the porcine lh/hcg receptor · 2001-07-07 · 10689 . 10690 the porcine lhihcg receptor frozen...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 19, Issue of September 5, pp. 10689-10697,1985 Printed in U.S.A.

The Porcine LH/hCG Receptor CHARACTERIZATION AND PURIFICATION*

(Received for publication, December 6, 1984, and in revised form, March 3, 1985)

Jayantha Wimalasena$Q, Pamela Moore$, John P. Wiebef, John Abel, Jr.4, and Thomas T. Chen$ From the $Department of Zoology, University of Tennessee, Knoxville, Tennessee 37916 and the §Hormonal Regulatory Mechanisms Laboratory, Department of Zoology, University of Western Ontario, London, Ontario N6A 5B7, Canada

Porcine luteal LH/hCG receptor (LH/hCG R) was solubilized with 70-80% recovery from the crude plasma membrane fraction by Triton X-100 in the presence of 25% glycerol and protease inhibitors. The solubilized receptor maintained 90% of original activity at -60 "C for 90 days. Equilibrium association constant (&) values of 1.92, 2.22, and 2.03 X 10" "' were observed for the whole homogenate, plasma membrane fraction, and solubilized LH/hCG R preparations, respectively. The specific binding capacity for the same fractions were 49, 70, 55 fmol/mg protein, respectively.

Complexes of LH/hCG R and Triton X-100 were resolved into two components with approximate M, = 2.7 x 10' and 5.4 X 10' by gel filtration on Sepharose 6B and two glycoprotein components by chromatography on concanavalin A-Sepharose. Solubilized porcine LH/hCG R was purified by two cycles of affinity chromatography on highly purified hCG-Sepharose with an overall recovery of 30-35% of the initial activity in the Triton extract. Purified porcine LH/hCG R had a specific binding capacity of 2300 pmol/mg protein and a K , = 1.5 X 10" M-'. Silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels demonstrated that the major protein in porcine LH/hCG R preparations has M, = 68,000. A weakly staining band at M, = 45,000 was also observed in the purified receptor preparation. Analysis of iodinated purified LH/hCG R by autoradiography has con- firmed these results. Porcine LH/hCG R was purified 40,000-fold by this method.

The luteinizing hormone (LH') human chorionic gonado- tropin (hCG) receptor (LH/hCG R) is the initial site of action of LH in the mammalian ovary. Physiological actions of LH include follicular development, ovulation, corpus luteum development, and control of steroidogenesis. Consequently elucidation of the molecular nature of LH/hCG R is of primary

* This study was supported by the NICHD (to J. W., T. T. C., and J . A.) and from Natural Sciences and Engineering Research Council of Canada (Canada) (to J. P. W.). Parts of this work were presented in preliminary form at the Seventh International Congress of Endo- crinology (July 1-7, 1984), Quebec City, Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertise- ment" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: LH/hCG R, luteinizing hormone/ human chorionic gondatropin receptor; PMSF, phenylmethylsulfonyl fluoride; ConA, concanavalin A; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PEG, polyethylene glycol; BSA, bovine serum albumin; FSH, follicle- stimulating hormone.

importance in elucidation of the role of LH in ovarian phys- iology. Previous studies have demonstrated that LH/hCG R of the rat ovary (1-7) is primarily localized in the plasma membrane fraction of ovarian cells and although a minor fraction of LH/hCG R could be extracted by low ionic strength buffers (8), nonionic detergents were required for solubilization of the major part of the membrane bound LH/hCG R. However, the majority of the receptors were not recovered in the soluble fraction after detergent-induced solubilization (7 , 9-11). Porcine LH/hCG R in the membrane bound form has recently been characterized (12, 13) but neither the characterization of solubilized LH/hCG R nor the purification of porcine LH/hCG R has been reported, to our knowledge. For detailed studies on structure and function of LH/hCG R and the development of monospecific receptor antibodies, purification of LH/hCG R from an abundantly available tissue such as porcine corpora lutea is of particular importance.

Herein we report the successful solubilization of the porcine LH/hCG R in a stable form with high yield, the partial characterization of the solubilized receptor and the purification of the receptor, using affinity chromatography.

EXPERIMENTAL PROCEDURES

Materials Highly purified hCG (CR 121, 13,450 IU/mg), human follicle-

stimulating hormone (hFSH, NIH, HSI), rat prolactin NIH iodination grade, were kindly supplied by the National Pituitary Agency, Baltimore, MD. Partially purified hCG, soybean trypsin inhibitor, phenylmethylsulfonyl fluoride (PMSF), concanavalin A-Sepharose 4B (ConA) were obtained from Sigma. Sepharose 6B, CNBr-activated Sepharose 4B, and molecular weight standards were supplied by Pharmacia Fine Chemicals, Piscataway, NJ. All other chemicals used in experiments were of reagent grade. Twenty-one-day-old immature female rats were bought from Charles River Laboratories, Wilming- ton, MA. They were rendered pseudopregnant as previously reported (7, 8). All materials for SDS-PAGE were purchased from Bio-Rad. Buffers used were: A, phosphate-buffered saline (PBS), 25% glycerol, pH 7.4; €3, PBS, 25% glycerol, 0.1% Triton X-100, pH 7.4; C, 10 mM PO,, pH 7.4, 25% glycerol, 0.05% Triton X-100, 0.5 M NaC1; D, same as C plus 0.025 M acetic acid adjusted to pH 3.8.

