- Email: [email protected]
[31] A receptor for prostaglandin F2α from corpora lutea
202
ENZYMES
AND
RECEPTORS:
PURIFICATION
AND
ASSAY
[31]
T h e r e are few previous studies in which the subceUular distribution of PG r e c e p t o r s has been investigated. 1-4 In these studies, h o w e v e r , neither the isolation o f all the subcellular organelles nor the critical a s s e s s m e n t of the intrinsic nature of intracellular organelles binding was accomplished. Acknowledgments The excellent help of Mr. F. R. Carman, Jr. in binding studies is gratefully acknowledged. This work was supported by Grants HD09577 and HD15177 from the National Institutes of Health.
[31] A Receptor By
for Prostaglandin Corpora Lutea SVEN
Fz~ f r o m
HAMMARSTROM
Prostaglandin F2~ is a luteolytic h o r m o n e in a n u m b e r of subprimate m a m m a l s . 1 It is p r o d u c e d b y the uterus at the end of the estrous cycle and is t r a n s p o r t e d via a venoarterial shunt m e c h a n i s m to the corpus luteum of the ovary. 2 Interaction of PGF2~ with a r e c e p t o r in the corpus luteum T M leads to a decrease in luteotropic h o r m o n e - s t i m u l a t e d cyclic A M P levels in the ovary. This reduces p r o g e s t e r o n e secretion, causes corpus luteum regression, and initiates a new estrous cycle. 4 D e t e r m i n a t i o n of PGF2~ R e c e p t o r s P r e p a r a t i o n o f T r i t i u m - L a b e l e d PGF2~. 5 Prostaglandin E , (6 mg, dissolved in 0.5 ml of methanol and kept at 0 °) is m i x e d with NaBZH4 (1 mg, ca 8 C i / m m o l , dissolved in 0.5 ml of methanol at 0 °) and left at 0 ° for 15 min and at 22 ° for 60 min. The mixture is then chilled to 0 °, and 5 mg of unlabeled N a B H , are added and allowed to react u n d e r the s a m e conditions. T w o v o l u m e s of w a t e r are added plus HC1 t o give a p H of 2 - 3 . The products are extracted with diethyl ether and separated by reversedphase partition c h r o m a t o g r a p h y on 4.5 g of Hyflo Supercel c o a t e d with 30 ml of c h l o r o f o r m - i s o o c t a n o l , 1 : 1 ( v / v ) equilibrated with 300 ml o f mobile p h a s e ( m e t h a n o l - w a t e r , 57:93, v / v ) . The elution v o l u m e s for
E.W.Horton and N. L. Poyser, Physiol. Rev. 56, 595-651 (1976). 2 j. R. Goding, J. A. McCracken and D. T. Baird, J. Endocrinol. 39, 37 (1967). a W. S. Powell, S. HammarstrOm, and B. Samuelsson, Eur. J. Biochem. 41, 103 (1974). aa W. S. Powell, S. HammarstrOm, and B. Samuelsson, Eur. J. Biochem. 56, 73 (1975). 4 j. p. Thomas, L. J. Dorflinger, and H. R. Behrman, Proc. Natl. Acad. Sci. U.S.A. 75, 1344 (1978). s E. GranstrOm and B. Samuelsson, Eur. J. Biochem. 10, 411 (1969). METHODS IN ENZYMOLOGY, VOL. 86
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181986-8
[31]
PROSTAGLANDIN Fz~ RECEPTOR
203
[9a-aH]PGF2~ and [9/3-aH]PGF~.~ are 78-100 and 100-150 ml, respectively. 5 Alternatively, reversed-phase high-performance liquid chromatography can be used to separate the diastereoisomeric products.6 The purity of the products can be determined by thin-layer chromatography (silica gel G; diethyl ether-methanol, 97:3, v/v; R e for PGF2~ methyl ester -- 0.25) or by gas-liquid radiochromatography (C value on SE-30 for PGFz~ methyl ester, tris-O-trimethylsilyl derivative is 24.5). To determine the specific radioactivity, a known amount (e.g., 0.5/zCi) of the labeled product is mixed with a known amount of deuterium-labeled PGF2~ (e.g., 3 nmol of [2H4]PGF2~). The degree of isotope dilution caused by the addition of radioactive product is determined by multiple ion analysis of the methyl ester, tri-O-acetyl derivative. 