GENERAL

AND

COMPARATIVE

Development

ENDOCRINOLOGY

35, 110-120 (1978)

and Application of a Radioligand-Receptor Assay for Thyrotropin J. WORKEWYCH AND K. W. CHENG

Department

of Physiology, Faculty of Medicine, Universi@ of Manitoba, Winnipeg, Manitoba R3E OW3, Canada Accepted November

28 1977

A membrane-bound receptor preparation for bovine thyrotropin (TSH) was obtained from fresh porcine thyroid glands after homogenization in 0.3 M sucrose and differential centrifugation at 10,000 and lOO,OOOg. Highly purified bovine TSH (30-40 ILJ/mg) was used as standard and was labeled with lz51by the chloramine-T method for use as tracer. The binding of Lz51-labeted TSH was dependent on the concentration of membrane protein, pH, time, and temperature of the incubation. Optimal specific binding was obtained at pH 7.0 after 1 hr of incubation at 37’ or 4 hr at 25’. A radioligand-receptor assay for TSH has been developed with a sensitivity of IO rig/ml. The binding of Y-labeled TSH was specific and not displaceable by other glucoprotein, protein, and polypeptide hormones up to concentrations of lOOO-fold excess. The bovine pituitary concentration of TSH was estimated to be 120.29 5 31.18 ng/mg by radiohgand-receptor assay as compared to 1 Il.39 ? 19.81 ng/mg by radioimmunoassay. The TSH activity in partially purified pituitary extracts of cow, dog, rabbit, rat, guinea pig, turkey, and chicken interacted in a manner parallel to that in the bovine TSH standard. By gel filtration on Sephadex G-100, the molecular size of pituitary TSH showed an interspecies similarity; but electrophoretic mobilities for TSH of various species varied slightly. This is a specific radioligand-receptor assay for TSH and can be employed to monitor the TSH activity of various species for biochemical and physiological studies.

Teng et al., 1975). In spite of numerous reports on the partial purification and characterization of TSH receptor in particulate membrane-bound preparations (Manley et al., 1974; Moore and Wolff, 1974; Verrier et al., 1974; Kotani et al., 1975; Tate, Schwartz et al., 1975b) as well as in solubilized preparations (Manley et al., 1974; Tate et a!., 1975a), little information is available on the development of specific radioligand-receptor assays for TSH, utilizing thyroid preparations of biologically specific receptors (Manley et al., 1974). This paper describes the development of a specific radioligand-receptor assay for TSH, utilizing 1251-labeled bovine TSH as tracer and a partially purified membrane fraction from porcine thyroid glands. Ap-

It has been established that the action of thyrotropin (TSH) on thyroid tissue is through its binding to the receptors in thyroid cell membranes (Wolff and Jones, 1971; Manley et al., 1972; Amir et al., 1973; Lissitzky et al., 1973), and adenyl cyclaselinked TSH receptors have been demonstrated in plasma membranes from thyroid cells (Verrier et al., 1974) as well as thyroid homogenates (Wolff and Jones, 1971; Manley et al., 1974; Kotani et al., 1975). Specific binding of radioactively labeled TSH to partially purified membrane-bound receptor preparations from thyroid homogenates has been reported for bovine (Moore and Wolff, 1974; Kotani et al., 1975), porcine (Verrier et al., 1974), and guinea pig (Manley et al., 1974; Smith and Hall, 1974; 00166480/78/‘0352-01

tO$Ol.OO/O

Copyright @ I978 by Academic Press, Inc. AU rights of reproduction in any form reserved.

1IO

RADIOLIGAND-RECEPTOR

plications of this assay system to the study of TSH activity in pituitary extracts of several species were also reported. MATERIALS

