Panel discussion: Purification, structure, cDNA probes

PANEL DISCUSSIONz PURIFICATION, !SIWJC.TURE,cDNA PROBES

Chairperson:

Panel:

Hilton A. Salhanick Center for Population Studies Harvard Uedical School 665 Huntington Avenue Boston, WA 02155, USA

Evan R. Simpson University of Texas Southwestern Medical School 5325 Harry Hines Boulevard Dallas, TX 75235, USA Yoshio Osawa Medical Foundation of Buffalo 73 High Street Buffalo, NY 14203. USA Larry E. Vickery University of California, Irvine Irvine, CA 92717, USA Peter F. Hall Prince of Wales Hospital High and Avoca Streets Randwick, NSW 2031, Australia

R. RABTER:

I

would ask that the panel members address several issues

raised in their presentations. suggested the sion on

biochemical

Perhaps one for them. species?

presence of

of the

Specifically, previous

aromatase isozymes.

purification

showed

no

This morning's discusevidence

for isozymes.

panel members could review the biological evidence

How different are the aromatases

studied in

various animal

For instance, Dr. Gloria Callard finds that the Km for aroma-

tase in goldfish brain is about 2 nmol, 15-30 nmol.

Would

whereas, in

the human,

it is

one of the panel members also address the issue of

specificity of the P-450

reductases?

Does

cause any specificity in the aromatase system?

STEROIDS

reports have

50 / l-3 1987

the

reductase component

Perhaps Dr. Osawa could

308

Salhanick et ai.

discuss the type I and type II aromatases sgo and

comment on

testosterone.

that he

their relative specificity for androstenedione and

In addition, could a

panel member

comments on the P-450 superfamily of genes? P-45&

fit into the

lating P-450s

proposed same years

scheme,

such as

and,

For instance, how do liver

specifically, the steroid-hydroxy-

those mediating

450s have greater homology

make some background

6p-hydroxylation? Do these P-

with placental

aromatase than

some of the

other p-450s in liver? In.VICIIKPY:

As

I said

in my

talk, we

see no evidence for multiple

forms.

Terminal sequences described by our group and by other investi-

g&OX-S

suggest

only

a

single

except for partial truncation. with all

intermediates and

species

in our purified preparation,

The preparation we

all substrates.

have seems

to work

I see no reason to invoke

isozymes at this time. UN~XIPPIFIED PARYICIPZWC:

May I ask a follow-up question?

Was anyone

isolated aromatases from tissues other than the placenta; and more specifically, has anyone looked at Northern blots of

the aramatase clones

in tumors, to see if the m.RHhs might be different? C

uBt?Dl?zsSBz

We

have

used our cDNA probe against human placental

aromatase to test a number of different tissues losa cells,

rat granulosa

Simpson showed, there are encode for

this protein

cells, and

no indication

three

placenta.

human ovary and granulosa cells, and We have

human placenta

approximately in the

including human granu-

in rat

itself.

species

of

As Dr.

mRNA that

We see the same species in granulosa cells

as well.

to date that there are aromatase mRNA-size dif-

STEROlDS 5011-3 1987

PANEL DISCUSSiON

ferences in different tissues.

309

Presumably, there are haloes

speciesof this protein. We believe that there is just one form. R.

EMMTER:

El.-cX:

What is the biologicalevidencefor isozymes? I don't think that there is anyone on this panel who

would propose that there is biologicalevidence for isozymes. Y, asnWA: What I describedas isozymesare isofunctional enzymes that are recognizeddifferentlyby our monoclonaland polyclonalantibodies. The epitope capable of suppressingenzyme activityby antibodybinding must be near or at the active site of the enzyme. The antibodiesare raised against the cytochrome P-450 portion.

