Molecular and Cellular Endocrinology, 60 (1988) 7-13 Elsevier Scientific Publishers Ireland. Ltd.

MCE 01929

Properties of fatty acyl-coenzyme A : estradiol-17P acyltransferase in bovine placenta microsomes

P. Martyn, D.L. Smith and J.B. Adams School of Biochemistry University of New Souih Wales, Sydney, N.S. W. 2033, Australia

(Received 16 February 1988; accepted 21 June 1988)

Key words: Estradiol-17b-fatty acid ester; Fatty acyl-coenzyme A : estradiol-17P acyltransferase; Lipoidal estrogen

Summary

The properties of the enzyme catalyzing the formation of non-polar derivatives of estradiol-17fi (E,) esterified to long-chain fatty acids have been investigated in microsomal preparations from bovine placenta cotyledons. A rapid enzyme assay has been developed which involves simple solvent partitioning.

The membrane-bound enzyme showed a pH optimum of 5.0 and addition of fatty acyl-coenzymes A (CoAs), such as oleoyl-CoA, palmitoyl-CoA and palmitoleoyl-CoA, increased [ 3H]E,-fatty acyl ester formation from [ 3H]E, by some 7-fold. Linoleoyl-CoA, linolenoyl-CoA and arachidonoyl-CoA were much

less effective as acyl donors. Only 17/%fatty acyl monoesters were synthesized in each instance. Similar results were obtained with microsomes or mitochondria from bovine endometrium. The apparent K, for

E, employing placenta microsomes was 8.0 &- 2.2 (SD) PM. Steroids such as testosterone, dehydroepi-

androsterone and 5-androstene-3fi,17/%diol acted as competitive inhibitors ( Ki values 79, 46 and 39 PM,

respectively). These, and other data to be reported separately, which showed that these steroids were substrates for the enzyme, demonstrate that the latter is not specific for E,. The [3H]E,-fatty acyl ester

fractions biosynthesized from [3H]E2 and bovine placental or endometrial tissue were analyzed by high pressure liquid chromatography (HPLC) and were found to have similar compositions characterized by a high percentage of unsaturated fatty acids.

Introduction

Schatz and Hochberg (1981) have reported the formation of non-polar estradiol-17P (E,) deriva- tives when [3H]E2 was incubated in vitro with

tissue segments from female rats. These metabo- lites, termed lipoidal derivatives, were found to be

esters in which the lipid component was linked to

be 17&position of E,. Although most tissues of the rat possessed the ability to form estradiol

lipoidal derivatives (E,-L) to some extent, much higher activity was found in estrogen target tissues

such as the uterus, and in mammary tumors in-

duced by 7,12_dimethylbenz[ alanthracene (DMBA). It was later demonstrated that the E,-L fraction, formed by incubating E, with bovine endometrium tissue in vitro, contained some ten components in which E, was esterified at the 17-position to long-chain fatty acids. Unsaturated

Address for correspondence: P. Martyn, School of Bio-

chemistry. University of New South Wales, Sydney, N.S.W.

2033, Australia.

fatty acids represented some 85% of the total fatty acids, with arachidonic acid predominant (Mellon- Nussbaum et al., 1982).

0303-7207/88/$03.50 0 1988 Elsevier Scientific Publishers Ireland, Ltd.

8

E,-L is also formed by incubating [‘H]E, with human and rat mammary tumor tissue in vitro (Abul-Hajj, 1982; Larner et al., 1985). However, the fatty acid components of the E,-L fraction,

formed from human mammary tumors in vitro, differed from that of bovine endometrium in that

saturated fatty acid esters were present in much

higher concentration and arachidonic acid ester was a minor component (Larner et al., 1985).