Methods Receptor Extraction-Porcine ovaries were collected in ice at a

local abattoir. Well vascularized corpora lutea were dissected from the ovaries and immediately frozen in a tray immersed in an acetone/ dry ice bath and stored at -60 "C until used. The individual corpora lutea used in these studies weighed 0.3-0.4 g. Corpora lutea were homogenized in Buffer A containing 1 mM PMSF and 1 mg/ml soybean trypsin inhibitor using a glass/glass homogenizer (Corning, Pyrex). The homogenate was processed according to Scheme 1. Whole pseudopregnant rat ovaries (100-120 mg/ovary) previously frozen at -60 "C were processed by the same method. Membrane-bound LH/ hCG R was solubilized by homogenizing the 27,000 X g pellet in an equal volume of Tris buffer (10 mM), pH 7.3, EDTA (5 mM), 2% Triton X-100, a n d 2 5 1 glycerol. The extraction medium also con-

10689

10690 The Porcine LHIhCG Receptor

Frozen corpora lutea with the ConA for 3 h at 4 "C. The gel mixture was poured into a Homogenized in PBS containing column and the supernatant was collected. The column was washed

100 pg/ml soybean trypsin inhibitor with 35 ml of Buffer B. The column was next eluted successively with and 1 mM PMSF 35 ml of 0.25 M a-methyl-D-mannoside, 30 ml each of 0.5 M and 0.75

M a-methyl-D-mannoside in Buffer B. Fractions of 2.0 ml were collected. Each fraction was assayed for protein and LH/hCG R

Preparation of hCG-Sepharose-Highly purified hCG was covalently linked to CNBr-activated Sepharose 4B by a modification of

27,000 supernatant the procedure recommended by the supplier (Pharmacia). Ten mg of hCG (CR121) was dissolved in 15 ml of 0.1 M NaHC03, 0.5 M NaCl,

100 ml of 1 mM HCl for 15 min at room temperature. The gel was then washed under suction with 500 ml of HC1. The gel was next

Dilute with PBS/-tglycerol (25%) washed with 0.5 M NaC1,O.l mM NaHC03, pH 8.3, and resuspended in the hCG solution. The mixture was shaken end to end for 3 h at room temperature in a 50-ml centrifuge tube. The gel was filtered under vacuum and resuspended in 300 ml of 0.5 M NaC1, 0.1 M NaHC03, pH 9. The mixture was filtered under vacuum and resus-

/Add ~lycerol \Add filtered under vacuum. Each acid and alkali wash was repeated three pended in 300 ml of 0.5 M NaCl, 0.1 M CHaCOONa, pH 4.5, and

times. The gel was next resuspended in 150 ml of 0.2 M glycine, pH PBS 8, and incubated at room temperature for 1.5 h. After collection by (") vacuum filtration the gel was incubated for 30 min at room temper-

ature successively with the acid and alkaline buffers. The gel was next washed with 1 liter of H20 and PBS until the pH of the suspension was 7.0-7.4. The gel was resuspended in PBS/O.l% Triton and stored until use at 4 "C. By adding tracer quantities of 9 - h C G to the unlabeled hCG, recoveries of hCG coupled to Sepharose 4B were calculated to be between 60 and 70% of added hCG. Each batch of hCG-Sepharose could be used for at least six cycles of affinity chromatography without any loss of LH/hCG R binding capacity. Before reutilization of previously used hCG-Sepharose the gel was washed with an excess of 0.1 M NaHCO,, 0.5 M NaCl, pH 9, and re-

tors. Affinity Chromatography of Receptor Extract on Highly Purified hCG-Sephurose-Three g of hCG-Sepharose in a total volume of 8

tained o.5 mM PMSF and mg/ml soybean trypsin inhibitor. Final ml of PBS was mixed with 12-15.0 ml of receptor extract (500-750 concentration of Triton in the mixture was 0.9%. ~ f t e ~ homogeniza- fmol of LH/hCG R activity). The suspension was mixed end to end tion in a glass/glass homogenizer the extract was incubated at 4 "C in a bath at room temperature (23 "') for and the

at 160,000 X gin a Beckman Ti-60 rotor in a Beckman ultracentrifuge. was poured into a and washed with 6o m1 Of Buffer and

(Millipore Corp., Bedford, MA) filters and the filtrate collected. The with 30 m1 Of buffer (lo, 40). from each fraction

Serves as the solubilized LH/hCG R preparation. ~ 1 1 steps in receptor were frozen at -60 "C until utilized for the second affinity column. extraction were performed at 4 "C. Column fractions with highest LH/hCG R activity were pooled and

prec+itation of F~~~ Receptors by Polyethylene Glycol-Soluble, Pad of the Pool was re-equilibrated with the affinity gel used for the hormone-free LH/hCG R was quantitatively precipitated by PEG first affinity chromatography Step described above (after washing gel)

R preparation, 50% of PEG in PBS was added to achieve the final and after collection of the supernatant the gel was washed with Buffer required PEG ~ f t ~ ~ incubation for 15 min at 4 =-c the C and eluted with Buffer D. Each fraction was assayed for LH/hCG precipitate was collected by centrifugation at 27,000 x g for 15 min. R activity. Protein was assayed in the second affinity chromatography The pellets were resuspended in Buffer B and aliquots were assayed fractions only intermittently as all acid-eluted fractions had unde- for LH/hCG R activity and At 25% PEG, 70-80% of tectable quantities of protein/0.2-ml fraction. Fractions containing

1 300 X g X 15 min pellet

27,000 X g X 30 min Supernatant activity.