Aromatase is a complex

enyzme system, and the cytochromeP-450 portion is only a part of it. The only isozymesthat we have found so far by this approach are isozymes among differentspecies. Initially,I expectedthat point mutations would have occurredduring the process of evolutionand that the amino acid sequencesw0tda differ among species. Yet, as far as the active site of aromataseis concerned, I thought that the amino acid sequencesat that region would be more closely conserved. I didn't expect that our monoclonal antibody would differentiate among the aromatase enzyme% present in differentspecies through differential suppressionof enzyme activity. However, the fact is that there is immunochemicalevidence for differences among species,at or near the active site of aromatasecytochromeP-450. Several years ago, we

reportedmultiple forms of arcmatasefrom

human placenta. We called these activitiesP2 and P3. We consistently observed,as for many P-450s studied,that if we solubilize and separ-

STEROIDS 50 / 1-3 1987

310

Salhanick et al.

ate the cytochrome P-450 fraction from the reductase fraction, there is no evidence for biochemical separation of the P-450s. solubilize

them

so

that

they

and P3 aromatase. the

P2

as I

When we

cannot

make

estriol,

a difference,

different

reductase.

oxido-reductases besides

One interconverts androgenic,

steroids; one

these data.

We see

the NADPH cytochrome P-450 17-keto-,

and 17P-hydroxy-

interconverts estrone and estradiol; and the last, which

is present in different animal species, estriol but

in that

while P3 will make estriol.

There are some complications in interpreting three

apply immunoaffin-

described, we get a single P-450 from both P2

In the active form, there is

aromatase

if we

retain the aromatase activity, we can

consistently separate the different forms. ity chromatography

However,

converts 16a-hydroxyestrone to

not estriol to 16a-hydroxyestrone. These can be separated

by the column chromatography used for separation of the multiple forms. One explanation

is that

the solubilised

active aromatase

has a good

I.l(+hydroxyestrogendehydrogenaae, but no 16a-hydroxyestrone reductase; therefore, there is no estriol formation.

16a-Hydroxytestosterone is a

very poor substrate to be directly converted to estriol. These species of

observations

do

cytochrome P-450.

not

address

the

question

We simply don't know.

of different

The P3 aromatase

carries an activity that reduces 16a-hydroxyestrone to estriol. oxido-reductase has

not been

fully characterized. The complex struc-

ture of lipid plus aromatase plus other enzymes bound

in

nature.

I

This

appears to

be tightly

could conceive that, biologically, the site to

which these oxido-reductases are complexed could make a difference.

STEROIDS

50 / l-3 1987

PANEL DISCUSSION

311

Another question relatesto the nature of the reductases. The liver microsomal NADPH cytochrome P-450 reductase frcnnother species efficientlycouples to purifiedaromataseP-450. However, ismunochemical suppression studies suggest speciesdifferences,particularlyin horse placentas. The efficiencyof couplinqof the solubilizedcytochrome P-450 of the P2 fractionalso shows a difference. The reductase that we isolate from human placenta couples best.

If we couple with

reductasefrom other sources such as porcine,rat, bovine, and rabbit liver, we see only S-20% of aromataseactivityeven if we increasethe amount of reductase. On that issue, Casper has shown that there is only one

E.SlMpNN:

reductasegene. This presumablycodes for the same reductaseno matter what organ it is in and this reductasepresumablycouples with every other cytochromeP-450 in the body. It is a

little hard to visualize

why placental reductasewould couple better than reductasefrom liver. What might be importantis the species differencerather than the organ difference. H. SAIJiMICK: Would you assume that the mitochrondrialreductasesare the same as the microsomalenzymes? E.SIWpSOW: No, these are different. ~~P~~

Could Dr. Hall comment on the ability of

lip-hydroxylase to 19-hydroxylatesteroid substrateand the possibility of contamination betweenmitochondrial Up-hydroxylase and microsomal aromatase?

STEROIDS

50 I l-3 1987

312

Salhanick

P. Hw:

et al.

There is no possibility of serious contamination between the

mitochondrial lip-hydroxylase and the not usually

expressed in

the same

bovine adrenal cortical mitochondria molecular model, from 18.

microsomal aromatase.

tissues.

They are

The lip-hydroxylase from

will also

19-hydroxylate.