7 Membrane-Bound Receptor Preparations. Corpora lutea from sheep or bovine corpora lutea, estimated to be 6-16 days in the estrous cycle, are dissected free from ovarian stromal tissue while still frozen. The luteal tissue is thawed in 4 volumes of 0.01 M Tris-HC1 buffer, pH 7.5 (4°) and homogenized (20 strokes) using a Potter-Elvehjem-type homogenizer. After centrifugation (1000 g, 20 min, 4°) the supernatant is passed through four layers of gauze bandage and recentrifuged (35,000 g, 45 rain, 4°). The sediment is resuspended in Tris-HC1 buffer (1.25 ml per gram of corpus luteum) and used for binding experiments. Binding Assays. Tritium-labeled PGFz~ is dissolved in Tris-HCl buffer, pH 7.5 at concentrations between 0.2 and 2 /~g/ml. Unlabeled PGF2~ is dissolved in ethanol at a concentration of 1.5 mg/ml. These solutions can be stored at - 2 0 ° for at least a month. Generally, four or five different concentrations of [3H]PGF~, in the range indicated above, are prepared. Separation of macromolecule-bound and free radioactivity is performed on 2-ml columns of Sephadex G-50, fine. The gel is swollen in 0.01 M Tris-HC1 buffer, pH 7.5, and packed in 2-ml disposable plastic syringes fitted with filter paper at the bottom. Filter papers are also placed on top of the columns, which are washed with a few milliliters of buffer prior to use and kept at 4 °. The columns can be prepared 8-12 hr in advance, provided they are washed shortly before use. Resuspended receptor preparation (0.2 ml), [aH]PGF2~ (10/~l) and ethanol (3.5 ~1) or unlabeled PGF2~ (3.5 ~l) are incubated with gentle shaking at 23 ° for 120 min. The incubation mixtures are put on ice, diluted with 0.2 ml of cold buffer, and immediately applied to Sephadex columns. As soon as the samples have entered the gel, 0.01 M Tris-HC1 buffer, pH 7.5 (0.85 ml) is added. The combined eluate (0.95 ml; "macromolecule 0 Column: C~s Nucleosil (250 x 4.6 mm); solvent: methanol-water, 6:4 (v/v) plus 0.01% acetic acid (1 ml/min); retention times: PGF2~, 25 min; PGF~, 30 min. 7 K. Gr6en, E. Granstr6m, B. Samuelsson, and U. Ax6n, Anal. Biochem. 54, 434 (1975).
204
ENZYMES AND RECEPTORS" PURIFICATION AND ASSAY
[31]
1 0.3
r'~ O ft. 0.2-
,u.,
0.1 D O
1'o
2'0
3o
FIo. 1. Scatchard plots of binding data at equilibrium for [9B-~H]prostaglandin F~ ( 0 - - 0 ) (2, 4, 10, and 20 ng) and [9¢l-~H]prostaglandin F~, (2, 4, 10, and 20 ng) plus prostaglandin E2 (51)1)ng) (©--O). Volume, 0.2 ml. From Powell et al. 3
bound PGF2~") is collected in a scintillation vial. The column is then eluted with an additional 3.5 ml of buffer, and the second eluate is collected in the same way as the first. The radioactivity in the two fractions (bound and free PGFz~, respectively) is determined by liquid scintillation counting after the addition of 10 ml of Instagel to each scintillation vial. Binding assays are done in duplicates to quadruplicates for total binding (no unlabeled P G F ~ added) and in duplicates for nonspecific binding (presence of unlabeled PGF2~). Data on specific binding (total minus nonspecific binding) are plotted according to Scatchard a for determination of receptor concentration and dissociation constant (Fig. 1). Properties of P G F ~ R e c e p t o r s
Affinity and Specificity. Binding of PGF2~ to membrane preparations from corpora lutea is saturable and reversible, and no metabolic alteration of the ligand takes place under the assay conditions described above. Fatty acids, steroids, nucleotides, luteotropic and follicle-stimulating hora G. Scatchard, A n n . N . Y . A c a d . S c i . 51, 660 (1949).