Extraction with Triton X-100 Incubate a t 4 "C, 45 min

I pH 8.3. Three g of CNBr-activated Sepharose 4B was incubated in

160,000 x g x 0.5 h

Triton pellet Soluble fraction

Filter through eight 0.45-p filters

Centrifuge at 160,000 x g x 1.5 h

Add

Soluble receptor SCHEME 1. Method for preparation of soluble pig LH/hCG recep- equilibrated with PBS.

for 45 min. The Triton extract was routinely centrifuged for 30 min supernatant was at O C by centrifugation. The gel

The supernatant was then filtered through two 0.45-r Millipore

filtration step was repeated another three times. The final filtrate were assayed for protein and LH/hCG R activity. The eluted fractions

two 8-m1 and seven 6-m1 fractions were LH/hCG was

6000-8000 (Sigma) by the following procedure. To soluble LH/hCG at room temperature for 2 h. The mixture was poured i n k the column

in the original soluble fraction was present in the PEG precipitate. LH/hCG R activity were Pooled and dialYzedovernight a t4 "c against ~ 1 1 of the LH/hCG R activity was precipitated at this PEG concen- 3 mM Po4 buffer, PH 7.0. The Pool Was next lyophilized in aliquots. tration. Aliquots used for SDS-PAGE were re-dialyzed against water and re-

Gel Filtration Chromatography of Receptors-Gel filtration was performed on a Sepharose 6B column (100 X 1.6 cm). The column was calibrated with ribonuclease (14,000 Da), chymotrypsinogen (25,000 Da), ovalbumin (45,000 Da), bovine serum albumin (BSA) (67,000 Da), aldolase (155,000 Da), myosin (200,000 Da), ferritin (460,000 Da), and thyroglobulin (669,000 Da). Before chromatography of soluble receptor extract the column was equilibrated with Buffer B. 1.5 ml of solubilized PEG precipitate, prepared as described above (containing 300 fmol of LH/hCG R activity), was loaded on the Sepharose 6B column, and the column was developed with Buffer B. Fractions of 1.5 ml were collected and each fraction was assayed for protein and LH/hCG R activity. Chromatography of soluble receptor extracts without concentration with PEG gave similar results (results not presented).

Concanavalin A-Sepharose 4B Chromatography of Receptors-Five g of ConA was washed with 100 ml of PBS and 100 ml of Tris buffer, 10 mM, pH 7.3, containing 1 mM CaClz and 1 mM MnC12. After washing, the gel was resuspended in 5 ml of the Tris buffer. 7.5 ml (200 fmol of LH/hCG R activity) of the receptor extract were mixed

lyophilized. SDS-PAGE Analysis-Purified receptor was analyzed by SDS-

PAGE on 7.5% gels according to a previously described method (39). Approximately 50-150 ng of LH/hCG R were applied per lane in a 0.75-mm slab gel polymerized in a Hoeffer Strudier electrophoresis apparatus. Gels were run at 20 mA for 3-4 h and were stained (14, 31) using the silver stain kit supplied by Bio-Rad. Phosphorylase b, BSA, ovalbumin, and lactic dehydrogenase (35,000 Da) were used as standards (100 ng of standards were applied per well).

Assay for LH/hCG R Actiuity-Soluble LH/hCG R activity was assayed as described (15) in triplicate with the following modifica- tions. For soluble receptors the assay buffer was 0.1% BSA in PBS and for affinity purified receptors the buffer was 30 mM NaCl, 10 mM PO,, pH 7.4, containing 0.1% BSA. Additionally, 20 pl of 5% BSA (in PBS) were added per assay tube (total volume 1 ml) when purified LH/hCG R was assayed. When membrane fractions were assayed for LH/hCG R activity the assay was terminated routinely by the PEG double precipitation method (15) or by filtration on 0.45-p filters (Millipore). In our experience pelleting of membranes by centrifuga-

The Porcine LHIhCG Receptor 10691

tion at 1,500 X g X 20 min on a table top centrifuge (Beckman TJ6) was only 50% as effective as PEG precipitation. The latter method was about 20% more effective than ultrafiltration on Millipore 0.45- p filters. The second PEG precipitate (15) was routinely washed with 1 ml of 0.1% BSA/PBS before counting precipitates in a Beckman 4,000 y counter. Nonspecific binding was assayed in the presence of 20 pg/ml of hCG (Sigma) and constituted 2% of added '9-hCG for membrane preparations and 3-4% of added 'T-hCG for solubilized preparations. Routinely 50,000-100,000 cpm (0.5-1.0 ng) of 'T-hCG was added per assay.

Iodination of Purified Receptors-Receptor fractions eluted from the second affinity step were dialyzed overnight as described above and lyophilized. 100-200 ng of lyophilized protein was iodinated with the Iodo-Bead solid phase reagent (N-chlorobenzenesulfonamide, Na salt) supplied by Pierce Chemical Co. The iodination method was essentially that recommended by Pierce as described below. Two Iodo-Beads were washed with 50 mM phosphate buffer, pH 7.2, and resuspended in 0.2 ml of ice-cold buffer. The lyophilized receptor preparation was dissolved in 300 pl of ice-cold phosphate buffer; this solution was added to the beads which were preincubated with 300 pCi of "'I at room temperature for 5 min. The receptor bead mixture was shaken for 2 min by hand. The supernatant was aspirated to a second tube kept in ice and the beads were washed with 0.2 ml of ice- cold phosphate buffer, this wash was transferred to the tube. 0.1 ml of 1 mM tyrosine was added to the tube and the mixture was next chromatographed on a 10-ml Sephadex G-25 column previously equilibrated with 0.1% Triton X-100 in PBS. 0.5-ml fractions were collected. Average specific activity was 15-20 pCi/pg. Aliquots of iodo- receptors were chromatographed on a Sephadex G-150 column (1.5 X 50 cm) which had been previously calibrated with ribonuclease, ovalbumin, BSA, and chymotrypsinogen. The column was either developed in PBS or 0.1% Triton in PBS after appropriate equilibration. 1.0-1.2-ml fractions were collected and 0.5-ml aliquots were counted. Recovery of applied radioactivity was approximately 70%.

Dialyzed iodo-receptor preparations were lyophilized and dissolved in H20. Aliquots of 40-50 X lo3 cpm were analyzed by SDS-PAGE as described above. Dried gels were exposed at -60 "C for 2-3 weeks before developing autoradiograms.

