STEREOSPECIl?ICITYOF ENZYMATIC REDUCTION OF PROSTAGLANDIN ** E2 To "2a Lawrence Levine,+ Kung-Yue Wu and Sheng-Shung Pong Department of Biochemistry, Brandeis University Waltham, Massachusetts 02154

ABSTRACT were prepared in 28 The serologic specificity of the immune reaction

Antibodies directed toward PGF rabbits.

was determined by inhibition of sodium borohydride-reduced (3H) PGE2 anti-PGF2B binding by several prostaglandins.

The

antibodies to PGF2S recognize the S-hydroxyl configuration in the cyclopentane ring of PGF2@.

With the use of both

anti-PGF and anti-PGFgB, the product of PGE2 reduction by 2a 9-ketoreductase purified from chicken heart was identified as PGF2,.

Guinea pig liver and kidney homogenates were

examined for PGE g-ketoreductase activity.

Although enzyme

activity was present, no evidence of PGF28 production was

Accepted April3, 1975 ** Supported by grant m-07966

from the National Institute

of Child Health and Development and grant IM-22M from The American Cancer Society.

+Lawrence Levine is an American

Cancer Society Professor of Biochemistry (Award No. PRP-21). Publication No. 995. PROSTAGLANDINS APRIL

1975

VOL. 9 NO. 4

531

PROSTAGLANDINS

INTRODUCTION Enzymes present

that convert prostaglandin

in cytoplasmic

in microsomal blood

(7).

fractions

fractions

of several tissues

of monkey

In addition,

(P~;)E to ~c;r'are (l-6),

liver (3) and in sheep

during metabolic

studies

in the

guinea pig where the main urinary product was found to be 58,

7a-dihydroxy-ll-ketotetranorprmtanoic

clear that -in vivo reduction had occurred

the product

has been identified

hydroxyl

of the C-o keto group of PtiD2

(8).

In sheep blood, (PGF2)

acid, it was

of the P;;E:,9-ketoreductase

chromatographically: (7).

group is in the a-configuration

laboratory

the products

activities

had been measured

since PGr',@ reacted

the oIn our

of PGE 9-ketoreductase serologically

only O.$

with anti-Ptir', 2a'

with the anti-Ptib and since 2a

at least with monkey liver cytoplasmic the yields of Ptil?2(z, fractions, a product

approached

833, it was unlikely

Nevertheless,

(3).

toward PGe'28 were available, enzymatic

conversion

partially

purified

PSza

since the @-hydroxyl

measure

and kidney.

serologic

analyses

was

directed of the

of P&i3;1 to rtib'2by PGti g-ketoreductase

and are reported

here.

In addition,

group is in a major urinary

in the guinea pig (a), attempts were made to

enzymatic

cytoplasmic

since antibodies

2.B

from chicken heart were made with anti-

and anti-PGFzf

metabolite

that PGk’

production

and microsomal No

of Pc;rzB from PcX2 by

fractions of guinea pig liver

such enzymatic

activity was observed.

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1975 VOL. 9 NO. 4

PROSTAGLANDINS

MATERIALS The prostaglandins

were obtained

of the Upjohn Company, (210 Ci/mmole)

Kalamazoo,

and (3H) PGF2,

from New England

Nuclear

Immunogenic was synthesized 5 mg PGF28,

AND METHODS

Michigan.

(3H) PtiB2

(178 Ci/mmole)

Corp.,

conjuqates.

PGr'2B-human albumin of Bauminger

co). and 3.5 mg of N-hydroxysuccinimide. mixture was stirred

in a Vortex Mixer

room temperature.

The precipitated

formed was removed by centrifugation

transferred

the intermediate

(Miles Research

containing

Products

addition

the reaction

Division,

for 30 minutes at dicyclohexyl-urea

albumin-PGF28

that

and the supernatant

Elkhart,

opalescent

the reaction

mixture usually

subcutaneously

1975

The reaction

was quantitatively

this same buffer.

Immunization.

APRIL

(Eastman-Kodak

M

Indiana).