On a

the 19-methyl is the same distance from 119 as lip is

The possibility would be that the substrate can fit

when you

get it out of the microsomal membrane, and perhaps it is a more permissive environment. site, and

The substrate might fit in two ways

I suppose

hydroxylation, it occur there.

That

that once

you have

is conceivable

into the active

started the process with 19-

that the

second hydroxylation would

would be almost routine for a P-450 that can start

the mechanism off. UNILZNTIFIED PARTICIPANT:

Is it possible that a

separate 19-hydroxyl-

ase enzyme exists? E.SIHPSON:

As you know, there is a very active 19-hydroxylase in the so I

adrenal of some species -- the gerbil, for example -it is

think that

quite a likely possibility that there is a 19-hydroxylase, which

is an independent entity from the aromatase as

such, in

some species.

It might be the second member of the cytochrome P-450 gene family XIX. To try to answer the question of the biosyn-

UNILZNTIFIED PARTICIPANT: thesis of 19-nor steroids, show that

presented at

this meeting to

porcine granulosa cells convert both androstenedione and 19-

hydroxyandrostenedione to aromatase

evidence was

inhibitors,

19-norandrostenedione. 4_hydroxyandrostenedione,

We

find

that the

aminoglutethimide

phosphate, and ketoconazole, all inhibit the conversion of both precur-

STEROIDS

50 / l-3

1987

PANEL DISCUSSION

sors to

313

19-norandrostenedione.We suggest that the formationof 19-

norsteroidsis not a cytochromeP-450-mediatedevent, even based on the evidencewe have. Whether this reaction is catalyzedby the aromatase enzyme, I can't say at the moment. L. VEKERY:

Some of these questionsmay be answered,in part, when the

Dallas group is able to express the recombinantprotein and then assay the activitiesof the recombinantproteinsproduced from a single gene. B.SIM?SM:

When we get a full-lengthclone, that will become a possi-

bility. p. SIm:

I have been fascinatedby the observationsof extraordin-

arily high levels of 19-norandrostenedione in ovarian follicularfluid. It seems to me that if there were a mechanismwhereby the aromatization process could be aborted at that step, this might be a very important controlpoint in terms of the amount of estrogenssecreted,or of a compoundwhich may have totally different properties that we don't recognizeat the present time. This, then, raises the questionwhether anyone can propose a mechanismthat would abort the aromatase so that 19-norsteroidis secreted. Y. OSAWA:

We have shown the enzyme system which we call non-aromatiz-

ing 19-hydroxylasein the dog and sheep adrenal.

These tissuesmay

have high androstenedione 19-hydroxylase activityand showed an apparent isotope effect of about 11.7, which is obviouslydifferentfrom that of the aromatase. We have not characterizedthe enzyme, but it proceeds to form the 19-aldehydeand also 19-norandrogen. It results in very little estrogen synthesis.

STEROIDS

50 I l-3 1987

I don't really understandthe

314

Salhanicket a/.

nature of this enzyme; it could be the same P-450 as the aromatase, but with

something

second stage. have shown

else

regulating

diverting

or

the process after the

Alternatively, it may be a totally different enzyme.

We

that it is a cytochrome P-450-dependent process, but beyond

that we don't know much about it. F._LrW:

I believe it of interest to show how powerful

antibody directed

against aromatase

localizing and understanding performed

the

can be,

synthesis

immunocytochemistry using

Dr.

particularly in terms of of

We have

In the upper

left (Fig-

the syncytiotrophoblasts are clearly marked, whereas the cyto-

trophoblasts are clear and the aromatase only

in the

nuclei

show

serum which

no

has apparently

our own

Finally, in Figure 3, one can see

the syncytiotrophoblasts, aromatase is

expect it, not in the nucleus but in shows an

polyclonal anti-

the same distribution, being found only in

the syncytiotrophoblasts (Figure 2).

The insert

There is

activity.

syncytiotrophoblasts. That can be seen by com-

paring it with estradiol dehydrogenase using

that in

aromatase.