AND METHODS

Purified bovine TSH (potency = 30-40 IU/mg) and bovine LH (potency = 2.0 X NIH-LH-Sl) and their a and /3 subunits and the rabbit anti-bovine TSH semrn were all gifts from Dr. .I. G. Pierce, UCLA, Los Ange!es, Calif. Purified bovine FSH (potency = I60 X NIH-FSH-Sl) and its a and /3 subunits were prepared as described previously (Cheng, 1976, 197s). Rat thyroid-stimulating hormone (Rat TSH-l-2; Potency = 35 IU/mg), bovine growth hormone (NIHGH-BIS), and ovine proiactin (NIH-P-SlO) were obtained from the National Institute of Arthritis, Metabolic and Digestive Disease, National Institute of Health, Bethesda, Md. Porcine insulin (Lot 1119) was from Connaught Laboratory, Ltd., Toronto, Ontario, Bovine serum albumin (fraction V) was purchased from Miles Laboratory, Kankakee, Ill. NaY (carrier free) was purchased from New England Nuclear, Boston. Mass. Bovine y-globulin and cbloramine-T were from Sigma Chemical Co., St. Louis, MO. Sephadex G-100 was from Pharmica (Canada). Ltd., Dorval, Quebec. All other reagents and chemicals .were reagent grade. Preparation of receptor membranes from porcine thyroids. Fresh porcine thyroids were collected in ice from a local slaughterhouse. The procedure for preparation of the particulate membranes from porcine thyroids for TSH was identical to the previously published method for preparing bovine testicular receptor for FSH (Cheng, 1975a, b). Freshly dissected reddish porcine thyroid glands, after trimming off all extraneous tissues, were rinsed with cold 0.85% saline, cut into small pieces, and homogenized in 0.3 M sucrose (Brinkman Polytron PT-10) at a concentration of 5 ml of buffer/g of tissue. The homogenate was filtered through four layers of cheesecloth, and the filtrate was then centrifuged at lO.OOOg (Beckman, J-21B refrigerated centrifuge, JA-:4 rotor) for 30 min at 4’, and the supernatant was further centrifuged at lOO,OOOg (Beckman, L5-65 ultracentrifuge, 60-Ti rotor) for 1 hr at 4’. The pellet was resuspended in 0.025 M Tris-HCI buffer, pH 7.0, containing 10 mM MgCIZ, at a concentration of 1 ml of buffer/g of the original weight of thyroid tissue. This crude preparation of receptor membranes was used in the radioligand-receptor assay for TSH and could be stored at -20° up to 6 months. Radioiodination qf bovine TSH. Iodination of bovine TSH was carried out by the chloramine-T method of Hunter and Greenwood (1962) with 5 pg of hormone and 0.5 mCi of Na’Y. Radioactively labeled hormone was separated from unreacted free ‘? by

ASSAY FQR THYROTRQPEN

111

gel filtration on Sephadex G-100, eluting with 0.01 &l sodium phosphate-buffered saline (PBS). pH 7.4. The specific activity of the Y-labeled bovine TSH obtained was 45-60 @/pg. Procedure for radio!igand-receptor assay. TO exk~ assay tube, 0.1 ml of standard bovine TSH or unknown samples in 0.025 M Tris-HCI at pH 7.0 (containing 10 mM MgC& and 0.1% labeled bovine TSH diluted with the same buffer (50,000 cpm) and 0.1 ml of thyroid receptor membranes (300-600 pg of protein) were added. Al! the above solutions were kept at 4’ for use. The assay tubes were then shaken vigorously and incubated at room temperature (25’) for 4 hr. Following incubation, the reaction was stopped by adding 3.0 ml of cold assay buffer. After centrifugation at 15OOg (IEC, PR 6000) for 30 min, the supernatant was drained and the remaining pellet was counted in an automatic gamma spectrometer. Calculations of spec$c bindinE. §peci!ic binding (percentage) is defined as (CB - Cx) x loo/CT. where CB is the counts per minute bound; Cx is the nonspecifically bound counts per minute, nondisplaced by lOOO-fold molar excess of unlabeled bovine TSH; and CT is the total counts per minute put into the tribe. For assay standard curves, the total specific binding in the absence of unlabeled hormone is taken as 100% bound Y-labeled TSH, and the specific bindir~g in the presence of standard TSH at different concentrations is expressed as a percentage of the iOO% bound Y-labeled TSH. i?adioimmunoaLssay for bovine TSH. The procedure for the radioimmunoassay for bovine TSH was identical to that for bovine FSH and its a and j3 subunits as reported (Workewycb and Cheng, manuscript in preparation). AlI dilutions were made with PBS containing 0.1% BSA. To each assay tube, 0. I ml of rabbit anti-bovine TSH serum (1:30,005 diiution)~ 0.1 rnj of ‘Z51-labeled bovine TSH (25,QOOcpm). and Q~1 ml of unknown samples or TSH standards were aldded and incubated for 24 hr at 25’ (room temperature). Separation of antibody-bound and free YY-labeled ‘TSH .was achieved by adding 0.2 ml of PBS buffer, 0.5 m! of 2.0% bovine y-globulin. and 1.0 rni of 22% polyethylene gIyco1. followed immediateiy by vertexing. After CentrifugatioE at 1500,q for 30 min at p, the superEatant was aspirated and the precipitate was counted in an automatic gamma spectrome?er” Gel jiltration on Sephade.x G-100. Ge! filtration on Sephadex G-100 (1 x IO@cm) was carried ost at 4 with 0.5% NH&ICOs. Distribution of proteins ir eluates after fractionation was monitored by the absorbance at 278 nm with a spectrophotometer. Polyacrylamide grl electrophoresis. WC gel electrophoresis was carried out according to .Davis (1964). Gels of 7.5% polyacrylamide and dimensions of 0.4 x 6.0 cm were used. After electrophoresis. gels were