It is possible that these novel estrogen deriva-

tives may serve as storage forms from which E,

can be rapidly generated by esterase activity (Abul-Hajj and Nurieddin, 1983; Larner et al.,

1985; Adams et al., 1986). Despite the potential role of E,-L formation and breakdown in the

regulation of free E, levels in cells, very little data is available on the enzymes governing these reac-

tions, fatty acyl-CoA : E, actyltransferase and fatty acyi-E, esterase. In this communication we de- scribe the properties of the former enzyme present

in microsomal preparations from bovine placenta

cotyledons. In addition, we have analyzed the E,-L fraction formed by incubating [3H]E2 with

this tissue for purposes of comparison with the

specificity towards fatty acyl-CoAs shown by the

microsomal enzyme.

Materials and methods

Materials

Unlabelled steroids and fatty acyl-CoA thioes- ters where obtained from Sigma Chemical Co. (St.

Louis, MO, U.S.A.). The thioesters were stored frozen in aqueous solution, pH 6.5, under N,.

[2,4,6,7-3H]E, (89 Ci/mmol) and [l-‘4C]oleoyl- CoA (50 mCi/mmol) were obtained from the Ra- diochemical Centre (Amersham, U.K.). Fatty acid chlorides were purchased from Nu Check Prep. (Elysian, MN, U.S.A.).

Preparation of estradiol-I 7fi-fatty acid esters This was an adaptation of the method de-

scribed by Mellon-Nussbaum et al. (1982). Briefly, E, (10 mg, 10 FCi) in anhydrous pyridine (100 ~1) was reacted with fatty acyl chloride (50 ~1) at 0 o C and then allowed to stand at room temperature overnight. Three ml of 0.3 M HCl was added, and the products extracted into ether. To the dried residue in benzene (0.1 ml) was added a solution

made by dissolving NaHCO, (50 mg) in water (0.6 ml) followed by the addition of methanol (10 ml) and stirring until clear. This was left overnight at

50” C, dried, water added (3 ml) and extracted with ether. The latter was dried and the residue partitioned between methanol-water (70 : 30, v/v)

and hexane to remove E,. E,-17/&Monoester in the hexane layer was separated from the 3-mono-

ester and E,-diester by column chromatography

on silica gel (Mellon-Nussbaum et al., 1982). Final purification was carried out by HPLC on a 3.9

mmx30 cm C,, p Bondapak column (Waters Associates) with acetonittile as solvent (flow rate 1

ml/mm) and monitoring at 280 nm. The E,-17B- acyl esters had higher R, values on thin-layer

chromatography (TLC) in solvent system A than the 3-esters and could also be distinguished by the

presence of a free phenolic group revealed by spraying with Turnbull’s reagent (equal volumes

of aqueous 1% FeCI, and 1% K,Fe(CN),). Fur- ther validation of the composition of the synthetic

17”-esters was carried out by mass spectrometry.

Mass spectrometry

Spectra were recorded using solid probe sample insertion into the chemical ion source of a Finni-

gan-MAT Quadrupole model 3200 mass spec- trometer. Data acquisition employed a Finnigan-

MAT Incas model 2300 data system. Methane was used as the chemical ion reactant gas (ion source

pressure 0.9 Torr) and samples were heated from room temperature to a nominal 350 * C. Standards

were identified from their [MH]’ ions and base

peak at m/z 255 [MH-RCOOH] ‘,

Fenta~~orobe~zy~ ethers of E,-l ?~-fatty acid esters This method was adapted from that of Netting

and Duffield (1985). The esters (0.1 pmol) were dissolved in dimethylacetamide (50 ~1). This solu- tion and pentafluorobenzyl bromide (Fluka, Buchs, Switzerland) (2 ~1) were simultaneously added to a reactivial containing tetraethyla~onium hy- droxide (4 ~1 of 0.4 M in methanol). The mixture was stirred for I min and hexane (100 ~1) added

followed by water (100 ~1). The hexane phase was removed, dried, taken up in hexane and applied to a silica Sep-pak cartridge (Millipore-Waters, Mil- ford, MA, U.S.A.). This was washed with hexane

9

and the penta~uorobenzyl ether eluted with di- chloromethane/hexane (1: 1, v/v).

