Enzymatic radioiodination of porcine thyroid-stimulating hormone

Enzymatic radioiodination of porcine thyroid-stimulating hormone

BIOCH1MIE, 1974, 56, 769-774. Enzymatic radioiodination of porcine thyroid-stimulating hormone. P h i l i p p e JAQUET (*), Georges HILNNEN (**) a n ...

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BIOCH1MIE, 1974, 56, 769-774.

Enzymatic radioiodination of porcine thyroid-stimulating hormone. P h i l i p p e JAQUET (*), Georges HILNNEN (**) a n d Serge LISSlTZKY (* **).

(*) Clinique Endocrinologique de la Facultd de M~decine, Hdpital de la Conception, 13005 Marseille, ( " ) Section d'Endocrinologie, D~partement de Clinique et de Sdm~iologie Mddicale, Institut de M~decine, Universit~ de Liege, Belgique. (*'*) Laboratoire de Biochimie M~dicale et Unit~ thyro~'dienne de I'INSERM, Facultd de Mddecine, 27, Bd. Jean-Moulin, 13385 Murseille. (11-1-197/D. Summary. - - Enzymatic radioiodination of highly purified porcine thyroid-stimulating hormone (37 I.U./mg) was performed using lactoperoxidase, hydrogen peroxide and Na1251. Preparations of the radioiodinated hormone with specific radioactivities of 54 to 240 :~Ci/~xg (1.2 to 5.6 iodine atoms per mole thyrotropin) were obtained. For these iodine contents, biological activity as shown by the property of the iodinatcd hormone to stimulate isolated thyroid cell reorganization into follicles was preserved (80 to 100 p. cent activity). In contrast, immunoreactivity of the radioiodinated hormone preparations decreased with increasing amounts of incorporated iodine. Purity of the preparations was controlled by polyacrylamide gel electrophoresis in sodium dodecylsulfate. In this analytical system radioiodinated thyrotropin showed an apparent molecular weight of about 42,000 as compared t o a b o u t 60,000 for the native hormone suggesting that molecular rearrangements consecutive to radioiodination and/or electrophoresis in sodium dodecylsulfate occured. Ion exchange chromatography of the enzymatic digest of radioiodinated thyrotropin preparations disclosed 125I-labelled mono- and di-iodotyrosines representing about 80 p. cent and 12 p. cent of the total hormonal radioactivity respectively whatever the amount of iodine incorporated up to 4.7 atoms per mole. INTRODUCTION.

MATERIALS AND METHODS.

Studies of h o r n m n a l b i n d i n g to cell receptors requires the availability of a labelled h o r m o n e p r e p a r a t i o n of high specific r a d i o a c t i v i t y r e t a i n ing full biologicaI potency. R a d i o i o d i n a t i o n of p o l y p e p t i d e h o r m o n e s is the more w i d e l y used p r o c e d u r e : it i s : g e n e r a l l y p e r f o r m e d u s i n g the c h l o r a m i n e - T method of i o d i n e oxidation [1]. Recently, Thorell a n d J o h a n s s o n [2] a n d Miyachi et al. [3] a d a p t e d the e n z y m a t i c method of i n m m n o g l o b u l i n r a d i o i o d i u a t i o n developed by Marchalonis [4] to r a d i o i o d i n a t e several p o l y p e p t i d e hormones. The system consisted of lactoperoxidase, h y d r o g e n p e r o x i d e a n d Na125I; it allowed to obtain i m m u n o r e a c t i v e h o r m o n e s of high specific r a d i o a c t i v i t y [2, 3] w i t h preserved biological activity [3~ :

MATERIALS. Lactoperoxidase (A412/A2so = 0.653) was obtained from CalbiochelnicaI Co, (Los Angeles, USA) a n d 30 p. cent h y d r o g e n p e r o x i d e from Merck (Darmstadt, West Germany). P o r c i n e TSH was purified from a side fraction d u r i n g the p u r i f i c a tion of l u t e i n i z i n g h o r m o n e [61). The TSH prep a r a t i o n was extensively purified by the method of Liao et al. [7]. The t h y r o s t i m u l a t i n g activity of the final p r o d u c t was 37 I . U . / m g as d e t e r m i n e d by the McKenzie bioassay [8]. Bovine s e r u m album i n was from Sigma (St. Louis, Missouri, USA). Rabbit a n t i s e r u m specific to P-TSH was p r e p a r e d as p r e v i o u s l y described [6] a n d goat a n t i s e r u m to r a b b i t i m m u n o g l o b u l i n (MR 66) was p u r c h a s e d from Wellcome (Great Britain). Carrier-f.ree Na125I w i t h o u t r e d u c i n g agent (0.2 mCi/jM, specific activity --~ 10 Ci/mg) was from the Commissariat h l ' E n e r g i e Atomique (Saclay, F r a n c e ) . A m o l e c u l a r weight of 28 000 for P-TSH was used for calculations.