112

WORKEWYCH

segmented at S-mm intervals and the protein in each segment was eluted with 0.5% NH4HC03. After appropriate dilutions, the TSH activity in eluates of each gel segment was monitored by the radiohgandreceptor assay.

RESULTS

Specific Binding of ‘251-Labeled Bovine TSH to Porcine Thyroid Plasma Membranes Membrane preparations from porcine thyroid glands, resuspended in 0.025 M Tris-HCI buffer, pH 7.0, containing IO m,44 MgQ, at concentrations of 1 g wet weight of tissue/ml of buffer, were measured to contain approximately 3-5 mg of protein/ml of buffer. To examine the binding of 1251labeled bovine TSH by thyroid membranes at different protein concentrations, the membranes were pooled, concentrated by centrifugation, and resuspended in buffer to yield membrane preparations of protein concentrations at 1200, 900, 600, 300, and 150 ,ug/tube (loo-p1 suspension). Figure I

AND

CHENG

shows bovine creased plasma

that the binding of 1z51-labeled TSH to thyroid membranes inproportionally to the amount of membranes present.

Effect of pH on Binding of ‘25i-Labeled Bovine TSH to Porcine Thyroid Membranes The specific binding of 1251-labeled bovine TSH to porcine thyroid membranes was dependent on the pH of the incubation medium as shown in Fig. 2. Maximal binding occurred at pH 6.8-7.0. Effects of Time and Temperature on the Binding of ‘251-Labeled Bovine TSH to Porcine Thyroid Membranes In order to determine the optimal conditions for the radiohgand-receptor assay, the specific binding of 1z51-labe1ed bovine TSH to porcine thyroid membranes was studied by incubating at various temperatures and time intervals, as shown in Fig. 3. At 3T, maximum binding was attained after only 1 hr of incubation, and a decrease was observed after 2 hr. At 2S’ (room tempera-

n=7

300

600

900

I 1200

6.5

6.8

7.1

7.4

7.7

8.0

PH ug

membrane

protein

FIG. 1. Specific binding (percentage) of ‘Ylabeled bovine TSH to porcine thyroid plasma membranes as a function of protein concentration. A constant amount of 051-labeled bovine TSH (50,000 cpm) was incubated with the indicated amounts of plasma membranes at 25’ for 16 hr.

FIG. 2. Effect of pH on the specific binding (percentage) of *z51-iabeled bovine TSH to porcine thyroid plasma membranes. A constant amount of 1z51-labeled bovine TSH (50,000 cpm) was incubated with 600 m of membrane protein in 0.025 M Tris-HCI buffers of the indicated pH at 2.5’ for 16 hr. Values are means of two experiments.

RADIOLIGAND-RECEPTOR

0

4

ASSAY

6

incubation

FOR

12

T~~R~T~G~~~

16

time

20 2.0

i4 24

(hours]

FIG. 3. Effect of time and temperature of incubation on the specific binding (percentage) of 1z51-labe$ed bovine TSH to porcine thyroid plasma membranes. A constant amount of 1z51-labeled bovine TSH (5~,~~~ cpm) was incubated with 400 ~g of membrane protein in 0.025 M Tris-HCl buffer, pH 7.0, for the indicated time intervals at 4 ( A ), 25 (.i ), or 37’ ( 0 ):