Preparation of microsomes

Bovine placenta was collected fresh from the abattoir and transported to the laboratory on ice.

Cotyledons were removed and stored at - 80’ C until used. No loss in enzyme activity occurred

upon freezing or holding the tissue frozen for

periods up to 3 months. Thawed tissue (25 g) was minced with scissors and homogenized in 100 ml of 50 mM Tris/HCl buffer, pH 7.4, containing 1

mM EDTA and 1 mM dithiothreitol, using an Ultraturrax instrument (Janke and Kunkel,

Ikawerk, Stansen I. Breisgau, F.R.G.). The homo- genate was filtered through two layers of cheese

cloth and centrifuged at 800 x g for 10 min. Mitochondria were removed by centrifugation of the supernatant at 10000 X g for 15 min and

microsomes pelleted from the supernatant by centrifugation at 100000 X g for 1 h. The pellet was resuspended in the homogenization buffer

and recentrifuged at 100000 X g for 30 min. Fi-

nally, this pellet was suspended in water, lyophi- lized, and stored at - 20 o C.

Enzyme assay procedure The incubation mixture contained 10 FM

[3H]E2 (5 x 10’ dpm added in 10 yl ethanol), 0.1 mM oleoyl-CoA, 0.5 mM dithiothreitol, 0.1 M

sodium acetate buffer, pH 5.0 and 1.6 mg mi- crosomes (- 1.0 mg protein). The total volume

was 1.0 ml. Reaction was carried out in glass- stoppered tubes at 37 o C for 30 min with shaking. Methanol (2.33 mt) was added, and after vortex-

ing, hexane saturated with 70% aqueous methanol (3.33 ml) was added and the tubes vortexed again.

After briefly centrifuging, the hexane layer was removed and back-washed with 70% aqueous methanol saturated with hexane. Radioactivity was then monitored on an aliquot of the final hexane fraction.

In kinetic experiments where the concentration of t3H]E2 was varied, a zero time control was carried out for each concentration of [3H]E2 em- pIoyed. Counts in these hexane fractions were then subtracted from those obtained from the incubated samples.

Ana~sis of the biosynthesized E,-L fraction

Conditions were based on those described by Mellon-Nussbaum et al. (1982). Minced bovine

placenta cotyledon tissue (4 g) was incubated with 13H]EL (1.2 pmol, 5.0 PCi in 0.13 ml ethanol) in 0.1 M sodium acetate buffer, pH 5.0 (10 ml) at

37°C in a shaking water bath for 3 h. For incuba- tions employing bovine endometrium, 0.5 pmol

(20 PCi) of [3H]E2 was employed. The tissue was filtered, washed with buffer, homogenized in 25

ml of chloroform/methanol (2 : 1, v/v), and filtered through nylon gauze. This process was

repeated twice more and the combined extracts passed through a paper filter before evaporating to dryness in vacua. The latter was partitioned

between 70% aqueous methanol and hexane. The

combined hexane fractions from four incubations were chromatographed on a silica gel column to separate the E,-17/3-monoacyl ester fraction from

other lipid fractions, as described above for the purification of synthetic E,-acyl esters. Individual components of the biosynthesized [ 3 H]E,-L frac-

tion were then analyzed by HPLC on a reverse- phase column again as described for the synthetic

esters. Prior to HPLC, the [3H]E,-L fraction in acetonitrile (1 ml) was chilled and crystalline de-

posits of cholesterol were removed by centrifuga- tion. Following calibration with authentic E,-acyl esters, individu~ labeiled components were moni- tored by liquid scintillation counting.

Thin-layer chromatography

This was carried out on foil-backed 0.25 mm silica gel plates (Kieselgel 60 FZs4, E. Merck,

Darmstadt, F.R.G.). Solvent systems were: A, chloroform/acetone (37 : 3, v/v); B, hexane/di-

ethylether/acetic acid (70 : 30 : 1, v/v/v). Scan- ning of plates for radioactivity was carried out on a Packard 7220/21/22 (Packard, Downers Grove, IL, U.S.A.) instrument.

