. Subcellular localization of 17B-estradioldehydrogenase activity revealed that the enzyme is predominantly particulate, i.e. associated with nuclei, mitochondria and microsomes. The specific activity of the enzyme from the nuclear and microsomal fraction was roughly ten times that of the 105,000 x g supernatant (see Table I). Recently, similar results (data not shown) have been obtained for sheep and horse term placentas, i.e. the specific activity of 17$-estradioldehydrogenase was higher in particulate

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fractions rather than cytosol. Table 1:

Fractiona

Nuclei Mitochondria Microsomes Supernatant

Subcellular localization of 178-estradiol dehydrogenase from bovine placenta

Volume

21 24 34 184

Total Activityb

147 40 204 155

Total Protein(mg)

126 110 204 1,795

Specific Activity 1.17 0.36 1.00 0.09 --

a. b.

Isolation of the subcellular fractions was according to published procedures (2,7). Activity is defined as pmol estrone formed per hour at 37'C. Examination of electron micrographs of isolated nuclei, mitochondria

and microsomes showed that the purity of each of these fractions was satisfactory. Since the nuclear fraction displayed the highest specific activity, it was used as a source to obtain a partially purified enzyme preparation (see Materials and Methods section). The optimum pH for 178-estradiol dehydrogenase activity from bovine placental nuclei was 9.5-10.5 (Fig. 1). The apparent Michaelis constants, Km, determined from LineweaverBurk plots, and based on initial linear velocities, for 178-estradioland NAD+ were 1.4 x 10m6fiand 5.5 x low51 respectively (c.f. Fig. 2A and ZB) Zinc at a concentration of 5 x 10W6M - was found to enhance the enzymatic activity by about 38% (Fig. 3). Higher concentrations lead only to about 20% activation. In contrast, MgCl2 and CaC12 at concentrations of 5 x 10q6M - and 2 x 10e5M - lead to about 25% and 30% inactivation, respectively.

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Fig. 1. pH activity curve of bovine placental 17&estradiol dehydrogenase. The buffers used were: 0.1 y sodium phosphate, pH 7.0; 0.1 MTris-HCl, pH 7.8; and 0.1 g sodium pyrophosphate, pH 8.5-10.5.

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[Estradiol]m’~10e4 M

@AD+]-‘40-’

Fig.

2.

M

Dot.&le reciprocal plot of velocity as a function of 17&estradioi concentration at a fixed concentration of NAD+ of 2 x lo-"M, At each experimental point, 1.4 x 1O-1o moles [&"Clestradial (about 13,000 cpm) was used. The molar concentration of 17$-estradiolwas at least 100 times that of radioactive estradiof. (B) DouI$lereciprocal plot of velocity as a function of IiAD concentration at a fixed concentration of 17& estradiol.of 3 x l.O-%, Experiments described in (A) and (B) were done at ieast two times.

(A)

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I 20

160-

60-

, 5

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Effect of ZnC12, A, CaC12, o, the activity of, 17$-estradiol bovine placenta.

and MgClZ,O, dehydrogenase

on from

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DISCUSSION

The results that

presented

176-estradiol

dehydrogenase

enzyme in contrast bound forms recent

of

years

metrial

to that

in the

tissue

somal

liver,

of

the

activity.

soluble

drogenase,

or a diffusion

The fact in the nuclear tempted

to put

is

the

cently

not

described

receptors

of

the

(sites)

might

forward

dependent associated extensively

these

receptors

appear

estrogen

injection

of

of

have

in uterine 17S-estradiol

17S-estradiol

NAD+-linked

differs

of

and some forms

as a function

enzyme its 11,

12).

re-

These nuclear

elements

subcellular

between

(1,

receptors

to the nucleus.

shown that cells,

(16)

One

although

(13-15).

structural

components

activity

a) the

acids

estrogen

dehy-

or microsomes.

specific

nucleic

the

dehydrogen-

significance.

literature with

and micro-

exhibited

gene expression,

the nuclear

in the

matrix

fraction

a second

of

speci-

was shown to be satis-

two postulates:

to be translocated

and Muldoon(16)

the nuclear

physiological

on the presence with

the highest

the highest

following

may be associated

such as the nuclear

radiolabeled

the

in

and endo-

enzyme from the nuclei

be of

membrane

identified

stage,

fractions

enzyme exhibited

cell,

Cidlowski

the

Several

(l-6).

with

represent

in the regulation

enzyme is

species

The supernatant

might

a particulate

testicular

pregnancy

these

demonstrate is

(10).