Other Methods-Highly purified hCG (CR 121) was iodinated by a lactoperoxidase procedure (16). Specific activity of Sephadex G-25 and ConA purified dialyzed hormone determined by self-displacement (17) was 40-60 Ci/g. Approximately 50-60% of the '%I-hCG was bindable to membrane bound and soluble LH/hCG R. Protein in crude preparations was determined by a previously described method (18). Protein in purified receptor preparations was determined by a micro Lowry procedure (41) with sensitivity at 250 ng of BSA. Absorbance was corrected for that due to non-protein contaminants. Receptor extracts were routinely stored at -60 or -70 "C.

RESULTS

Membrane Bound Receptor Actiuity-Porcine corpora lutea and whole rat ovaries were processed according to Scheme 1. The 27,000 x g supernatant of porcine corpora lutea was further fractionated into a 1.6 x lo5 X g pellet and supernatant (soluble fraction) (n = 4). For rat ovaries the last fractionation was performed twice. Distribution of porcine and rat (Table I) LH/hCG R activity was basically similar except for the significantly higher activity in the 27,000 x g supernatant in the porcine system 0, < 0.01). Furthermore, the percentage of activity in the 1.6 X lo5 X g pellet in the porcine system was higher than in the rat system while the LH/hCG R activity spontaneously soluble in the rat system was %fold (13%) of that in the porcine system. The specific activity of LH/hCG R in the porcine subcellular fractions were similar in all fractions except for that of the whole homogenate and the soluble fraction. In contrast, there were significant differences in the specific activities of rat LH/hCG R in the subcellular fractions. The high speed (27,000 X g) pellet had significantly higher LH/hCG R specific activity than the whole homogenate, low speed pellet, and the 27,000 X g supernatant. The specific activities of LH/hCG R in the rat system were 5-%fold higher than those in the porcine system except for the specific activity of the 27,000 X g supernatant.

Solubilization of LH Receptor Actiuity-The 27,000 x g pellet was extracted with Triton as described under "Meth- ods" and soluble preparations, S1 to S4 (Scheme 1 and Fig. l), were tested for LH/hCG R activity. As shown by S1 (Fig. l ) , optimal recovery of membrane bound LH/hCG R in the soluble form was realized when glycerol was added both during Triton extraction and at dilution of the initial Triton extract. Recovery was poor if glycerol was not added before centrifugation for 1.5 h (Sz uersus S4). By ultrafiltration of the centrifuged (30 min) extract, SI could be prepared in 45 min as opposed to the 2.0 h required to prepare S4 by the conven- tional method. Recovery of LH/hCG R in SI was 67% as depicted in Table I; in more recent experiments recovery has averaged 80 k 5%. LH/hCG R activity solubilized by all four methods was stable at -60 "C. SI and S P can be stored at -60 "C for 3 months with retention of 90% activity. All the soluble preparations retained over 80% of the original activity at 4 "C for at least 48 h, however, a substantial part of activity was lost between 72 and 96 h at 4 "C (Fig. 1).

In the rat system using the same procedures the major part of membrane bound LH/hCG R activity has been solubilized in a stable form (Table I) and in our more recent studies an average yield of 80% of membrane bound LH/hCG R activity has been recovered in the soluble extract. In contrast to the porcine system (Table 11), the specific activity of rat soluble LH/hCG R is significantly higher than that of the 27,000 X g membrane pellet (Table I). It should be noted that LH/hCG R activity not solubilized by Triton was recovered in the pellet resulting from the centrifugation at 1.6 X lo5 X g. Thus all of the LH/hCG R activity in the crude plasma membrane pellet was recovered either in the soluble fraction or Triton-insoluble pellet for either rat or porcine preparations.

Quantitative Analysis of Receptor Actiuity-Receptor activity of whole homogenate (HI, crude plasma membranes sedi- mented at 27,000 x g (P), and the soluble fraction (S) were measured simultaneously in the presence of increasing quantities of unlabeled highly purified hCG. This data is described in Table 11. The K,, of homogenate, membrane, and soluble fractions are similar. About 40% of the binding capacity of the corpus luteum resides in the crude plasma membrane fraction. The soluble fraction contained the majority of the LH/hCG R activity present in the membrane fraction, but this activity was significantly less than in the plasma membrane fraction. When the soluble fraction was prepared with or without enrichment with 25% glycerol immediately prior to centrifugation at 1.6 x lo5 x g for 1.5 h and the resulting soluble fractions were used to generate Scatchard plot data, glycerol was observed to have a substantial effect on both K, and N (Table 11). Thus glycerol increased both parameters nearly %fold. Similar results were obtained if the Triton extraction was performed with and without glycerol and ap- propriately diluted prior to the 30-min centrifugation and ultrafiltration. The results were, with glycerol, N = 160.0, K, = 2.22; without glycerol, N = 73.3, K, = 1.11.

Specificity of the Receptor-When lP51-hCG was bound to soluble and crude plasma membrane preparations of LH/hCG R in the presence of increasing concentrations of highly purified hormones only hLH was equipotent with unlabeled hCG in competition for "'I-hCG in the binding reaction (data not shown). Human FSH and rat prolactin and hGH were ineffective as competitors. This data demonstrates that both membrane bound and soluble LH/hCG R is highly specific for hCG and hLH.

Gel Filtration Chromatography-When the Triton extract containing soluble LH/hCG R activity is diluted to decrease the Triton concentration, the resulting LH/hCG R activity


TABLE I Subcellular distribution and specific activities of porcine and rat LHIhCH receptor activity

The following values are significantly different at p < 0.01 * and $, 8 and 1, 11 and **; 11 and $$, $3 and $§, § and **. Number of separate experiments are indicated in parenthesis..

Porcine LH/hCG R Rat LH/hCG R

% f S.E. Specific activity f S.E. % f S.E.

fmollmg protein fmollmgprotein

Specific activity 2 S.E.