Other methods Liquid scintillation counting was carried out on

a Beckman LS3801 instrument equipped with ex- ternal standardization. Protein was determined by the method of Lowry et al. (1957).

10

Results

Assay of acyl-CoA : E2-1 7P-trunsferase

Bovine placenta cotyledons proved to be a con-

venient source of the enzyme and the tissue could

be stored frozen without loss of activity. In paral- lel with earlier reports that the bovine adrenal

enzyme (responsible for formation of lipoidal de- rivatives from steroids such as pregnenolone) was

present in both mitochondria and microsomes (Mellon-Nussbaum and Hochberg, 1980), this has

proved to be the case for E,-L formation in the placenta cotyledons. We have chosen to investi-

gate the microsomal enzyme, and unless otherwise stated, the results have been obtained with resus- pended lyophilized microsomes.

The assay took advantage of the greatIy differ- ing polarities between E, and E,-L. Zero time controls carried out with [“H]E, revealed that

only 0.11 it 0.07 (SD)% (n = 65) of the total counts partitioned into hexane from 70% aqueous methanol, under conditions described in Materials

and Methods. Similarly, 99.8 + 2.1 (SD)% (n = 5) of [ 3H]E,-17/&oleate partitioned into hexane. Ad-

dition of oleoyl-CoA (0.1 mM) to incubations containing [3H]E2 (10 FM) and microsomes in- creased the level of radioactivity in the hexane

fraction by some 7-fold. TLC of the [3H]E,-L

fraction, formed in the presence or absence of oleoyl-CoA, revealed one peak only on scanning for radioactivity. The R, of this peak was identi- cal to that of authentic E,-17&oleate. Upon elu-

tion and treatment overnight at room temperature with aqueous-ethanolic KOH, the [3H]Ez-L was cleaved to [ 3H]E,, characterized by TLC and by recrystallization to constant specific activity. Thus

the [‘H]E,-L has the properties of 17/3-fatty acid esters of E,.

Influence of time, protein concentration and pH The pH optimum was 5.0 and E,-L formation

in the presence of 0.1 mM oleoyl-CoA and 10 FM E, was linear for microsomal protein concentra- tions up to approximately 1.0 mg/ml. Reaction rate was linear for some 40 min employing 10 mg/ml microsomal protein.

Optimum concentration of fatty aeyl-CoA An experiment was carried out with [‘“Cl-

oleoyl-CoA and [ ‘H]E, to determine if a stoichiometry existed between the yield of E,-L determined by assessment from each isotope.

[‘4C]Oleoyl-CoA was varied over the range lo-100 PM at a fixed concentration of 10 PM [‘H]E,.

The hexane fraction was subjected to TLC in

solvent systems A (X 1) and B (X 2) and the area corresponding to E,-oleate was eluted and

counted. Approximate stoi~hiom~t~ was estab- lished for [r4C]oleoyl-CoA concentrations up to 30

I_IM only. Beyond that concentration the apparent

yield of E,-L, estimated from 14C incorporation, increased over that estimated from 3H incorpora-

tion. Evidently the [i4C]oleoyl-CoA is converted into other lipids such as diglycerides which chro- matograph close to E,-L and whose yield in- creases as [‘4C]oleoyl-CoA concentrations rise above 30 PM.

When the concentrations of unlabelled oleoyl- CoA was varied, formation of [3H]Ez-L was ob-

served to increase up to oleoyl-CoA concentra- tions of 0.2 mM. At higher concentrations severe inhibition occurred, possibly due to the known

detergent action of fatty acyl-CoAs (Spector et al., 1979).