Simpson and Dr. Mendelson's

antiserum to study human placental aromatase. ure l),

a tool the

area of

the rough

found where one might endoplasmic‘reticulum.

golgi with no evidence of immunopositive Thus, aromatase -in vitro

material being processed within it.

can be

seen to be limited to the microsomes. uNIDEt?rIFIEDPARTICIPANT: mediate production. first

studied,

the

I would like to bring up the topic of inter-

Back in the 1950s and biosynthetic

hydroxylation for two reasons.

1960s when

aromatases were

activity was thought to involve 19-

First, the 19-hydroxy and 19-oxo deriv-

STEROIDS

50 / l-3

1987

PANEL DISCUSSION

315

1. Photomicrographs of human first trimester placenta Wostained for arcmatase cytochrome P-450. (a) Iranunostaining in the syncytiotrophoblast cytoplasm while the nuclei are consistently unstained. (b) Consecutive section to (a) showing complete abolition of irnrmnostaining after incubation with antiserum preadsorbed with 500 pg purified cytochrome P-450,,,. (c) Section immunostained for aromatase cytochrome P-450 and counterstained with Mayer's hematoxylin. Cytotrophoblast cells are easily recognized (arrows) and appear free of staining. (d,e) The inununoperoxidase has been replaced by a goldsilver intensification technique. Ivununoreactiveproduct appearing as a dense black precipitate is seen in the syncytiotrophoblast cells only. The unstained nuclei appear as small white dots. Orisinal magnifications: a,b,c,e, x100_ a, x20. Reprinted with permission of the authors and publisher (J CLIN ENDOCRINOL METAB 65:757, 1987). FIaJRE

STEROIDS

50 / l-3 1987

316

Salhanick et a/.

PIGOREZ2. Photomicrographs of human first trimester placenta immunostaining for E2DH. (a) General view of the chorionic villi, showing intense immunostaining of the syncytiotrophoblast layer only. Note the homogeneous and dense distribution of the peroxidase reaction product that, when seen in the cytoplasm in these thick sections, could account for apparent immunostaining over the nuclei. Some unstained nuclei (b) Higher magnification, emphasizing the appear as white dots. absence of staining of the cytotrophoblast cells (arrows). Original Reprinted with permission of the magnifications: a, x20; b, x100. authors and publisher (J CLIN ENDOCRINOL METAB 65:757, 1987).

STEROIDS

50 / 1-3 1987

PANEL DISCUSSION

317

Electron micrographs of human first trimesterplacenta EMJRB 3. irmrunostained for aromatasecytochromeP-450. (a) General appearanceof a syncytial cell (St). Peroxidasereactionproduct is concentratedin the cytoplasmand covers the endoplasmicreticulum(arrows). Note the absence of stainingof the microvilli(mv) and adjacentcytotropboblast cells (ct). fb) Higher magnificationof the area outlined in a, showing the absence of stainingof the Golgi apParatusand secretorygranules. Bar scales f 1 pm. Reprinted with permission of the authors and publisher (J CLIN VINYL HaTAR 65:757, 1987).

STEROIDS 50 I I-3 1987

318

Salhanick

atives

were

et al.

detected in the medium in relatively large quantities early

and appeared sequentially. Now, when

substrates.

Second,

we examined

they

were

data from

efficiently

the purified enzyme (I

think I first noticed this in Dr. Peter Hall's publication then in

Dr. Dwain

Hagerman's paper

and Dr.

used as

in BBRC and

Osawa's studies using 2-

dimensional TLC), it seemed to me that there was very little intermediate produced.

Could the speakers comment on that?

L. VICKERY:

There is a possibility that this may be due to the rate of

delivery of

reducing

equivalents.

When

the

delivery

of reducing

equivalent to the enzyme is slow, there is a greater probability of the intermediate products dissociating or trations of the substrate.

being displaced

by high concen-

I think Dr. Peter Hall has carried out some

very elegant experiments with the 17-hydroxylase, showing that have release

of 17-hydroxy intermediates from the hydroxylase lyase at

limiting reductase concentrations.