Homogenate 100 f 4.1” (9) 12.1 f 1.1 (lo)* 100 * 7.5b (5) 75.6 f 5.01 (6)

300 X g pellet 22.8 f 1.6 (10) 20.0 f 1.2 (lo)$ 23.6 f 2.4 (6) 101 f 10.11 (6)

27,000 X g pellet 44.2 2 1.6 (10) 22.5 f 1.4 (lo)$ 49.8 f 3.2 (6) 165 f 14.711 (6)

27,000 X g supernatant 26.4 f 1.1 (9) 19.6 f 1.5 (9)$ 18.0 f 0.95 (6) 34 f 2.0** (5)

1.6 X lo5 X g pellet 18.7 f 1.9 (4) 21.5 2 2.0 (3) 5.0 (2)

1.6 X lo5 X g supernatant 4.3 t 0.9 (4) 11.0 f 3.0 (3) 13.0 (2)

Recovery in soluble frac- 67.0 f 3.0 (10) 22.8 f 1.9 (9) 67.0 If: 2.9 (6) 291 f 15.5$$ (6) tion‘

Recovery in Triton insol- 25.0 f 2.8 (9) 21.9 f 1.9 (8) 26.4 ? 2.9 (6) 127 f 10.71s ( 5 ) uble pellet‘

100% = 157 fmol of hCG bound per corpus luteum. * 100% = 386 fmol of hCG bound per ovary. e The LH/hCG R activity in the 27,000 X g pellet is taken as 100% and the soluble and pellet fractions refer to

Triton X-100 extraction of the 27,000 X g pellet.

that could be loaded onto a Sepharose 6B column was low due to volume restrictions in gel filtration. Therefore, the receptor extract was concentrated by PEG precipitation as described under “Methods” (data not shown). Concentrated receptor extract which had a specific activity similar to the original Triton extract, was chromatographed on the Sepha- rose 6B column as described under “Methods.” Two major peaks of LH/hCG R activity were observed with a smaller peak at the void volume (data not shown). The K., values of the two detergent-LH/hCG R complexes were 0.32 k 0.02 and 0.42 f 0.02 (S.E.) (n = 4). These K,, values correspond to M , = 5.4 and 2.7 X lo5, according to the calibration curve developed for the column (data not shown). The recoveries of LH/ hCG R averaged 50%, however, the major part of the protein coeluted with LH/hCG R activity, therefore gel filtration chromatography was not useful as a tool in purification of LH/hCG R.

Concanavalin A-Sepharose 4B Chromatography-We have previously demonstrated that group affinity chromatography of rat LH/hCG R extracts on ConA resulted in significant purification of the receptor (9). Therefore porcine soluble LH/hCG R preparations were chromatographed on ConA as an initial step in receptor purification as described under “Methods.” Results from a typical experiment (n = 3) are depicted in Fig. 2. After washing the gel the column was developed with 0.25, 0.5, and 0.75 M a-methyl-D-mannoside. As shown in Fig. 2, two major peaks of LH/hCG R activity were eluted with 0.25 and 0.5 M sugar. About 40-60% of applied LH/hCG R activity was not absorbed to ConA. Con- siderable quantities of protein were coeluted with the receptor and the purification achieved was only 4-10-fold. Recoveries of LH/hCG R were poor (30-40%).

Purification of Receptor by Affinity Chromatography-hCG- Sepharose was prepared and equilibrated with solubilized LH/ hCG R activity as described under “Methods.” After washing the affinity gel with Buffer C or Buffer C minus Triton, the gel was eluted with phosphate buffer/glycerol adjusted to pH 3.8 with 0.025 M acetic acid as previously described (10). With

this protocol only 2% of bound LH/hCG R activity was eluted. Similar results were obtained if the elution was performed with Buffer D without 0.5 M NaCl or 0.05% Triton. However, upon elution with Buffer D a substantial part of the LH/hCG R activity equilibrated with the affinity gel was eluted in the first three fractions where the pH changed from 7.3 to 4.0 as depicted in Fig. 3A. A typical yield at this step for receptor activity was 45% (n = 4). The major part of the protein mixed with the affinity gel was eluted in the supernatant (see “Meth- ods”) and the wash fractions as indicated in Fig. 3A. Protein recoveries were above 70% of applied protein. Further purification was achieved by pooling the highest activity fractions containing LH/hCG R activity and rechromatographing this pool on a second affinity column, the results of which are depicted in Fig. 3B. Recovery from the second column was 70-80% of the activity used for the second step. Data from typical purification experiments is summarized in Table 111. Possible reasons for the loss of 50-60% LH/hCG R activity at the first affinity step are discussed under “Discussion.”

Characterization of the Purified Receptor-Pooled LH/hCG R from the second affinity column was dialyzed against 3 mM phosphate buffer, pH 7.0, and lyophilized. Lyophilized LH/ hCG R was analyzed by SDS-PAGE as described under “Methods.” A typical result is depicted in Fig. 4A. SDS-PAGE autoradiography of iodo-receptor preparations also revealed a M, = 68,000 band and weaker bands at M , = 45,000 and between M , = 45,000 and 68,000 (Fig. 4B).

Gel Permeation Chromatography of Iodo-LH/hCG R-Iodi- nated LH/hCG R was chromatographed on a calibrated Seph- adex G-150 column (Fig. 5) as described under “Methods.” When the column was developed with PBS, the majority of macromolecular bound 1251 eluted at Vo and about 5% of macromolecular ‘‘‘I eluted with a M, = 64,000 k 6,000. In the presence of 0.1% Triton, and V, peak was shifted to a M , = 110,000 f 8,000 and the smaller molecular weight peak was no longer observed. These results suggest that LH/hCG R complex in the presence of Triton may be a dimer of the M , = 64,000 protein. Alternatively the detergent-LH/hCG R


30 t

(3 0

\

IO 1

'A

0 I I 1 1

I 2 3 4 'e TIME (days)

FIG. 1. In vitro stability of soluble hCG receptors. Porcine receptors were solubilized from corpora lutea membranes and sepa- rated from the insoluble material by filtration through 0 . 4 5 ~ filters or centrifugation in the absence and presence of 25% glycerol. SI, preparation made 25% glycerol after filtration. Sp, glycerol was added to the centrifuged extract to a final concentration of 25% before ultracentrifugation for 1.5 h. Ss, preparation diluted with buffer only after filtration. S4, buffer was added to the centrifuged extract before ultracentrifugation for 1.5 h.