[31]
PROSTAGLANDIN Fz,~ RECEPTOR
205
mone do not interfere with the binding reaction. T M Dissociation constants determined by Scatchard plot analyses of binding data are of the order of 50 nM for bovine and 100 nM for ovine preparations. A somewhat lower value (28 nM) is obtained from the rate constants for association and dissociation. 3a The affinities of other prostaglandins and prostaglandin analogs can be determined using unlabeled compounds. Binding assays for Scatchard plot analyses are performed as described above. In one set of experiments, various concentrations of tritium-labeled PGF2~ are incubated in the presence or the absence of unlabeled PGF2~ in the usual way. In other sets of experiments a fixed concentration of the unlabeled prostaglandin or prostaglandin analog is added in addition to [3H]PGFz~ (plus or minus
TABLE I EFFECTS OF STRUCTURAL MODIFICATIONS OF PROSTAGLANDIN F2~ ON THE DISSOCIATION CONSTANT FOR BINDING TO BOVINE CORPUS LUTEUM RECEPTOR
Structural modification CO~H CH2CO2H 5,6-CH~CH 9a-OH
~ ~ ~ ~
CH2OH CO2H 5,6-CH2CH2 9fl-OH a 9-oxo 1 lct-OH ) 1 l-oxo 1 3 , 1 4 - C H ~ C H ~ 13,14-CH2CH2 13,14-C~C 15-OH ) 15-oxo 15-epi-OH ~ 15-OAc 15-OMe 15-H ) 15-Me 15-Pentyl > 15-(1,1-dimethylpentyl) 16-Butyl ) 16-(m-chlorophenoxy) 16-(1-butenyl) 17-Propyl ) 17-phenyl 20-H ) 20-ethyl Inversion o f all a s y m m e t r i c c e n t e r s (carbons 8, 9, 11, 12, and 15) c Inversion of all a s y m m e t r i c centers except carbon-15 c
Increase in Kd (Kd analog/Kd PGF2~) 108 52 40 146 54 9.2 4 4.2 180 12 9.2 1.2 1.3 1.7 0.40 3.4 0.54 2.4 6200 30
a Ovine corpora lutea. b The reference compound is 15-methyl-PGE2 m e t h y l ester. c The reference compound is 13,14-didehydro-PGF2~.
206
ENZYMES AND RECEPTORS: PURIFICATION AND ASSAY
it !
I
9tucosernlnldase (soluble)
I
(soluble)
2
I f, CYr~uu~trnec
$
d~ ~o5-
1
0
[31]
~- Glucuronidase
, I
t !t
cytochrorne c reductase
,
35t
6-
~..~ P Prostaglandin 3
2
6p
35b
35b r~ I
dehydrogenase
6p 0 0
50
100 0 Protein in fraction (% total)
50
100
FIG. 2. Distribution patterns of prostaglandin F ~ receptor and marker enzymes in fractions obtained from homogenates of bovine corpora lutea by differential centrifugation. Designations: 6p, 80p, 270p indicate 6,000, 80,000, and 270,000 g pellets; 35b, 35t indicate bottom and top layers of 35,000 g pellet; 270s, 270,000 g supernatant. From Powell e t al. 9
unlabeled PGF~). The unlabeled compound under investigation causes a decrease in the apparent dissociation constant, determined by Scatchard plots (Fig. 1). The dissociation constant for the unlabeled compound is calculated from the apparent dissociation constant, the dissociation constant for PGF2~, and the concentration of the unlabeled compound. Table
2-
A
B
!
o
.,-r-n--t-'N__ 5uccinate dehydrogenase
10-
5-
~o ._®>
7 5'-Nucleotfdase1~3 4 56
8
__F] _ o r
4
10t 80p 10
v
n
i
0
!
i
50
100
i
Protein in fractioo
!