The NAD+-dependent

fraction

be involved

activity

lowing

late

of

fraction

that

placenta

placental,

animal

clearly

have been

enzyme was associated

ase in the

b)

of

The purity

specific

might

kidney,

by electronmicroscopy.

lowest

is

dehydrogenase

placenta

fractions.

factory

from bovine

from a number of

activity

communication

from human placenta

17Pestradiol

In the bovine fic

in this

the of

For example,

distribution of

time,

ovariectomized

of foland

estrogen primed ovariectomized animals. In the castrate rats, binding of estrogen traces the following route: cytoplasmjnucleus

4

microsomes--j nucleus; in the primed castrate animals, the path of movement is:

cytoplasmjn~~leus~(microsomes)~cytopIasm.

Treatment of the enzyme from particulate fractions with 0.5% Triton X-100 resulted in about 20% decrease of specific activity of the mitochondrial fraction, and about 40-50% decrease of specific activity of the nuclear and microsomal fractions, respectively. Aoshima and Kochakian (1) have demonstrated the competitive inhibition by excess Triton X-100 of estradiol dehydrogenase from pig liver. This could mean that detergents of this type can bind at the steroid binding region of the enzyme. Whatever the case, the possible attachment of 178-estradiol dehydrogenase with membranes or phospholipids, although no empirical data are available from the present study, might provide the spatial arrangement essential for integration of the steroid dehydrogenase activity with its specific role in the metabolism of the cell. Similar to most other 17@-estradiol dehydrogenases,bovine placental 17B-estradiol dehydrogenase exhibits a relatively high p1-l optimum. The apparent Km with respect to 17&estradiol and NAD+ are similar to those from human placenta. The activation of the placental enzyme by zinc by about 40% might imply a role of this metal at the active site of the enzyme. Such a role has been established for some other dehydrogenases such as horse liver alcohol dehydsogenase (17) whereby a zinc atom is coordinated to 2 cysteinyl residues, one histidyl residue and a water molecule at the active site of the enzyme.

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Finally, it is worthy to comment here that our inability to detect 17a-estradiol dehydrogenase activity in the bovine placenta can be due to the absence of the enzyme, or that it exists in extremely trace amounts. ACKNOWLEDGEMENTS This work was supported by NIH grant AM 15708-07 and NIH postdoctoral fellowship 50055 (M.C.). The authors wish to thank Mrs. June Manning for typing the manuscript. REFERENCES 1. Aoshima, Y. and Kochakian, C.D., ENDOCRINOLOGY,72, 106 (1963). 2. Davis, B.P., Rampini, E. and Hsia, S.L., J. BIOL. CHEM., 247, 1407 (1972). 3. Pollow, K., Runge, W. and Pollow, B., Z. NATURFORSCH.,30, 4 (1975). 4. Inano, H. and Tamaoki, B., EUR. J. BIOCHEM., 5, 13 (1974). 5. Tseng, L. and Gurpide, E., AM. J. OBSTET. GYNECOL., 114, 995 (1972). 6. Tseng, L. and Gurpide, E., ENDOCRINOLOGY,94, 419 (1972). 7. Canick, J.A. and Ryan, K.J., STEROIDS, 32, 499 (1978). 8. Folin, 0. and Ciocalteu, V., J. BIOL. CHEM., 2, 627 (1927). 9. Spurr, A.R., J. ULTRASTRUCTURE RES., 2&, 31 (1969). 10. Warren, J.C., Mueller, J.R. and Chin, C.C., AM. J. OBSTET. GYNECOL., 129, 788 (1977). 11. Lee, D.C., McKnight, G.S. and Palmiter, R.D., J. BIOL. CHEM., 253 3493 (1978). 12. Harris, J. and Gorski, J., ENDOCRINOLOGY,103, 240 (1978). 13. Ruh, T.S. and Bandendistel, L.J., ENDOCRINOLOGY,102, 1838 (1978). 14. Baulieu, E.E., Atger, M., Best-Belpomme,M., Corvol, R., Courvalin, and de J.C., Mester, J., Milgrom, E., Robel, P., Rochefort, I-1. Catalogne, D., STEROID HORMONE RECEPTORS. VITAMINS AND HORMONES, 33, 649 (1975). 15. Chan, I. and O'Malley, B.W., NEW ENGL. J. MED., 294, 1322 (1976). 16. Cidlowski, J.A. and Muldoon, T.G., BIOLOGY OF REPRODUCTION,5, 234 (1978). 17. Argos, P., Garavito, R.M., Eventoff, W., Rossmann, M.G. and Branden, C.I., J. MOL. BIOL., 126, 141 (1978).