The

on addit.ion of

of about 0.5 ml of 5mM phosphate

against

(DMF)

to the albumin, was kept at 4O C for '2 hours.

(pH 7.5, 0.15 M NaCl), dialyzed

To

12.5 mg of human albumin

mixture, which becomes

the intermediate After

et al (9).

with 0.1 ml of DMF to 0.5 ml of cold 0.1

sodium bicarbonate

reaction

immunogen

in 0.1 ml of dimethylformamide

was added 3 mg of dicyclohexyl-carbodiimide

fluid containing

were purchased

Boston, Mass.

by the procedure

dissolved

from Dr. Udo Axen

buffer.

mixture was exhaustively During

this dialysis,

clarifies.

A single rabbit was immunized

at multiple conjugate

VOL. 9 NO. 4

sites with 3 mg of the human

in complete

Freund's

adjuvant.

PROSTAGLANDINS

Five weeks later the rabbit was boosted by the same route with the same dose.

Eight weeks later the rabbit was

again boosted with 3 mg of the conjugate, this time intramuscularly in the form of an emulsion with incomplete E'reund'sadjuvant.

The rabbit was bled each week after

the booster injections and the plasma (the blood was collected in EDTA) from the bleeding following the second boost was used in this study. Radioimmunoassay. as follows:

Radioimmunoassays were performed

For PGF2a analyses, 100 ~1 of rabbit antiserum

diluted 1:5000 in TRIS buffer (0.01 M Tris, pH 7.5, 0.14 M NaCl containing 0.1% gelatin) and 100 ~1 (3H) PGF2, (14,000 CPM) were incubated in the presence and absence of unlabeled prostaglandins for 1 hour at 37' in a total volume of 0.3 ml.

After addition of 100 ~1 of normal

rabbit serum (diluted l:lOO), 100 ~1 of goat anti-rabbity-globulin was added and the reaction mixture was incubated overnight at 2-4O C.

The antibody-bound (3H) PG was

collected as a precipitate by centrifugation at 1,000 x g for 30 minutes.

The precipitate was dissolved in 0.2 ml

of 0.1 N NaOH and counted in a modified Bray's solution (5 4 ppo,

100 g napthelene per liter of dioxane).

Of

the added 14,000 CPM, 5,200 CPM were precipitated with immune serum and about 150 CPM were precipitated by the same amount of non-immune rabbit serum. For PGF2S analyses, the same experimental conditions

534

APRIL

1975 VOL. 9 NO. 4

PROSTAGLANDINS

l,,?ere used except that 100 111 of the rabbit anti-P& diluted

1:X3

was used.

About

2:B

4-,000 CPM of the added

lLi,OOLiCPM of Fir (Pc;F,c plus POY,~) were precipitated this 0.5 $1 of immune plasma, while precipitated

mixture

plasma.

of ('H) PGF. ('H) PGr'2-+ (actually L_)?

of (jH) PW2o

(0.01

lui TRIS, $3 7.5,

After

adjustment

G.14

in 3.0 ml of TRIS buffer

M LiaCl containing

O.l$ gelatin).

15 mg of

of the pl to 10.2 with NaOH,

NaBHh was added and the reaction

This

mixture was kept at room

at which time 50 mg of pyruvic

for 2.5 hours,

acid was added.

a

and c3H) ~&'2~] was made as follows:

4 &Ci of ('H) PCS2 was diluted

temperature

18C to 200 CPM were

by L7.5 ~1 of non-immune

Preparation

by

('H) PGF2 preparation

at -10' and diluted appropriately

was stored

for use in the

radioimmunoassay. Measurement was measured 1 1mM NADPH,

of Enzymatic

in a reaction

or enzyme

bath

acetic acid before

APRIL

1975

O.l$ gelatin

solution

The pH was adjusted analysis

VOL. 9 NO. 4

and 0.14 M

of the pH to 12.8 with 0.1 N

of the diluted

for 5 min.