We), maximum binding was observed after 4 hr of incubation. Under both conditions, specific binding decreased after reaching its maximal level due to increases of nonspecific binding with time. However, the binding at 4’ increased very slowly and reached a level of 2% after 24 hr. Speci$city, Sensitivity, and Precision of the Radioligand-Keceptor Assay for TSH A radioligand-receptor assay for TSH was set up using 300-600 pg of membrane protein per tube (Fig. l), depending on the binding capacity of the particular preparation of thyroid membranes, to yield 6-7% specific binding of lz51-labeled bovine TSH. TQ obtain maximal specific bindings, 0.025 A4 Tris-HCl buffer at pH 7.0 was used (Fig. 2)V and incubation was carried out for either 1 hr at 37’ or 4 hr at 2.5’ (Fig. 3). The specific binding of lz51-labeled bovine T§H by porcine thyroid membrane was displaced in an identical dose-dependent manner by highly purified bovine TSH and rat TSH of equal biological potencies (Fig. 4)~ However, highly purified bovine LH,

FSI-I, growth hormone? ovine proIactin, and porcine insulin, as well as the a and /3 subunits of bovine TSII, up to ~O~~-~old higher concentrations did not inhibit the binding of lz51-labeled bovine T’S thyroid membranes (Fig. 4). From the standard curves bovine and rat TSH (Fig. 4), the limit of detection of the present assay system for TX-I was 10 nglml, which could b cantly discriminated from zero at t confidence level on the basis of 2 the zero value. The intra-assay and interassay precision was carried out by rneas samples of 50 ngiml within assays an tween assays; and the precision of this imately &IO% (n assay system was app &15% (n = 18) = 18) within assays a between assays as expressed by the coefficient of variation Specisc ~~dioi~~unoa§$ay TSH

for

Figure 5 shows t of radioimmunoassay for bovine TSI-I and ~r~lQ~les. other protein and poly~ep~id~

114

WORKEWYCH

AND

CHENG

FSH TSHM TSHfi GH INS PRL

r TSH

t 100

10

10000

1000 w/ml

HORMONE

FIG. 4. Inhibition curves for bovine and rat TSH and other protein and polypeptide hormones in the radiohgand-receptor assay, using rz51-labeled bovine TSH and porcine thyroid membranes. Membranes (600 pg) were incubated with 1z51-labeled bovine TSH (50,000 cpm) in 0.025 A4 Tris-HCl, pH 7.0, at 2Y for 4 hr. (O), purified bovine TSH, or rat TSH; (m) bovine LH, bovine FSH, bovine TSH-cx and -6 subunits, bovine growth hormone, ovine prolactin, and porcine insulin.

1

100

Ii8

rig/ml

1,000

lO,liOO

hormone

FIG. 5. Dose-response curves for bovine TSH and other protein and polypeptide hormones in the radioimmunoassay, using rabbit anti-bovine TSH serum and 1z51-IabeIedbovine TSH. ( 0 ) Bovine TSH; ( 0 ) bovine TSH-fi; ( n ) bovine TSH-a; ( 0 ) bovine LH; ( A ) bovine FSH and bovine LH-0 and -@; ( A ) bovine growth hormone, ovine prolactin, and porcine insulin.

RADIOLIGAND-RECEPTOR

ASSAY

FOR

T~~RQTR~P~~

115

TABLE 1 The detection range of the assay for purified COMPARISON OF THE X!W CONCENTRATION IN bovine TSH was I- 100 rig/ml; and both puBOVINE ANTERIOR PITUITARY EXTRACTS BY rified bovine TSH-fi and TSH-a crossRADIOLIGAND-RECEPTOR ASSAY (RRA) reacted in a parallel manner at levels of 10 AND RA1310~~kfu~0.4~s.4~ (RIA)a and l%, respectively. The specificity of the Bovine TSH activity radioimmunoassay for bovine TSH was WW~ shown by cross-reactions of less than 0.1% Bovine pituitary by bovine LH, FSH, growth hormone, extract RRA RIA RRAIRIA ovine prolactin, porcine insulin, and the cx I 92.8 123.2 0.75 and ,6 subunits of bovine LH (Fig. 5).