Apparent K, of E,

By varying [‘H]E2 at a fixed concentration of

0.1 mM oleoyl-CoA, Michaelis-Menten kinetics

were observed (apparent K, 8.0 + 2.2 (SD) PM, n = 6). The apparent K, was not significantly altered when palmitoyl-CoA and linolenoyl-CoA were used in place of oleoyl-CoA. Steroids possessing a 17P-hydroxyl group (testosterone), a 3P-hydroxyl group (dehydroepiandrosterone), or both groups (5-androstene-3@,17/3-diol), all acted as competitive inhibitors with K, values of 79, 46 and 39 PM, respectively. Estrone, which itself was

not acylated in this system, was also a competitive inhibitor (Ki 46 PM).

Specificit,v of fatty acyl-CoAs Fig. 1 compares the relative yields of [‘H]E,-L

formed from 10 FM 13H]E2 and a number of individual fatty acyl-CoAs (0.1 mM). Microsomal preparations from both placenta cotyledons and bovine endometrium were employed. As can be seen, both microsomal preparations exhibited very similar profiles characterized by low acyl incorpo-

11

t3

nn P *u lJ L l.e A, c P PO 0 L LB I\r c P 0 *r c

PLACENTA ENDOMETRIUM ENDOMETRIUM

MICROSOMES MlCROSOMES MITOCHONDRIA

A: Bovine placenta cotyledon

microsomes; B: bovine endometrium microsomes; C: bovine

endomettium mitochondria. Values are means of duplicate or

triplicate assays. P, palmitoyl-CoA; PO, palmitoleoyl-CoA; 0,

oleoyl-CoA; L, linoleoyl-CoA; Le, linolenoyl-CoA; Ar,

arachidonoyl-CoA; C, control (no added fatty acyl-CoA).

ration from the essential fatty acids, linoleic, lino- lenic and arachidonic acid. Mitochondria obtained from bovine endometrium behaved similarly (Fig.

1). The apparent inability of arachidonoyl-CoA to

act as substrate for the placenta microsomal pre-

paration at 0.1 mM concentration was possibly due to inhibition of enzyme by excess substrate.

Accordingly, assay was carried out at concentra- tions ranging from 2.5 PM to 100 PM. However, no increase over the control carried out in the

absence of arachidonoyl-CoA, was observed. Analysis of the stock arachidonoyl-CoA solution showed that hydrolysis to free CoA was minimal

(1.5%). In addition, arachidonoyl-CoA at a con-

centration of 0.04 mM did not inhibit [3H]E,-L formation when oleoyl-CoA was employed in the

1500

E s 1000

B

i

: a 500

UN

PLACENTA

0 0 5 10 15 20 25

RETENTION VOLUME (ml)

Fig. 2. Composition of [ 3H]E,-L formed by incubating [ ‘H]E,

with bovine placenta cotyledon tissue. The [‘H]E,-L fraction

was obtained by partitioning into hexane and separated from

other lipids by column chromatography on silica gel. Compo-

nents were then analyzed by HPLC on a reverse phase column.

concentration range 0.01-0.10 mM (data not shown).

Composition of E2-L formed from endogenous fatty acids

The E,-L fraction obtained by incubating

bovine placenta cotyledon tissue with [ 3H]E,, was purified by column chromatography and compo- nents analyzed by HPLC. Data is shown in Fig. 2. Similar analyses were made of the E,-L fraction

obtained from incubations of bovine endometrial tissue with [ 3H]E, under identical conditions (Fig.

3). Our data for bovine endometrium is in good

agreement with that previously reported (Mellon-

Nussbaum et al., 1982) and is characterized by a

high percentage of unsaturated fatty acids. The

3000 - 18:s 204 ENDOMETRIUM 4ODO- l&9

20:4 ENDOMETRIUM r 8 I&?

(non-pregnant cow) 0 (pregnant cow) 0 2ooa 16:1 3Doo-

0 0 5 10 15 20 25 0 5 10 15 20

RETENTION VOLUME (ml) RETENTION VOLUME (ml)

Fig. 3. Composition of [‘H]E,-L formed by incubating [3H]E, with bovine endometrium tissue (see Fig. 2).