0
!
50
100
protein recovered from gradient
(°1o total)
(°/o total)
C
0.4 1.255
•
-
-0.3
1.20-
~~
E c 8
E
'0'2~
1.15-
j~
-0.1 1,10
-
o
1;
~ ~ume
~
o
(m()
FIG. 3. Distribution patterns of prostaglandin F ~ and marker enzymes in fractions obtained from homogenates of bovine corpora lutea by differential (A) and density gradient (B)
centrifugations. (C) Density gradient fractionation of the top layer of the 10,0011 g pellet (10t) obtained by differential centrifugation;
208
ENZYMES AND RECEPTORS: PURIFICATION AND ASSAY
[31]
TABLE II PHYSIOCHEMICAL PROPERTIES OF TRITON X-100-SOLUBILIZED PROSTAGLANDIN F2a RECEPTORa
Rs (A)
s20.w x 101~ (cm/sec • dyne)
v (cm3/g)
% Triton (w/w)
63
4.6
0.78
26
MWcomple x (g/mol) 144000
Triton bound MWreceptor (mol/mol (g/mol) complex) 107000
58
fifo 1.6
a From Kyld~n and Hammarstr6m. 1°
I summarizes the effects of various structural modifications of PGF~, on the dissociation constant. Subcellular Distribution. 9 Subcellular fractions prepared from homogenates of corpora lutea have been used to compare the distributions of marker enzymes with that of PGF2~ receptor (Fig. 2). The results show that the distribution of the receptor is the same as that of the plasma membrane marker, 5'-nucleotidase. On the other hand, marker enzymes for mitochondria (succinate dehydrogenase), endoplasmic reticulum (NADPH-cytochrome c reductase) and lysosomes (soluble N-acetyl-flglucosaminidase and ~-glucuronidase) have distinctly different distribution patterns. The receptor is purified 6-fold (over the homogenate) in the top layer of the 35,000 g sediment (fraction 35t in Fig. 2). A 32-fold purification can be achieved by a combination of differential and sucrose density gradient centrifugations (Fig. 3). In this case, the top layer of the 10,000 g sediment (10t; 4-fold purification of receptor) is fractionated on a sucrose density gradient. Fraction 5 contains 32-fold purified receptor and 120-fold purified 5'-nucleotidase, suggesting that the enzyme is more stable than the receptor to purification. Physical Properties. 10The binding of PGFz~ to the receptor is inhibited by low concentrations of detergents. However, preformed hormone-receptor complex is stable to much higher detergent concentrations and can be solubilized by, e.g., deoxycholate or Triton X-100. By using tritium-labeled PGFz~, a convenient marker for the hormone-receptor complex is obtained. The molecular size of the complex is determined by a combination of Sepharose 6B chromatography and density gradient centrifugations on sucrose-H20 and sucrose-ZH20 gradients. The former analysis gives the Stokes' radius, and the latter gives the sedimentation coefficient 9 W. S. PoweU, S. HammarstrOm, and B. Samuelsson, Eur. J. Biochem. 61, 605 (1976). lo U. Kyld~n and S. Hammarstr0m, Eur. J. Biochem. 109, 489 (1980).
[31]
PROSTAGLANDIN F2,~ RECEPTOR
209
and the partial specific volume. F r o m these parameters, the molecular weight o f the receptor and the amount of detergent bound to the r e c e p t o r can be calculated (Table II). 1° Acknowledgment The work was supported by a grant from the World Health Organization.