Isn't that correct?

p. HALL:

It is certainly true that we don't think of

P-450 as

rate-limiting in

tions.

limiting.

another cycle

the reduction of

the ordinary liver-drug hydroxylation reac-

When the enzyme has more than

it requires

you can

one hydroxylation

to carry out,

of reduction and then reductase does become

If you start, for example, with the C-21 side-chain cleavage

enzyme and incubate with progesterone, you can measure both 17a-hydroxylation and the lyase reaction which cleaves off the acetate. Vmax values

for that last step are always much lower than if you start

with 17a-hydroxyprogesterone as the substrate. of reduction

Now, the

may be

I think

that the rate

a factor in allowing the release of intermediates

STEROIDS

50 / l-3

1987

PANEL DISCUSSION

319

which may not occur when the reductase activity is adjusted in the microsome-in vivo. We don't really know very much about that yet. Dr. Leo Samuelsperformeda series of

elegant experiments with microsomes

from testis showing that when the 17a-hydroxyprogesterone is added to the water phase it does not compete well

with the 17-hydroxyprogester-

one generated on the surface of the enzyme.

Once you get it out of the

microsomal membrane, all sorts

of intermediates would be

of spillage

possible.

L.vIclcKRY: I think Dr. Bellino suggested some time ago that reductase can be partially rate-limiting in the microsomal system tion.

In fact,

we have

reductase, at least

in

almost

rate

double

the

found that

the of

by supplementing microsomes with

detergent-reconstituted

and adding

tase will cause the rate to go up at least 50%. reductase may

system,

we can

aromatization without adding more P-450.

Simply dispersing the microsomes in detergent

that the

for aromatiza-

be partially

extra reduc-

There is some evidence

rate-limiting -in

situ, and this

could lead to more spillover of the intermediates. P. SIIYERI:

I

would like to cosunentthat we showed clearly, in 1974,

that the ratio of metabolites that one observes is totally dependent on the substrate

concentration at the start.

It was first suggested that

the rate-limiting step was, in fact, 19-hydroxylation, because very low substrate levels period of time.

were used and it was totally consumed in a very short We showed

that, with

very high

fact, the third hydroxylation is rate-limiting. what the rates are -in vivo, the enzyme

STEROIDS

50 / l-3 1987

is far

substrate levels, in If you try to estimate

from saturation

in the

320

Salhanick et a/.

placenta.

One would expect that the reductase may or may not be rate-

limiting, but clearly you don't see a lot or 19"norandrostenedione in pregnancy.

of ~9-hydro~androstenedione So

it is

running very effi-

ciently.

P. HALL:

Dr. Cisawa,I would like to reopen the "19-nor" question.

you able

to say anything about the way in which the aldehyde moiety is

lost from the f9-formyl or the 19-aldehyde in hydroxylase?

the case

of the adrenal

Does it come off as formic acid or COz?

Y. OSAWA:

Your

mechanism.

I am not prepared to answer that question

trying to

Are

question relates

to the non-aromatizing 19-desmolase as yet.

We are

meke an androgen labeled with I*C at the 19 position so that

we can identify the fate of that 19-carbon for sure. on tritium-labeled

substrates, I

would guess

stead of formaldehyde is lost in the case

From

our studies

that carbon dioxide in-

of non-aromatizing 19-desmo-

lation. D. PUETT:

I

wanted to ask about Dr. Hall's Km values, which seemed a

bit high. P. HALL:

These are all

aqueous phase

in

Vmax over

liposomes with

and phosphatidylcholine. between

which the

enzyme is

with no special detergent present.

fold lower and the enzyme

values in

preparations

3 times

higher if

present in an

The Km values are 5you reconstitute the

an equal mixture of phosphatidylethanolamine Based on this,

that

we

have

I think

that the differences

seen today may be related to the

detergent that is still present after purification or that is

added in

the reconstitution procedure.