The reaction

of 1 ml of cold TRIS-HCl

containing

NaCl) and, after adjustment NaOH, incubation

chicken heart PGE

for 10 min. and the enzymatic

was stopped by addition

(0.01 M, pH 7.4

Enzyme activity

of 0.1 ml containing

fraction.

at 37’

mixtures were incubated

buffer

mixture

2 kg PGE2 and purified

9-ketoreductase

reaction

Activity.

in a boiling water

to pH 7.5

by radioimmunoassay.

with 1 N Analyses

PROSTAGLANDINS

of the enzyme reactions chromatography

for Pi;FzaV?~y thin layer

have confiriiledresults

for PGPz, by radioinmunoassay

obtained by analyses

without

chronatographic

separation (4). RESULTS A-ND DISCU5SIOid Although

the extent of cross-reaction

anti-P"k' depends " 2a course,

on the animal being immunized:

dose, and route of immunization,

toward PGF~, do recognize

this cross-reaction lo;&.

to PGY

has varied

Reduction PGk&

anti-PGPpa

that antibodies

with i?Gb

The mixture bound

than did (%I) PGP. Za'

(‘13)

did

PG~',,~. The binding L

and heteroloyous

(%I) prostaylandins

and are expressed

activity,

less effectively

increments

with

mixture

with the anti-PGP'2p curves by the homologous are shown in Figure bound by increments

so that their binding

be about the same when expressed

1

of

the same

profiles

as molecules

would

bound by

of antiserum.

The inhibition

data with the pure homologous

heterologous

prostaylandins,

in Pig. 2.

PGb'2B cross-reacts

536

in a mixture of

They are labeled to essentially

antisera. specific

as radioactivity

directed

2a'

The unresolved

of (%I) PGF2 bound more effectively than

a confiyuration.

made in our laboratory

of PtiE2 with NABH4 results

and PGE'2@.

directed

from as little as 0.2;b to

It was expected

toward PGP2B would cross-react

2cf

and the

antisera

the g-hydroxyl's

With several of the antisera

as much as

of PGF. with ;19

PGY2a and PGF2@,

are shown

with anti-PGk2a

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and

1975

about 27;

VOL. 9 NO. 4

PROSTAGLANDINS

Anti-PGF2,

Anti-PGFzB

/I

Figure 1.

Antiserum

Binding of (%I) PGF 2a (14,000

CPM) and a

PGFzB \14,OOO CPM) to mixture of (+-I) PGY~, and (31-1) anti-PGFza (left) and anti-PGk28 (right).

APRIL

1975

VOL. 9 NO. 4

537

PROSTAGLANDINS

and PGP

cross-reacts with anti-PGF 10%. PGE 2a 2 28 inhibits binding to an appreciable extent, 0.1s with

anti-PGPea and 1%

with anti-PGFeB, but the alkaline-

dehydration product (PGB2) reacts only slightly (Fig.2). Both of these antisera were used for radioimmunoassay of the PGF2 produced by enzymatic reduction of PGE~ by the 9-ketoreductase purified from chicken heart (3,4).

Because

with anti-PGP2B, in these 2 experiments residual substrate (PGE2) is converted to PGB~

of the cross-reaction of PGE

by alkaline treatment.

Thus, only the product (PGF2)

is being measured in the radioimmunoassay.

The results of

one such experiment are summarized in Table I;

the PGF2

levels were calculated from the standard curves shown in Fig. 2.

The inhibition of immunobinding observed for the

product with anti-PGF2a can either be accounted for by 0.320 But when the same sample 28' the inhibition can be accounted

kg of PGF2a or 17 .O cLgof PGE

is assayed with anti-PGF 28' for by 0.370 clgof PGF2, or by 0.041 (*gof PGF

28' As can be seen in Table I, large discrepancies exist

calculated from the 28 However, when inhibition obtained with the two antisera. in the concentrations of PGF

estimated as PGF2a the concentrations agree very well. The agreement in levels of PGr‘2a (or any immunoreactive material) when obtained by antisera of different serologic Specificities validates the radioimmunoassay procedures: number of such antisera, the identification.