ENZYMES
AND
RECEPTORS:
PURIFICATION
AND
ASSAY
[31]
T h e r e are few previous studies in which the subceUular distribution of PG r e c e p t o r s has been investigated. 1-4 In these studies, h o w e v e r , neither the isolation o f all the subcellular organelles nor the critical a s s e s s m e n t of the intrinsic nature of intracellular organelles binding was accomplished. Acknowledgments The excellent help of Mr. F. R. Carman, Jr. in binding studies is gratefully acknowledged. This work was supported by Grants HD09577 and HD15177 from the National Institutes of Health.
[31] A Receptor By
for Prostaglandin Corpora Lutea SVEN
Fz~ f r o m
HAMMARSTROM
Prostaglandin F2~ is a luteolytic h o r m o n e in a n u m b e r of subprimate m a m m a l s . 1 It is p r o d u c e d b y the uterus at the end of the estrous cycle and is t r a n s p o r t e d via a venoarterial shunt m e c h a n i s m to the corpus luteum of the ovary. 2 Interaction of PGF2~ with a r e c e p t o r in the corpus luteum T M leads to a decrease in luteotropic h o r m o n e - s t i m u l a t e d cyclic A M P levels in the ovary. This reduces p r o g e s t e r o n e secretion, causes corpus luteum regression, and initiates a new estrous cycle. 4 D e t e r m i n a t i o n of PGF2~ R e c e p t o r s P r e p a r a t i o n o f T r i t i u m - L a b e l e d PGF2~. 5 Prostaglandin E , (6 mg, dissolved in 0.5 ml of methanol and kept at 0 °) is m i x e d with NaBZH4 (1 mg, ca 8 C i / m m o l , dissolved in 0.5 ml of methanol at 0 °) and left at 0 ° for 15 min and at 22 ° for 60 min. The mixture is then chilled to 0 °, and 5 mg of unlabeled N a B H , are added and allowed to react u n d e r the s a m e conditions. T w o v o l u m e s of w a t e r are added plus HC1 t o give a p H of 2 - 3 . The products are extracted with diethyl ether and separated by reversedphase partition c h r o m a t o g r a p h y on 4.5 g of Hyflo Supercel c o a t e d with 30 ml of c h l o r o f o r m - i s o o c t a n o l , 1 : 1 ( v / v ) equilibrated with 300 ml o f mobile p h a s e ( m e t h a n o l - w a t e r , 57:93, v / v ) . The elution v o l u m e s for
E.W.Horton and N. L. Poyser, Physiol. Rev. 56, 595-651 (1976). 2 j. R. Goding, J. A. McCracken and D. T. Baird, J. Endocrinol. 39, 37 (1967). a W. S. Powell, S. HammarstrOm, and B. Samuelsson, Eur. J. Biochem. 41, 103 (1974). aa W. S. Powell, S. HammarstrOm, and B. Samuelsson, Eur. J. Biochem. 56, 73 (1975). 4 j. p. Thomas, L. J. Dorflinger, and H. R. Behrman, Proc. Natl. Acad. Sci. U.S.A. 75, 1344 (1978). s E. GranstrOm and B. Samuelsson, Eur. J. Biochem. 10, 411 (1969). METHODS IN ENZYMOLOGY, VOL. 86
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181986-8
[31]
PROSTAGLANDIN Fz~ RECEPTOR
203
[9a-aH]PGF2~ and [9/3-aH]PGF~.~ are 78-100 and 100-150 ml, respectively. 5 Alternatively, reversed-phase high-performance liquid chromatography can be used to separate the diastereoisomeric products.6 The purity of the products can be determined by thin-layer chromatography (silica gel G; diethyl ether-methanol, 97:3, v/v; R e for PGF2~ methyl ester -- 0.25) or by gas-liquid radiochromatography (C value on SE-30 for PGFz~ methyl ester, tris-O-trimethylsilyl derivative is 24.5). To determine the specific radioactivity, a known amount (e.g., 0.5/zCi) of the labeled product is mixed with a known amount of deuterium-labeled PGF2~ (e.g., 3 nmol of [2H4]PGF2~). The degree of isotope dilution caused by the addition of radioactive product is determined by multiple ion analysis of the methyl ester, tri-O-acetyl derivative. 