STEROIDS 50 / 1-3 1987

PANEL DISCUSSION

H.

Regarding

BRODIB:

the

question

Simpson or Dr. Hall might speculate tions and

what they

expect to

321

of regulation, I wonder if Dr.

about post-translational modifica-

find with respect to the regulation of

aromatase? EE.SIHExm:

Since

this is,

of course,

an enzyme

of the endoplasmic

reticulum, one presumably would not expect to find any sequence modification in terms of loss of a precursor in the the enzyme

into the

endoplasmic reticulum.

process of

insertion of

From that point of view,

the product of _in vitro translation appears to be identical that found

in the

cell at

cleavage of a leader protein of

any given

moment.

sequence, which

the endoplasmic

is what

reticulum.

cell.

can In

also

occur

terms

glycosylation, I

of

if

There appears to be no you would

expect for a

Of course, the standard post-

translational modification of these enzymes is This

in size to

the insertion

of heme.

you express the cDNA in a non-steroidogenic

other

guess that

post-translational modifications this issue

has not

such as

been addressed, so I

have no knowledge of that. P. EALL:

It would have to be a guess,

but we

couldn't find

any evi-

dence of the aromatase being a glycoprotein. B

PARTICIPANT:

back to the old days. with placental

Another

All the

microsomes, but

question can be raised that goes

discussants this it has

morning have started

been reported

and observed in

many different laboratories that half of the total activity in placenta comes down

with the

mitochondrial fraction.

STEROIDS

50 / l-3 1987

Is that just due to con-

322

Salhanick et al.

tamination of the lower fractions with endoplasmic reticulum or are you confident that there is not a mitochondrial enzyme in placenta as well? I can comment on that.

L. VICKERY:

I don't know that we can rule out

other locations of the enzyme, but it is difficult to remove microsomes from nuclear debris and from mitochondrial pellets.

I know some of the

early isolation procedures actually threw away the microsomes the

debris

fractions

because

sedimented at low-g force. chondrial pellets of the activity is

so

much

and took

of the endoplasmic reticulum

If one takes the

debris pellets

and mito-

and rehomogenizes them and recentrifuges them, a lot then released

into the

so-called microsomal frac-

tion. P. HALL:

It certainly can be difficult to release endoplasmic reticu-

lum from the mitochondrial fraction. membrane shares

direct continuity

In fact,

the outer mitochondrial

with the endoplasmic reticulum.

least in our hands, I can say that less than 10% of

At

the total activity

of aromatase could be found in the mitochondrial fraction. LlNIDFN!IFIEDPARTICIPANT:

This

isn't a

the distribution of the aromatase. that it

is primarily

found in

In human placenta, it

the microsomes.

cently been examining sheep placenta. specific activity

of aromatase

question, it is a comment on

We find

in homogenate

However, we have re-

that if

we measure the

compared to that in the

classical microsomal fraction, the specific activity is only one-fourth that in the microsomes.

would appear

in the homogenate

One could predict from this

that there is aromatase activity in other fractions. When we looked at the so-called

heavy-light mitochondria-peroxisomal fraction, there was

STEROIDS

50 / l-3

1987

PANEL DISCUSSION

a considerableamount of aromataseactivity there.

323

It could well be

that, in other tissues, there ie aromatasedistributedthroughmany membrane fractions. I think it will be staining studies to

interesting when people do

see if other fractions,specificallyin other

tissues like sheep placenta, and not just in human placenta,contain aromatase. It is still an open question,I think. F.NAFTOLIN: On careful examinationof electronmicrographsof inxnunohistochemicallystainedmaterial,the outer mitochondrialmembrane does stain, whereas the mitochondriathemselvesdo not -- neither the cristae nor any of the spaces within the mitochondria. The endoplasmic reticulumis really packed with aromatase-staining material. If aromatase is a mitochondrialenzyme: (a) it is evanescent,and be present in a relativelysmall amount.

STEROIDS 50 / f-3 1987

(b) it must