Comparison of Measurements of TSH Concentratiun in Pituitary GZands of Various

Species

by

Radioligand-Receptor Assay and RadiQimm~~oas5ay for Bovine TSH To test the accuracy of this radioligandreceptor assay, 11 extracts of bovine pituitary glands were assayed and compared with the values obtained by an established radioimmunoassay for bovine TSH. The anterior pituitary lobes, after rinsing with normal saline, were homogenized in 0.025 M Tris-HCL at concentrations of 100 mg wet weight/ml of buffer. After centrifugation at 10POOOg,the supernatants were assayed at apm-opriate dilutions by both radioligand-receptor assay and radioimmunoassay for bovine TSH (Table 1). The tissue concentration of TSH in bovine pituitaries was estimated to be 120.29 ? 31.18 and Ill.39 2 19.81 ng/mg by radiohgand-receptor assay and radioimmunoassay, respectively. The ratio of radiohgand-receptor assay/radioimmunoassay for bovine pituitary TSH was calculated to be 1.Og 2 0.19. Assuming that the biological potency of bovine TSH was 35 LIJ/mg, the concentration of TSH would be 4*21 & I.09 mKJ/mg wet weight of pituitary tissue by radiohgand-receptor assay. Since purified rat TSH of similar biological potencies imhibited the binding of lz51labeled bovine TSH to the thyroid membranes m a manner identical to that of bovme TSH (Fig. 4), pituitary extracts of og, rabbit, rat, gumea pig, turkey, and icken were prepared to examine the pos-

2 3 4 5 6 7 8 9 IO 11

Mean k SD

96.0

168.0

86.4 121.6 86.4 179.2 137.6 118.4 115.2 121.6 120.29 ? 31.18

115.5

0.83

150.4 88.0 107.0 78.0 129.6 110.4 105.6 1is.4 99.2

1.;2 0.98 i.14 1.08 1.38 I.25 i.12 0.97 J.23

111.39 2 19.81

I.08 2 .19

’ Values are expressed in nanograms of TSH per milligram of wet pituitary tissue; each value is the mean of two separate determinations for both assay systems. (Reference standard: highly purified bovine TSH, 30-40 IU/mg.)

sibihty of utihzing this radioliga~d-receptor assay for bovine TS to monitor the T activity of various ecies. The pituit extracts in Tris-HCl were dimted appropriately and subjecte to both ~ad~o~~~a~dreceptor assay and d~oimm~~oassay for bovme TSH, as summarized in Table 2. Activity was detected in pituitary extracts of all species studied by radiohga receptor assay; however* by ~adi~~~~~noassay, activity was detected only in the dog, other than the cattle, to a small amount of cross-rea relatively large quantity of dog in the extract (Table 2). Ge/ Fihation of Pituitary Extracts from Various §pecies ori Sephadex G-!OO A bovine pituitary extract 4.32 and 4.40 pg of TSWmL as by radiohgand-receptor assay a immunoassay, respectively. via5 fraction-

116

WORKEWYCH

TABLE

AND

2

COMPARISON OF THE TSH CONCENT~TION IN PITUITARY EXTRACTS OF VARIOUS SPECIES BY RADIOLIGAND-RECEPTOR ASSAY (RRA) AND RADIOIMMUNOASSAY (RIA) FORBOVINE TSHO

TSH activity

h$w~ Pituitary extract Cattle

Dog Rabbit Rat Guinea pig Turkey Chicken

RRA

RIA

120.4 701.2 64.4 90.3 81.0 70.6 24.2

111.4 19.7 N.D.b N.D. N.D. N.D. N.D.

u Values are expressed as nanograms of TSH per milligram of wet pituitary tissue; each value is the mean of two separate determinations for both assay systems. (Reference standard: highly purified bovine TSH, 30-40 Wmg.) b Not detectable.

ated on a Sephadex G-100 column and the eluates were assayed for TSH by radioligand-receptor assay and radioimmunoassay as shown in Fig. 6. The TSH activity, as detected by both assay systems, was in fractions 17-28, and the total activity recovered in the eluates was 3.35 pg by

10

FRACTION

20

CHENG

radioligand-receptor assay (77.5% recovery) and 3.28 pg by radioimmunoassay (74.5% recovery). Similarities in elution volumes and percentages of recovery of activities by both assay systems indicated that the radiohgand-receptor assay was monitoring the same TSH as was the radioimmunoassay. In order to determine whether TSH in various species was similar in molecular size, 1.0 ml each of pituitary extracts of dog, rabbit, rat, guinea pig, turkey, and chicken was chromatographed individually onto the same column of Sephadex G-100, and the eluates were monitored for TSH activity by radioligand-receptor assay. The elution patterns of proteins, as well as TSH activity (fractions 17-28) for the pituitary extracts of all the species studied (not shown) were very similar to those of the bovine pituitary extract (Fig. 6), indicating an interspecies similarity in the molecular size of TSH. Inhibition of the TSH Activity from Various Species in the Radioligand-Receptor Assay The lyophilized fractions (elutants 17-28, Fig. 6), containing the TSH activity after