538

less

the larger the

equivocal the

Nevertheless, even though most of the APRIL

1975

VOL. 9 NO. 4

PROSTAGLANDINS

Anti-PGF,#

Anti-PGFp,

0.1

0.0 I

1.0

IO

100

1000

Nanograms

Figure

2.

Inhibition

of (%)

P(;L"~~anti-PGr' C' 3

(top) and ('2) ptirzw anti-p&'.~a binding increnents

of PW

Experimental

APRIL

1975

(bottom) by

, PGk'23 and alkaline-treated '2a -./

conditions

VOL. 9 NO. 4

binding

are given in Materials

PtiE2 IBM!). and Methods.

539

PROSTAGLANDINS

product appears to be PGF2c, the serologic specificities of these two antisera are such that it is difficult to rule out the presence of lO$ or less of PGF product.

in the PGF 2 2f3 The stereoselectivity of other PGE g-keto-

reductases when assayed with anti-PGF can be assumed, 2a especially when yields of PGF2, approach 65% (3). However in the major metabolite found in guinea pig urine (8) and in a minor metabolite in rat urine (10) the $I-ketogroup has been reduced to a p-hydroxyl group.

We

attempted to look for PGF28 generated by microsomal and cytoplasmic fractions of guinea pig kidney and liver in the presence of PGE2 and NADH or NADPH.

When measured with

the two antisera, anti-PGF2a and anti-PGF no evidence 28' production was found; at least 908 (the limit for PGF2!3 of resolution with the antibodies used in this study) of the product was identified as PGF2,.

The cytoplasmic

fraction of guinea pig liver was also subjected to DWESephadex chromatography and each eluate fraction was assayed with both antisera (Fig.2) for reductase activity on PGE 2 in the presence and absence of NADPH.

The protein

and yields of PGF2a shown were calculated from

PGk

2a. standard inhibition curves obtained with both antisera.

Only one peak of PGE~ 9-ketoreductase activity was detected and the PGF,_ levels obtained with each antiserum were the cu

same.

NADPH as opposed to &DH

cofactor for this activity.

was

Neither

homogenates reduced PGE2 to PGl'2@. 540

the

more

the Other

APRIL

effective

kidney

or

ograns

1975

of

liver the

VOL. 9 NO. 4

PROSTAGLANDINS

0.6

0.6 I.5

6

g E 0.4

s ‘I: x

1.0 0.2

20

60

40 Fraction

60

Numbrr

Figure 3. DSAE-Sephadex chromatography of a guinea pig liver cytoplasmic preparation: six g fresh guinea pig liver in 18 ml of ice cold phosphate buffer (0.092 k potassium phosphate, pH 7.3 plus 0.1 mM dithiothreitol) was macerated in a Sorvall homogenizer for 4 min. After centrifugation at 10,000 xg for 20 min. the supernatant fluid was centrifuged at 100,000 xg for 1 hour. Five ml of this supernatant fluid was applied to a DEAESephadex column (1.6 x 22 cm) that had been previously equilibrated with the phosphate buffer. After washing with 25 ml of the same phosphate buffer, the column was eluted with 1.60 ml of phosphate buffer containing a gradient of KC1 from 0 to 1 M. Fractions of 1.8 ml were collected. For assay of PGE g-ketoreductase activity the reaction mixtures contained 2 IJ.gPGE2, 1 r@l NADPH, 0.092 M phosphate buffer, pH 7.3, and 30 ~1 of aliquot from DEAE-Sephadex column in a volume of 0.1 ml. Amount measured by anti-PGP (0) or anti-PGE'29 (a)* of PGF A280 (87; KC1 gradient (_f?

APRIL

1975

VOL. 9 NO. 4

541

PROSTAGLANDINS

TABLE I CHARACTERIZATION OF PGF2 PRODUCED BY ENZYMATIC CONVERSION OF PGE2.*

PGF2, Antiserum

!-q/ReactionMixture

Anti-PGF20

0.320

Anti-PGY2B

0.370

*PGE2 (2

pGF2B W/Reaction Mixture 17.0 0.041

P-g), 1.0 mM NADPH and purified chicken heart

PGE 9-ketoreductase were incubated in a total volume of 0.1 ml.