7 Membrane-Bound Receptor Preparations. Corpora lutea from sheep or bovine corpora lutea, estimated to be 6-16 days in the estrous cycle, are dissected free from ovarian stromal tissue while still frozen. The luteal tissue is thawed in 4 volumes of 0.01 M Tris-HC1 buffer, pH 7.5 (4°) and homogenized (20 strokes) using a Potter-Elvehjem-type homogenizer. After centrifugation (1000 g, 20 min, 4°) the supernatant is passed through four layers of gauze bandage and recentrifuged (35,000 g, 45 rain, 4°). The sediment is resuspended in Tris-HC1 buffer (1.25 ml per gram of corpus luteum) and used for binding experiments. Binding Assays. Tritium-labeled PGFz~ is dissolved in Tris-HCl buffer, pH 7.5 at concentrations between 0.2 and 2 /~g/ml. Unlabeled PGF2~ is dissolved in ethanol at a concentration of 1.5 mg/ml. These solutions can be stored at - 2 0 ° for at least a month. Generally, four or five different concentrations of [3H]PGF~, in the range indicated above, are prepared. Separation of macromolecule-bound and free radioactivity is performed on 2-ml columns of Sephadex G-50, fine. The gel is swollen in 0.01 M Tris-HC1 buffer, pH 7.5, and packed in 2-ml disposable plastic syringes fitted with filter paper at the bottom. Filter papers are also placed on top of the columns, which are washed with a few milliliters of buffer prior to use and kept at 4 °. The columns can be prepared 8-12 hr in advance, provided they are washed shortly before use. Resuspended receptor preparation (0.2 ml), [aH]PGF2~ (10/~l) and ethanol (3.5 ~1) or unlabeled PGF2~ (3.5 ~l) are incubated with gentle shaking at 23 ° for 120 min. The incubation mixtures are put on ice, diluted with 0.2 ml of cold buffer, and immediately applied to Sephadex columns. As soon as the samples have entered the gel, 0.01 M Tris-HC1 buffer, pH 7.5 (0.85 ml) is added. The combined eluate (0.95 ml; "macromolecule 0 Column: C~s Nucleosil (250 x 4.6 mm); solvent: methanol-water, 6:4 (v/v) plus 0.01% acetic acid (1 ml/min); retention times: PGF2~, 25 min; PGF~, 30 min. 7 K. Gr6en, E. Granstr6m, B. Samuelsson, and U. Ax6n, Anal. Biochem. 54, 434 (1975).
204
ENZYMES AND RECEPTORS" PURIFICATION AND ASSAY
[31]
1 0.3
r'~ O ft. 0.2-
,u.,
0.1 D O
1'o
2'0
3o
FIo. 1. Scatchard plots of binding data at equilibrium for [9B-~H]prostaglandin F~ ( 0 - - 0 ) (2, 4, 10, and 20 ng) and [9¢l-~H]prostaglandin F~, (2, 4, 10, and 20 ng) plus prostaglandin E2 (51)1)ng) (©--O). Volume, 0.2 ml. From Powell et al. 3
bound PGF2~") is collected in a scintillation vial. The column is then eluted with an additional 3.5 ml of buffer, and the second eluate is collected in the same way as the first. The radioactivity in the two fractions (bound and free PGFz~, respectively) is determined by liquid scintillation counting after the addition of 10 ml of Instagel to each scintillation vial. Binding assays are done in duplicates to quadruplicates for total binding (no unlabeled P G F ~ added) and in duplicates for nonspecific binding (presence of unlabeled PGF2~). Data on specific binding (total minus nonspecific binding) are plotted according to Scatchard a for determination of receptor concentration and dissociation constant (Fig. 1). Properties of P G F ~ R e c e p t o r s
Affinity and Specificity. Binding of PGF2~ to membrane preparations from corpora lutea is saturable and reversible, and no metabolic alteration of the ligand takes place under the assay conditions described above. Fatty acids, steroids, nucleotides, luteotropic and follicle-stimulating hora G. Scatchard, A n n . N . Y . A c a d . S c i . 51, 660 (1949).