TABLE I1 Quuntitatiue data on porcine LH/hCG R binding parameters

KA, equilibrium association constant in 10'' M - ~ . N , specific binding capacity in femtomoles/corpus luteum. SBC, specific binding capacity in femtomoles/mg of protein. n, number of experiments.

K. +S.E. N +S.E. SBC +S.E. n

Homogenate 1.92 0.3 550 49.0 4.5 2 27,000 X g pellet 2.22 0.21 214" 11.5 69.8 3.5 3 Soluble (+glycerol)b 2.03' 0.06 181' 6.1 55.2' 3.4 6 Soluble (-glycerol) .1.10' 0.13 106' 6.8 30.0' 2.8 4

Significantly different at p < 0.05. Triton X-100 solubilized preparation containing 25% glycerol.

e Significantly different at p c 0.01.

complex may be much larger than LH/hCG R itself. Highly purified LH/hCG R was used to bind '251-hCG in

the presence of increasing quantities of unlabeled pure hCG. Scatchard analysis of this data is depicted in Fig. 6. A typical one-site plot was observed with K, = 1.5 x 10" M" (S.E. f 0.5; r = 0.97) and an estimated binding capacity of 2330 pmol/ mg. Binding of '251-hCG by purified LH/hCG R was not inhibited by 100 ng/ml of hFSH or rat prolactin but was partly inhibited by rLH as depicted in Fig. 6. The Scatchard plot for displacement of '251-hCG by rLH yielded a K, = 0.5 X lo9 M" and binding capacity = 15,900 pmol/mg. Last, addition of an excess of unlabeled hCG to the corpora lutea

FRACTION NUMBER

FIG. 2. Chromatography of porcine receptor on concanavalin A-Sepharose. A receptor preparation was chromatographed on a concanavalin A-Sepharose column. The column was washed with PBS, 0.1% Triton, and glycerol and eluted with the same buffer containing increasing quantities of methyl-D-mannoside as indicated. Each fraction was sampled for receptor activity by 1261-hCG binding and for protein (total fraction).

extract as described in legend to Fig. 7 inhibited the binding of the solubilized receptor to the hCG-affinity column.

DISCUSSION

The significant differences in the specific activities of LH/ hCG R among rat subcellular fractions but not among porcine subcellular fractions may indicate differences in the localiza- tion of LH/hCG R among rat corpora lutea and porcine corpora lutea. In both porcine (13) and bovine corpora lutea (19, 20), the microsomal membrane fraction may contain a significant quantity of LH/hCG R activity in contrast to the rat system.

Our results clearly demonstrate that porcine LH/hCG R can be efficiently solubilized in a stable form from the crude plasma membrane fraction with the retention of the same K.. The recovery of LH/hCG R in the soluble fraction and the Triton-insoluble pellet was 100% both in porcine and rat systems in marked contrast to previous studies on the rat (5, 9, 11, 15) and bovine systems (21, 22). Notably, the specific activity of soluble rat LH/hCG R was about two times that of the crude plasma membrane pellet. The large increase in soluble LH/hCG R activity resulted from the enrichment of all buffers with glycerol and to a lesser degree by a large reduction in the time required for soluble LH/hCG R preparation, from 2 h to 45 min. The beneficial effect of glycerol on FSH receptor solubilization was previously demonstrated (23). However, in contrast to the results obtained with FSH receptors where only binding capacity of FSH receptors increased, glycerol increased both K,, and binding capacity about 2-fold in the case of porcine LH/hCG R. Similarly, specific activity of rat LH/hCG R observed in this study is at least 2- fold of that previously reported (5, 9, 11). It is known that glycerol may prevent the possible unfolding of protein struc- tures in aqueous solution (24) but whether glycerol has a similar effect on LH/hCG R is unknown. While the present manuscript was in preparation, Ascoli (25) reported that glycerol increased the yield of soluble LH/hCG R activity from testicular cells.

Gel filtration demonstrated two forms of LH/hCG R-detergent complexes, the approximate M, of the two complexes, 2.7 X lo5 and 5.4 X IO5, may suggest that the larger complex


I I 15

FRACTION NO

4

E ‘y

*11

0 \

v) e K a 0 K 0

” 3 I

W

0 t-

K n

0

5 10 15

FRACTION NO FIG. 3. Purification of the rat LH/hCG receptor by hCG-

Sepharose affinity chromatography. Receptor preparation from porcine corpora lutea was shaken with a suspension of hCG-Sepha- rose (8 ml of packed gel) for 2 h a t room temperature. The suspension was packed into a column and the supernatant was drained and collected. The column was washed with 40 ml of Buffer C. The receptor activity was eluted with 30 ml of Buffer D. Each eluted fraction was monitored for pH and immediately adjusted to pH 7.2 with 1 M NH,OH. Fractions were sampled for receptor activity and protein. Upper panel (A) indicates the first chromatographic step. The fractions with the highest receptor activity were pooled and rechromatographed in the second affinity purification step ( B ) (bottom panel). Ordinate indicates ‘*‘I-hCG bound.

TABLE 111 Purification of porcine LHIhCG R

Data in the table is averaged from two experiments. They do not correspond quantitatively to those in Fig. 4. Fig. 4 represents results from a typical affinity purification experiment.

