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
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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
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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
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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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