[31]
PROSTAGLANDIN Fz,~ RECEPTOR
205
mone do not interfere with the binding reaction. T M Dissociation constants determined by Scatchard plot analyses of binding data are of the order of 50 nM for bovine and 100 nM for ovine preparations. A somewhat lower value (28 nM) is obtained from the rate constants for association and dissociation. 3a The affinities of other prostaglandins and prostaglandin analogs can be determined using unlabeled compounds. Binding assays for Scatchard plot analyses are performed as described above. In one set of experiments, various concentrations of tritium-labeled PGF2~ are incubated in the presence or the absence of unlabeled PGF2~ in the usual way. In other sets of experiments a fixed concentration of the unlabeled prostaglandin or prostaglandin analog is added in addition to [3H]PGFz~ (plus or minus
TABLE I EFFECTS OF STRUCTURAL MODIFICATIONS OF PROSTAGLANDIN F2~ ON THE DISSOCIATION CONSTANT FOR BINDING TO BOVINE CORPUS LUTEUM RECEPTOR
Structural modification CO~H CH2CO2H 5,6-CH~CH 9a-OH
~ ~ ~ ~
CH2OH CO2H 5,6-CH2CH2 9fl-OH a 9-oxo 1 lct-OH ) 1 l-oxo 1 3 , 1 4 - C H ~ C H ~ 13,14-CH2CH2 13,14-C~C 15-OH ) 15-oxo 15-epi-OH ~ 15-OAc 15-OMe 15-H ) 15-Me 15-Pentyl > 15-(1,1-dimethylpentyl) 16-Butyl ) 16-(m-chlorophenoxy) 16-(1-butenyl) 17-Propyl ) 17-phenyl 20-H ) 20-ethyl Inversion o f all a s y m m e t r i c c e n t e r s (carbons 8, 9, 11, 12, and 15) c Inversion of all a s y m m e t r i c centers except carbon-15 c
Increase in Kd (Kd analog/Kd PGF2~) 108 52 40 146 54 9.2 4 4.2 180 12 9.2 1.2 1.3 1.7 0.40 3.4 0.54 2.4 6200 30
a Ovine corpora lutea. b The reference compound is 15-methyl-PGE2 m e t h y l ester. c The reference compound is 13,14-didehydro-PGF2~.
206
ENZYMES AND RECEPTORS: PURIFICATION AND ASSAY
it !
I
9tucosernlnldase (soluble)
I
(soluble)
2
I f, CYr~uu~trnec
$
d~ ~o5-
1
0
[31]
~- Glucuronidase
, I
t !t
cytochrorne c reductase
,
35t
6-
~..~ P Prostaglandin 3
2
6p
35b
35b r~ I
dehydrogenase
6p 0 0
50
100 0 Protein in fraction (% total)
50
100
FIG. 2. Distribution patterns of prostaglandin F ~ receptor and marker enzymes in fractions obtained from homogenates of bovine corpora lutea by differential centrifugation. Designations: 6p, 80p, 270p indicate 6,000, 80,000, and 270,000 g pellets; 35b, 35t indicate bottom and top layers of 35,000 g pellet; 270s, 270,000 g supernatant. From Powell e t al. 9
unlabeled PGF~). The unlabeled compound under investigation causes a decrease in the apparent dissociation constant, determined by Scatchard plots (Fig. 1). The dissociation constant for the unlabeled compound is calculated from the apparent dissociation constant, the dissociation constant for PGF2~, and the concentration of the unlabeled compound. Table
2-
A
B
!
o
.,-r-n--t-'N__ 5uccinate dehydrogenase
10-
5-
~o ._®>
7 5'-Nucleotfdase1~3 4 56
8
__F] _ o r
4
10t 80p 10
v
n
i
0
!
i
50
100
i
Protein in fractioo
!