Fraction Protein Activitp Specific binding capacity

mg fml pml lmg Whole Homogenate 60 1260 0.049 Crude plasma membrane 34 546 0.069 Triton extract 19 423 0.055 pH 4 eluate from first 190

pH 4 eluate from second 0.0015 152 2300

a Activity measured at 0.5-1 ng of ‘%I-hCG, used for calculation of

affinity column

affinity column

recovery.

h

97

66

45

is a dimer. However, the molecular weight estimations based on gel filtration may be inaccurate if the receptor-detergent complex is not globular. Estimations of M, by gel filtration for bovine LH/hCG R gave a value of 2.8 x 10‘ (22) and for *251-hCG-receptor complexes solubilized from porcine granulosa cells, a M , 250,000-370,000 was estimated (26). Analysis of subunit molecular weight as discussed below indicated that the porcine LH/hCG R-detergent complexes are probably multimeric forms of smaller subunits. The porcine LH/hCG R appears to be at least partly glycoprotein in nature. Heter- ogeneity of LH/hCG R based on the carbohydrate moiety in the glycoprotein was also observed in the rat system (9).

Affinity chromatography using immobilized crude hCG has previously been applied to purification of LH/hCG R (10-12,

29 FIG. 4. SDS-PAGE analysis of purified receptor. Lyophilized

aliquota of receptor prepared as described under “Methods” were analyzed on a 7.5% SDS-PAGE slab gel (panel A). The gel was stained by the silver stain method. Standard proteins used were phosphorylase, BSA, ovalbumin, and carbonic anhydrase. (Molecular weights of standard proteins are expressed in thousands, e.g. 97 = 97 X lo3. Panel B depicts results from SDS-PAGE autoradiographic analysis of ‘261-receptor preparation prepared as described under “Methods.” A dried 7.5% slab gel was silver stained and autoradi- ographed by standard procedures as described in the text. Molecular weight markers were illustrated for autoradiography by the addition of a drop of ’=I-hCG by a micropipette onto each protein band. The dark area at the bottom of the autoradiograph resulted from a trace of ‘“I-hCG contaminating the sample lanes by accident.


e X

Q z t- o a o jb . Y

a 20 40 60 80 U FRACTION NO. -

' $

Q t- o - a o a U : 20 FRACTION 40 NO. 60

z o -Triton v'.

FRACTION NO. FIG. 5. Gel filtration analysis of iodo-receptors. An aliquot

of lZ5I-receptor preparation was chromatographed on a Sephadex G- 150 column as described in the text. In the lower panel the column was developed with PBS, 0.1% Triton, in the upper panel the column was developed with PBS. Large triangle in lower panel indicates elution of nonaggregated receptor.

21, 42). However, only two reports (10, 40) have published binding profiles resulting from affinity chromatography procedures. In results of Dufau et al. (40), continued elution of rat testicular LH/hCG R activity occurred in marked contrast to the elution of activity in 2-4 fractions we have consistently observed (Fig. 4) with porcine LH/hCG R (n = 4). We have observed that unless precautions are taken artifactual apparent specific binding of lZ5I-hCG to purified receptors is observed when receptor protein is present in nanogram quantities in the assay system. We have used a number of methods for: 1) binding of soluble LH/hCG R to purified hCG-Sepha- rose and 2) elution of bound LH/hCG R activity. Binding of LH/hCG R to hCG-Sepharose was time- and temperature- dependent. Only Buffer D containing both Triton and NaCl was successful in eluting more than 5% of bound LH/hCG R. Elution with Buffer D at pH 5.5 was not effective in eluting LH/hCG R activity. These observations may explain the low (5%) recovery observed in the rat system (11). The loss of approximately 50-60% of LH/hCG R activity applied to the affinity gel remains unexplained. Since 70-80% of activity loaded during the second affinity step is recovered in the acid eluate, i t is unlikely that: (a) interaction of the affinity gel with detergent extract inactivates LH/hCG R or (b ) some LH/hCG R is bound extremely tightly to the immobilized hCG and is not eluted by detergent/acid/salt. Recovery of 25% of loaded receptor activity in the wash after incubation for 2 h at 4 "C or upon incubation at 37 "C for 1 h in addition to the 40% activity eluted in the acid buffel3 suggests that

J. Wimalasena, P. Moore, J. P. Wiebe, J. Abel, and T. T. Chen, unpublished results.

0 ' 2 4 6 8

BOUND hCG pM

\CG 0

1 2 3 4

LOG HORMONE PM

FIG. 6. Competitive displacement of lZ6I-hCG binding to purified receptor by unlabeled hormones. Top, data obtained by inhibition of lZ6I-hCG binding with increasing quantities of unlabeled hCG is plotted according to the Scatchard equation. Bottom, binding of lZ6I-hCG to purified receptor was performed in the presence of increasing quantities of unlabeled hormones. rLH, rat luteinizing hormone NIH rLH; PRL, rat prolactin.

receptor inactivation during the equilibration is both temperature- and time-dependent. Similar results were observed during the purification of rat LH/hCG R (45). Bovine luteal LH/hCG R was partially purified by ConA and hCG affinity chromatography (21) and highly purified by methods other than affinity chromatography (22) with a recovery of 15%.

The purified receptor had a K. similar to that of the Triton extract or the membrane fraction. The estimated specific binding capacity of 2330 pmol/mg suggests that porcine LH/ hCG R has been purified ahout 40,000-fold above the Triton extract (Table 111). According to SDS-PAGE analysis and autoradiography more than 80% of the protein in the purified preparation had a M, = 68,000. Sephadex G-150 chromatography suggested an apparent M, = 110,000-120,000 for iodo- receptor, the latter may be a dimer of the M, = 68,000 protein. The expected specific binding capacity of purified LH/hCG R is approximately 11,700 pmol/mg protein, a value 5-fold higher than the estimated number, if the M, value is taken as 68,000. This disparity could be due to a number of reasons (27). It is possible that a large fraction of the protein in purified LH/hCG R is in fact inactivated receptor as suggested by the 30-35% recovery of binding activity. Furthermore, the molecular weight of the hormone binding molecule of LH/ hCG R is unknown. The M , = 68,000 subunit could be a proteolytic degradation product of a larger hormone binding molecule. The 200,000-fold purification apparently required for complete purification of LH/hCG R has been achieved only with the adrenergic receptor (28-30). In addition to the