30

40

50

NUMBER

FIG. 6. Distribution of proteins and TSH activity of bovine pituitary extracts after gel filtration on Sephadex G-100 ( I x 100 cm). Protein was monitored by absorbance at 278 nm ( 0 ), and TSH activity was determined by both radioligand-receptor assay ( ... .... ) and radioimmunoassay ( --- ). The void volume (VJ and salt peak were determined by chromatographing blue dextran and NaY.

RADIOLIGAND-RECEPTOR

ASSAY

FOR

THYROTROPIN

11-Y

gel filtration on Sephadex G- 100 of the pituitary extracts from various species, were redissolved at concentrations of 100 pg dry weight/ml of buffer and assayed at different dilutions by this radioligand-receptor assay. Figure 7 shows that TSH activity in these artially purified fractions from cow, dog, rabbit, rat, guinea pig, turkey, and chicken interacted in a manner parallel to that of the purified bovine TSH standard, indicating that TSH from various species interacted in a similar manner with the porcine thyroid receptor. Similarly, crude pituitary extracts of cow, dog, rat, and turkey also inhibited in a manner parallel to bovine TSH standard (not shown).

pig, and turkey were further analyze by polyacrylamide gel electrophor ure 8 depicts the e~ectrophore~ic tion of the pituitary TSH activity fr ous species. One major peak ofT§ ity of relatively. similar electr mobilities was observed for ~it~~tar~es of cow? dog, rabbit9 and rat (Fig* 8); however, the peak of TSH activity in guinea pig was in gel segment 3; whereas, the turkey TSH activity was relatively equally divided between gel segments .5 and 7 (Fig. Q5 in lectrophoretic mob~l~t~es of of different species varied slightly.

Electrophoretic Mobility of the TSH Activity from Various Species

Numerous studies have been reporte the characterization of the birding of Ylabeled bovine TSH to particulate preparations of thyroid plasma mem ley et af., 1974; Moore and Verrier et al.*

The lyophilized fractions (elutants 17-28, Fig. 6), containing TSH activity after gel filtration on Sephadex G- 100 of the pituitary extracts from cow, dog, rabbit, rat, guinea

100Q00

10000

1000

100 q/

IO

mf

FIG. 7. Comparison of dose-response curves of radioligand-receptor assay for purified bovine TSH standard ( @ ) and the TSH activity in partially purified pituitary extracts after gel filtration on Sephadex G-100 (Fig. 6) from rat ( q ), guinea pig ( 0 ), cow, and rabbit ( !Z ), dog ( A ), chicken ( A ), and turkey (

118

WORKEWYCH

AND

ELECTROPHORETIC

CHENG

MOBILITY

rabbit

,th 5

, , , , (81 6

IL guinea

pig

12

turkey

t

GEL

6

12

SEGMENT

FE. 8. Comparison of efectrophoretic mobilities by disc gel electrophoresis of the TSH activity in pituitary extracts after gel filtration on Sephadex G-100 (Fig. 6) from cow, dog, rabbit, rat, guinea pig, and turkey. TSH activity in each gel segment was monitored by radioligand-receptor assay and expressed as a percentage of the total activity in the gel column.

Tate et ul., 1975b). The optimal pH for the bmding of 1z51-labefed TSH to porcine thyroid membranes has been demonstrated to be 6.8-7.0 (Fig. 2), in good agreement with that for guinea pig thyroid membranes (Manley et af., 1974), but that for the binding of bovine thyroid membranes has been reported to be pH 5.5 (Moore and Wolff, 1974) or pH 6.0 (Tate ef al., 1975b). The interaction between 1z5f-labeled TSH and porcine thyroid membranes has been

shown to be relatively fast at 37’ (1 hr) and moderately fast at 2Y (4 hr); whereas at O0 the interaction was very slow (Fig. 3). Our data for the effects of temperature and time on the binding between TSH and its receptor in porcine thyroid membranes were very similar to the finding of Kotani et al. (1975) for bovine thyroid plasma membranes. In contrast, Tate et al. (1975b) reported that maximal binding for 1z51-labefed TSH to bovine thyroid membranes oc-