After 10 minutes at 37O, 0.9 ml TRIS

buffer (0.01 M TRIS HCl pH 7.4,

0.14 M NaCl) was added After

and the pH was brought to 12.8 with 0.1 N NaOH.

immersion in a boiling water bath for 5 minutes, the PH was adjusted to pH 7.5

with 1N acetic acid.

In this

experiment for radioimmunoassay with anti-PGF2, and anti-PGF28 (Fig.2). 100, 10, 1.0 and 0.1 1-11 (added as 100 ~1 of the appropriate dilutions) were analyzed. Controls containing PGE2 plus NADPH and enzyme plus NADPH are treated identically.

They contain no

immunoreactive material.

542

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1975

VOL. 9 NO. 4

PROSTAGLANDINS

guinea pig may have such an enzyme, or as stated by Samuelsson (ll), other metabolic pathways reduction

leading to

of the 9-keto group of PGE may be present.

REFERENCES 1.

Hamberg, M, and Israelsson, U.

Metabolism of

Prostaglandin E2 in Guinea Pig Liver. of Seven Metabolites.

I. Identification

J. Biol. Chem. 245: 5107-5114

(1970). 2.

Leslie, C.A. and Levine, L. Evidence for the Presence of a Prostaglandin E2 9-ketoreductase in Rat Organs. Biochem. Biophys. Research. Commun. z:

3.

717-724 (1973).

Lee, S.C. and Levine, L. Prostaglandin Metabolism I. Cytoplasmic NADPH-Dependent and Microsomal NADHDependent Prostaglandin E 9-ketoreductase Activities in Monkey and Pigeon Tissues.

J. Biol. Chem. &:

1369-1375 (1974). 4.

Lee, S.C. and Levine, L. Purification and Regulatory Properties of Chicken Heart Prostaglandin E 9-ketoreductase.

5.

J. Biol. Chem. (1975) in press.

Kaplan, L., Lee, S.C. and Levine, L. Partial Purification and Some Properties of Human Erythrocyte Prostaglandin 9-ketoreductase and 15-hydroxyprostaglandin Dehydrogenase.

Arch. Biochem. and Biophys. (1975),

in press. 6.

Lee, S.C., Pong, S.S., Katzen, D., Wu, K.Y. and Levine, L.

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Distribution of Prostaglandin E 9-

1975 VOL. 9 NO. 4

543

PROSTAGLANDINS

ketoreductase and Types I and II 15-hydroxyprostaglandin Dehydrogenase in Swine Kidney Medulla and Cortex. Biochemistry -14: 7.

142-145 (1975).

Hensby, C.N. Reduction of Prostaglandin E2 to Prostaglandin F2o by an Enzyme in Sheep Blood. Biophys. Acta 348:

8.

145-154

Biochem.

(1974).

Hamberg, M. and Samuelsson, B.

The Structure of a

Urinary Metabolite of Prostaglandin E2 in the Guinea pig. 9.

Biochem. Biophys. Res. Commun. a:

22-27 (1969).

Bauminger, S., Zor, U., and Lindner, H.R. Radioimmunological Assay of Prostaglandin Synthetase

10.

(1973)

Prostaglandins 4:

Green, K.

Metabolism of PGE2 in the Rat.

Biochemistry 10: 11.

313-324

Activity.

1072-1086

.

(lg71),

Samuelsson, B. Biosynthesis of Prostaglandins. Federation Proceedings Jl_: 1442-1450 (1972).

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1975

VOL. 9 ND. 4

Stereospecificity of enzymatic reduction of prostaglandin E2 to F2alpha.

Antibodies directed toward PGF2beta were prepared in rabbits. The serologic specificity of the immune reaction was determined by inhibition of sodium ...
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