FIG. 7. Control experiment for affinity chromatography. Four ml of Triton extract was prepared as described under “Methods.” The extract was incubated with 3 pg/ml of CR 121 hCG overnight a t 4 “C (11). After removal of an aliquot for binding assay, the extract was mixed with 5 ml (packed volume) of CR 121 (hCG)-Sepharose and affinity chromatography was performed as described above. No iodo-hCG binding activity was eluted from the column either in the wash or acid eluate. (In the whole extract, iodo-hCG binding was reduced by 85%.) The first two fractions with a pH 4.3 were pooled and subjected to a second cycle of affinity chromatography. The first two pH 4.3 fractions from the second cycle (no binding activity observed) were pooled, dialyzed, and lyophilized. Lyophilizates from this experiment (receptor minus) and affinity purified receptor from another 4 ml of unincubated extracts (receptor plus) were simultaneously analyzed by 7.5% SDS-PAGE disc gels under reduced dena- turing conditions and gels were stained with Coomassie Brilliant Blue. Results demonstrated a strong band at M, = 70 X lo3-72 X lo3 in the receptor plus gels but no corresponding protein was observed in the receptor minus extract. In the receptor plus extract two faint diffuse bands were also observed at 130 X lo3 and 220 X lo3, these bands were absent from the receptor minus extract.

M, = 68,000 peptide, in a number of studies a faintly staining band a t M, = 45,000 was observed by silver staining and autoradiography. Whether this band is a receptor constituent is uncertain at the present time. Since highly purified hCG was used as the binding ligand all components in the purified receptor preparation may be components of porcine LH/hCG R. These results, however, do not rule out the possibility that

the purified LH/hCG R has other peptides (e.g. the M, = 45,000 peptide) which are not stained by the silver technique and are not labeled by the present iodination techniques. Labeling of peptides may be limited by unavailability of surface tyrosine residues as observed with the relatively low iodination of the p subunit of intact hCG.

The molecular weight of pure LH/hCG R is unknown a t present; crude rat testicular and ovarian receptor appears to have M, = 180,000-200,000 (4, 7, 15). Recently, detergent- free and detergent-extracted rat ovarian LH/hCG R was shown to consist of five hCG binding species (8, 9) and testicular detergent-free LH/hCG R appears to have a hormone binding species with M , = 43,500 (32). Rat testis and ovarian LH/hCG R purified by affinity chromatography were proposed to have a M , = 80,000 (12) and M , = 90,000 (ll), respectively. Bovine LH/hCG R purified by methods other than affinity chromatography (22) demonstrated three protein bands on SDS-PAGE gels, with M , = 160,000, 57,000, and 44,000. Dattatreyamurty et al. (22) measured the molecular weight of 900-fold purified bovine LH/hCG R following boiling in SDS by gel filtration chromatography and l2‘1-hCG binding. Under these conditions a 5.9 x lo6 multimer was dissociated into M , = 280,000 oligomer which in turn appeared to dissociate to M, = 120,000-140,000, 85,000, and 38,000 subunits. Studies on photoaffinity labeling of LH/hCG R in cultured porcine granulosa cells (26,33,34) and cross-linking of bound hCG to cells of testicular origin (35, 36) have suggested that testicular LH/hCG R has a subunit M , = 90,000-100,000 (36) and that three to four ovarian peptides MI = 58,000-96,000 were covalently attached to hCG. Very recently 1251-hCG was covalently cross-linked to rat ovarian membranes and SDS-PAGE autoradiography revealed peptides with MI = 46,000, 58,000, and 89,000 for receptor components (43) while photoaffinity labeling suggested peptides of M, = 86,000,65,000, and 60,000 out of which only the M , = 86,000 component was suggested to be the receptor (44). Therefore proteins of M, = 50,000-80,000 are apparently components of LH/hCG R, in agreement with the M, = 45- 68 X lo3 peptides we have observed in the purified receptor.

Recent studies have shown that a number of hormone receptor molecules which behave as large receptor-detergent complexes on gel filtration are multimers or aggregates of smaller subunits. Notable examples are the insulin receptor (27,37,38) and the p adrenergic receptor (28-30). I t is known that the subunit of the purified /3 adrenergic receptor can bind adrenergic receptor specific ligands with the appropriate specificity (30). Evidence for M, of intact hormone binding sites on other hormone receptor subunits is not yet available. Porcine LH/hCG R may be present in the membrane as an oligomer of the M, = 68,000 peptide perhaps closely associated with lipids; such complexes might explain the high molecular weights ( M , 270,000 and 540,000) observed in the present study. The larger LH/hCG R complexes may be required for the physiological function of the receptor.

In conclusion we have characterized the soluble porcine corpus luteum LH/hCG R and purified the receptor approximately 40,000-fold with a recovery of 30% of the initial activity. The receptor preparation is composed predominantly of a MI = 68,000 protein, however, we cannot rule out the presence of other proteins in the purified receptor which were not detectable by our methodology. The purification method described herein is currently used by us to generate pure receptor for structural and immunological studies.

Acknowledgments-We are grateful to R. Melchin for her assist-


ance in the preparation of the manuscript and P. Sieg for technical 21. Khan, F. S., Rathman, P., and Saxena, B. B. (1981) Biochem. J . assistance. 1 9 7 , 7

22. Dattatrevamurtv. B.. Rathnam, P.. and Saxena, B. B. (1983) J.

1. 2. 3. 4.

5.

6. 7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18. 19.

20.

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Biol. Reprod. 30, Suppl. 1, Abstr. 201

the porcine lh/hcg receptor · 2001-07-07 · 10689 . 10690 the porcine lhihcg receptor frozen...

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