0
!
50
100
protein recovered from gradient
(°1o total)
(°/o total)
C
0.4 1.255
•
-
-0.3
1.20-
~~
E c 8
E
'0'2~
1.15-
j~
-0.1 1,10
-
o
1;
~ ~ume
~
o
(m()
FIG. 3. Distribution patterns of prostaglandin F ~ and marker enzymes in fractions obtained from homogenates of bovine corpora lutea by differential (A) and density gradient (B)
centrifugations. (C) Density gradient fractionation of the top layer of the 10,0011 g pellet (10t) obtained by differential centrifugation;
208
ENZYMES AND RECEPTORS: PURIFICATION AND ASSAY
[31]
TABLE II PHYSIOCHEMICAL PROPERTIES OF TRITON X-100-SOLUBILIZED PROSTAGLANDIN F2a RECEPTORa
Rs (A)
s20.w x 101~ (cm/sec • dyne)
v (cm3/g)
% Triton (w/w)
63
4.6
0.78
26
MWcomple x (g/mol) 144000
Triton bound MWreceptor (mol/mol (g/mol) complex) 107000
58
fifo 1.6
a From Kyld~n and Hammarstr6m. 1°
I summarizes the effects of various structural modifications of PGF~, on the dissociation constant. Subcellular Distribution. 9 Subcellular fractions prepared from homogenates of corpora lutea have been used to compare the distributions of marker enzymes with that of PGF2~ receptor (Fig. 2). The results show that the distribution of the receptor is the same as that of the plasma membrane marker, 5'-nucleotidase. On the other hand, marker enzymes for mitochondria (succinate dehydrogenase), endoplasmic reticulum (NADPH-cytochrome c reductase) and lysosomes (soluble N-acetyl-flglucosaminidase and ~-glucuronidase) have distinctly different distribution patterns. The receptor is purified 6-fold (over the homogenate) in the top layer of the 35,000 g sediment (fraction 35t in Fig. 2). A 32-fold purification can be achieved by a combination of differential and sucrose density gradient centrifugations (Fig. 3). In this case, the top layer of the 10,000 g sediment (10t; 4-fold purification of receptor) is fractionated on a sucrose density gradient. Fraction 5 contains 32-fold purified receptor and 120-fold purified 5'-nucleotidase, suggesting that the enzyme is more stable than the receptor to purification. Physical Properties. 10The binding of PGFz~ to the receptor is inhibited by low concentrations of detergents. However, preformed hormone-receptor complex is stable to much higher detergent concentrations and can be solubilized by, e.g., deoxycholate or Triton X-100. By using tritium-labeled PGFz~, a convenient marker for the hormone-receptor complex is obtained. The molecular size of the complex is determined by a combination of Sepharose 6B chromatography and density gradient centrifugations on sucrose-H20 and sucrose-ZH20 gradients. The former analysis gives the Stokes' radius, and the latter gives the sedimentation coefficient 9 W. S. PoweU, S. HammarstrOm, and B. Samuelsson, Eur. J. Biochem. 61, 605 (1976). lo U. Kyld~n and S. Hammarstr0m, Eur. J. Biochem. 109, 489 (1980).
[31]
PROSTAGLANDIN F2,~ RECEPTOR
209
and the partial specific volume. F r o m these parameters, the molecular weight o f the receptor and the amount of detergent bound to the r e c e p t o r can be calculated (Table II). 1° Acknowledgment The work was supported by a grant from the World Health Organization.