25

12

TABLE 1

COMPOSITION OF ESTRADIOL FATTY ACID ESTERS @z-L) FORMED BY BOVINE PLACENTA AND ENDOMETRIUM

Fatty acid

C22 : 5 docosahexaenoate

C20 : 4 arachidonate

Cl8 : 3 hnolenate >

Cl8 : 2 linoleate

Cl6 : 1 palmitoleate

Cl8 : 1 oleate

Cl6 : 0 palmitate

Cl8 : 0 stearate

Placenta

(%)

4.3

27.6

28.9

7.2

2.5

Endometrium (%)

Pregnant

7.0

30.7

26.1 b

7.8

Non-pregnant

6.0

32.6

13.9

10.6

8.1

Literature a

9.9

27.6

0.0

10.1

8.5

18.0

5.4

9.0

a Mellon-Nussbaum et al., 1982. The composition of I&,-L from bovine endometrium was carried out by HPLC and gas-liquid-

chromatography (GLC)-mass spectrometry and contained 3.2%, 4.2% and 4.0% of docosapentaenoate (C22 : 5). and eicosatrienoate

(C20 : 3) and adrenate (C22 : 4), respectively. These components were not resolved by the method employed here.

’ This fraction (see Fig. 2) was resolved (placenta, non-pregnant endometrium) by HPLC of the pentafluorobenzyl ethers on a 5p

Brownlee siiica column employing hexane/d~chloromethane (70 : 30, v/v).

composition of E,-L from the placenta was in turn very similar to that of the endometrium. [3H]E,-

oleate and palmitate were not separable on the reverse phase column. This peak was resolved by conversion of the components to penta~uoroben- zyl ethers followed by HPLC on a silica column.

Table 1 gives the percentage composition of E,-L

from placenta and endometrium for comparison with published data (E,-L from endometrium).

Discussion

We have attempted to develop a model system for study of fatty acyl-CoA : E,-17P transferase.

The assay system involving solvent partition is both rapid and simple and can be used for the study of fatty acid ester synthesis from labelled

steroids other than E,. Although the microsomal enzyme will acylate

E, specifically at the 17fl-position, other steroids

bearing 3/3- or 17~-hydroxyl groups can act as competitive inhibitors. Such steroids are them- selves acylated by the microsomal enzyme, al- though they have lower affinities (apparent K, values 30-50 PM) than E, (details will be pub- lished separately). Therefore the data suggests that a single enzyme of wide specificity is involved. It is not unusual for a steroid-metabolizing enzyme to exhibit this behavior. For example, hydroxy- steroid sulfotransferase (EC 282.2) from human

adrenal glands, which was isolated in a highly purified state (single band on normal and SDS-gel electrophoresis), can accept a steroid at the sulfurylation site either via ring A or ring D

(Adams and McDonald, 1981). A similar situation occurs with 3tu,20/3-hydroxysteroid dehydrogenase

(EC 1.1.1.53) in the reduction of both 3-keto and 20-keto groups (Sweet and Samant, 1980). On the

basis of the distribution of enzyme in tissues of

the rat (Schatz and Hochberg, 1981). and on the determined apparent K, values, it could be tenta- tively concluded that E, is the normal substrate

for the placental enzyme. The microsomal enzyme of the placenta showed

a relatively low utilization of linoleoyl-CoA and linolenoyl-CoA, and negligible utilization of arachidonoyl-CoA as compared to palmitoyl-CoA, palmitoleoyl-CoA and oleoyl-CoA. This was also the pattern exhibited by microsomes and

mitochondria prepared from endometrium. The fact that no evidence for arachidonoyl-CoA utili- zation was obtained over a wide concentration range was unexpected in view of the high con- centration of arachidonate present in E,-L. By contrast, palmitoyl-CoA which was a good sub- strate both with the membrane-bound enzyme (Fig. 1) and for the solubilized and reconstituted form of the enzyme (Lee and Adams, 1987) nev- ertheless was present in relatively low levels as E,-palmitate in the biosynthesized E,-L fraction