RADIOLIGAND-RECEPTOR

ASSAY

curred within minutes at OoeIt is interesting to observe that the displacement of 1z51-labeled tracer by inreceptor-bound creasing concentrations of standard bovine TSH leveled off at about 45% (Fig. 4). This finding indicated the possibility of the existence of two binding sites with different affiniFies for TSH in porcine thyroid plasma membrane. Recently, two binding sites with very different affinity constants (KJ of 1~047 x IO8 and 0.57 x 106 AC1 have been demonstrated in partially purified bovine thyroid plasma membranes (Sate et Al., 1977). This might also be an explanation for the conflict of differences in the optimal pH and temperaFLue for the binding of lZ51labeled TSH to different preparations of thyroid plasma membranes reported by various investigaFors (Manley et ul., 1974; Moore and Wolff, 1974; Kotani et CL~.?197.5; Tate et ai., 1975b). However, a close relationship between TSH receptor binding and activation of adenyl cyclase has been demonstrated with porcine, bovine, and guinea pig thyroid plasma membranes (Manley et CZI!.,1974; Moore and Wolff, 1974; Verrier er al., 1974: Kotani et ul., Manley et LZ/. (1974) reported a specific and. sensitive radioligand-receptor assay for using guinea pig Fhyroid plasma mernbranes. In their studies on thyroid tissues from guinea pig, mouse, rat, ox, and sheep, they pointed out that only guinea pig thyroid possessed the favorable combination of high affimty and high capacity for TSH suiFable for radioligand-receptor asanley et al.. 1974); however, their guinea pig had been treated wiFh proplythiouracil for 3-12 months. Results from oFher studies on bovine thyroid plasma membranes (Kotani et u/., 1975; Tate et al., 1975b) also suggested Fhat membranes from bovine thyroids were not suiFable fQr radioligand-receptor assay because microgram quantities of purified bovine TSI-I were required for displacemenF of recepFor-bound lz51-labeled TSH. The presently reported radioligand-receptor assay

FOR

THYROTROPIN

119

utilizing porcine thyr es has been shown to able and specific assay system ( limit of detection for the assay imately 0.3 mIJ/ml; similar in the assay system reported by Manley et r~l. (1974) using guinea pig Fhyroids~ The source of porcine thyroids is readily avaihbk for preparaFions of relatively large amounts of receptors, and the procedure for preparing the particulate receptor fraction is relaFively simple as it does not require either sucrose density gradient ce~tr~fu~~~~o~ (Amir et ok!., 1973) or cell culture techniques (Iissitzky et a/., 1973). ~~rtb~rrno binding activity of the porcine plasma membranes was stable for a months upon storage at -20”. This radioreceptor assay bas been shown to be highly specific for TSH since both bovine and rat TSH of similar biological potencies inhibi the binding of r23labeled bovine T %o the porcine thyroid membrane receptor eclually well on a weight basis (Figs 4). All other glycoprotein, m-otein, and polypeptide hormones. including bovine TSH a and /3 subun concentrations of 1000-fol displace the binding of t Fracer (Figs 4). TSH activity has been deFected in l?iFuitary extracts species studied, inchding dog, guinea mg, turkey> and chick dose-response curves for t purified pituitary extracts (Fig 7)T as well as for the crude extracts (unpu servation), were all parallei %o standard bovine pituitary TSII of various species interacted with the thyroid membrane receptor in a very similar or identical manners Furthermore, utilizing this radio1 say, the molecular size 0 various species has bee be very similar identical (Fig. 6); however, in eIectrop retie mobility> slight variations between species were observed (Fig. 8).

120

WORKEWYCH

Since this radioligand-receptor for bovine TSH is activity specific, the assay system should prove very useful for comparative studies on the biochemistry and physiology of TSH activity in various species. This assay is suitable not only for measurements of activity in fractions during isolation of mammalian as well as nonmammalian TSH, but also for studies on structure-function relationships of the TSH molecule (Cheng et al., 1973). ACKNOWLEDGMENTS J. Workewych is a recipient of a graduate studentship from the University of Manitoba and K. W. Cheng is a scholar of the Medical Research Council of Canada. This research is supported by MRC (Canada) Grant MA-5110. We would like to express our appreciation to Mrs Herminia Sy and Miss Glenda Lagadi for technical assistance and to Miss Janet Greer for typing the manuscript.

REFERENCES Amir, S. M., Carraway, T. F., Jr., and Kahn, L. D. (1973). The binding of thyrotropin to isolated bovine thyroid plasma membranes.J. Viol. Chem. 248, 4092-4100. Cheng, K. W. (1975a). A radioreceptor assay for follicle-stimulating hormone. J. Clin. Endocrfno/. MetuboL 41, 581-589. Cheng, K. W. (l975b). Properties of folliclestimulating-hormone receptor in cell membranes of bovine testis. &o&em. J. 149, 123-132. Cheng, K. W. (1976). Puritication and properties of bovine pituitary follitropin. Biochem. J. 159, 65 l-659. Cheng, K. W. (1978). Isolation and characterization of the subunits of bovine follitropin. Biochem J., in press. Cheng, K. W., Glazer, A. N., and Pierce, J. G. (1973). The effects of modification of the COOH-terminal regions of bovine thyrotropin and its subunits. J. Biol. Chew. 248, 7930-7937. Davis, B. J. (1964). Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-427. Hunter, W. M., and Greenwood, F. C. (1962). Preparation of iodine-131 labelled human growth hor-

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mone of high specific activity. Nafure (London) 194, 495-496. Kotani, M., Kariya, T., and Field, J. B. (1975). Studies of thyroid-stimulating hormone binding to bovine thyroid plasma membranes. !t4erubo/ism 24, 959-971. Lissitzky, S., Fayet, G., Vet-tier, B., Hennen, G., and Jacquet. P. (1973). Thyroid-stimulating hormone binding to cultured thyroid cells. FEBS Lett. 29, 20-24. Manley, S. W., Bourke, J. R., and Hawker, R. W. (1972). Reversible binding of labelled and nonlabelled thyrotropin by intact thyroid tissue ia vitro. J. Endocrinol. 55, 555-563. Manley, S. W., Bourke, J. R., and Hawker, R. W. (1974). The thyrotropin receptor in guinea-pig thyroid homogenate: General properties. J. Endocrinol. 61, 419-436. Moore, W. V., and Wolff, J. (1974). Thyroidstimulating hormone binding to beef thyroid membranes. Relation to adenyiate cyclase activity. J. Biol. Chern. 249, 6255-6263. Sate, A., Zakarija, M., and McKenzie, J. M. (1977). Characteristics of thyrotropin binding to bovine thyroid plasma membranes and the influence of human IgG. Endocrine Res. Commun. 4,95-113. Smith, B. R., and Hall, R. (1974). Thyroid-stimulating immunoglobulins in Graves’ disease. Lancet 2, 427-431. Tate, R. L., Holmes, J. M., Kohn, L. D., and Winand, R. J. (1975a). Characteristics of a solubilized thyrotropin receptor from bovine thyroid plasma membranes. J. Biol. Chem. 250, 6527-6533. Tate, R. L., Schwartz, H. I., Holmes, J. M., Kahn, L. D., and Winand, R. J. (l975b). Thyrotropin receptors in thyroid plasma membranes. Characteristics of thyrotropin receptor activity by tryptic digestion. J. Biol. Chem. 250, 6509-6516. Teng, C. S., Smith, B. R., Anderson, J., and Hall, R. (1975). Comparison of thyrotropin receptors in membranes prepared from fat and thyroid tissue. Biochem. Biophys. Res. Commun. 66, 836-841. Verrier, B., Fayet, G., and Lissitzky, S. (1974). Thyrotropin-binding properties of isolated thyroid cells and their purified plasma membranes. Relation of thyrotropin-specific binding to adenylatecyclase activation. Eur. J. Biochem. 42, 355-365. Wolff, J., and Jones, A. B. (197I). The purification of bovine thyroid plasma membranes and the properties of membrane-bound adenyl cyclase. J. Bfol. Chem. 246, 3939-3947.

Development and application of a radioligand-receptor assay for thyrotropin.

GENERAL AND COMPARATIVE Development ENDOCRINOLOGY 35, 110-120 (1978) and Application of a Radioligand-Receptor Assay for Thyrotropin J. WORKEWYC...
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