13

(Table 1). Arachidonoyl-CoA did act as a sub-

strate for the solubilized and reconstituted form of the enzyme, but the apparent K, value was the highest among the acyl-CoAs evaluated (Lee and Adams, 1987). It is evident that the composition

of E,-L formed by whole tissues, or cells in cul-

ture, depends on (i) availability of acyl-CoA es-

ters, and (ii) the balance between the opposing reactions of synthesis and breakdown. When hu-

man mammary cancer cells were exposed to [ 3H]E, in culture, rapid accumulation of [3H]E,-L oc- curred within the cells, but upon transfer to

medium lacking the hormone, breakdown of the

E,-L to free E, followed (Adams et al., 1986).

Both during the time of exposure to [3H]E2, and in the subsequent hormone withdrawal period, the relative abundance of E,-esters comprised of es-

sential fatty acids were observed to decrease

(Martyn et al., 1987). Furthermore the percentage of E,-arachidonate and E,-linolenate within the

total E,-L fraction formed by the cancer cells over a similar time period was lower than that reported

here for placenta and endometrium. A differential turnover of components of the E,-L fraction could help to explain the anomaly between the kinetic

data obtained with the fatty acyl-CoA transferase, and the actual composition of E,-L formed by intact tissue in vitro. An esterase, capable of hy-

drolyzing a range of estradiol-17P-fatty acid esters,

has been found in the cytosol of bovine placenta and human mammary cancer tissue cells, and is currently under investigation.

In conclusion, we have established that the formation of E,-L is carried out by an enzyme which is not specific for E,. This suggested that steroids other than E, could be converted into

fatty acid esters. Such a situation has been realized

and the formation and turnover of fatty acid esters of 5-androstene-3/?,17P-diol in human mammary cancer cells in culture will be described

elsewhere (Adams et al., 1988). Steroid fatty acid

esters may be involved in transport through mem- branes and/or as storage forms capable of being reconverted to free steroid as has been established for E,-L in human mammary cancer cells in cul-

ture (Adams et al., 1986; Martyn et al., 1987).

Acknowledgements

We wish to thank Dr. A. Duffield for the mass spectrometry and Dr. A. Netting for help with the

HPLC analyses. This work was supported by grants from the National Health and Medical

Research Council and the New South Wales Cancer Council.

References

Abul-Hajj, Y.J. (1982) Steroids 40, 149-156.

Abul-Hajj, Y.J. and Nurieddin, A. (1983) Steroids 42,417-426.

Adams, J.B. and McDonald, D. (1981) B&hem. Biophys. Acta

664, 460-468.

Adams, J.B., Hall, R.T. and Nott, S. (1986) J. Steroid Biochem.

24, 1159-1162.

Adams, J.B., Martyn, P., Smith, D.L. and Nott, S. (1988)

Steroids (in press).

Lamer, J.M., Eisenfeld, A.J. and Hochberg, R.B. (1985) J.

Steroid Biochem. 23, 637-641.

Lee, F.T. and Adams, J.B. (1987) Biochem. Biophys. Res.

Commun. 144, 569-575.

Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randal, R.J.

(1957) J. Biol. Chem. 193, 265-275.

Martyn, P., Smith, D.L. and Adams, J.B. (1987) J. Steroid

B&hem. 28, 393-398.

Mellon-Nussbaum, S. and Hochberg, R.B. (1980) J. Biol. Chem.

255. 5566-5572.

Mellon-Nussbaum, S., Ponticorvo, L., Schatz, F. and Hoch-

berg, R.B. (1982) J. Biol. Chem. 257, 56785684.

Netting, A.G. and Duffield, A.M. (1985) Biomed. Mass Spec-

trum 12, 668-672.

Schatz, F. and Hochberg, R.B. (1981) Endocrinology 109,

697-703.

Spector, A.A., Mathur, S.N. and Kaduce, T.L. (1979) Prog.

Lipid Res. 18, 31-53.

Sweet, F. and Samant, B.R. (1980) Biochemistry 19, 978-986.