F o r the p u r p o s e of i n v e s t i g a t i n g the b i n d i n g of TSH to specific t h y r o i d cell receptor sites [5] we used this e n z y m a t i c method to p r e p a r e biologically active t25I-labelled p o r c i n e TSH of high radioactivity.

Abbreviations. P-TSH, porcine thyroid-stimulating hormone ; MIT, 3-iodotyrosine ; DIT, 3,5-diiodotyrosine.

METHODS. 1. Radioiodination. R a d i o i o d i n a t i o n was performed a c c o r d i n g to Thorell a n d J o h a n s s o n [2] 5O

770

P. J a q u e t , G. H e n n e n a n d S. L i s s i t z k y .

w i t h slight modifications. Lactoperoxidase (0.6 × 10 -5 M) and H20 2 (4.4 × 10 5 M) were dissolved i n 0.5 M phosphate buffer pH 7.4. One m i n u t e later, 10 ~1 of this solution was i n t r o d u c e d into a 1~0 × 75 m m glass tube c o n t a i n i n g 5 ~g of P-TSH freshly dissolved i n the same buffer at a c o n c e n t r a t i o n of 1 m g / m l . Variable a m o u n t s of carrier-free Na12~I (from 0.5 to 2 mCi) were then a d d e d to o b t a i n a final volume of 36 ~l. I n c u b a t i o n time at room t e m p e r a t u r e was 1 m i n u t e after w h i c h the reaction was stopped by a d d i t i o n of 0.4 ml of the same buffer. 2. Purification of EI~.5I]TSH. The i o d i n a t i o n m i x t u r e was i m m e d i a t e l y l a y e r e d on a 1 × 30 cm Sephadex G-50 c o l u m n e q u i l i b r a t e d w i t h 0.25 M Tris-C1 pH 8.0 a n d previously w a s h e d w i t h 1 ml of 1 p. cent b o v i n e serum a l b u m i n . Aliquots of 1 ml were collected i n tubes c o n t a i n i n g 0.1 ml of 1 p. cent bovine serum a l b u m i n . The fractions eluted i n the void volume c o n t a i n i n g about 50 p. cent of the r a d i o a c t i v i t y of t h e peak top f r a c t i o n or more were pooled (3 to 4 ml) a n d refiltered on a Sephadex G-2~00 c o l u m n (1 × 100 cm) e q u i l i b r a t e d w i t h 0.1 M p h o s p h a t e buffer pH 7.4 c o n t a i n i n g 0.5 p. cent b o v i n e s e r u m a l b u m i n . F r a c t i o n s of 1 ml were collected. All o p e r a t i o n s were p e r f o r m e d at + 2°C. Radioactivity d i s t r i b u t i o n i n effluent a n d b a l a n c e of losses in columns, gels a n d i o d i n a t i o n tube were o b t a i n e d by c o u n t i n g i n a well-type gamma s c i n t i l l a t i o n spectrometer w i t h m o d i f i e d geometry. 3. Methods of control of radioiodinated P-TSH,

c. Distribution of iodotyrosyl residues in [1~5i] TSH. It was d e t e r m i n e d a c c o r d i n g to Rolland et al. [12] by s e p a r a t i o n of MIT a n d DIT o n a Dowex-50 × 4 c o l u m n after digestion of the labelled h o r m o n e w i t h p r o n a s e and l e u c y l a m i n o peptidase i n the p r e s e n c e of 1 mg c a r r i e r h u m a n 19 S t h y r o g l o b u l i n . Aliquots of 2 ml were collected a n d counted. d. Radioimmunoassay. The p e r c e n t of [125I]TSH b o u n d to anti P - T S ~ antibodies was estimated by the double a n t i b o d y t e c h n i q u e u s i n g an excess of a highly specific anti P-TSH r a b b i t a n t i s e r u m a n d goat a n t i s e r u m to r a b b i t i m m u n o g l o b u l i n G as described p r e v i o u s l y

[9]. e. Biological activity. The p r o p e r t y of TSH to stimulate specifically the r e o r g a n i z a t i o n into follicles of c u l t u r e d isolated t h y r o i d cells [13-14] was used to control the biological activity of [l:25I]TSH p r e p a r a t i o n s [15]. An aliquot of the TSH solution w h i c h served for i o d i n a t i o n , was used as reference. I n s t a n d a r d conditions, the m i n i m u m a m o u n t of TSH detectable by this in vitro bioassay was about 0.5 to 1 n g / m l a c c o r d i n g to cell p r e p a r a t i o n s . Results are expressed as m e a n __ s.e.m. RESULTS. 1. IODINATION CHARACTERISTICS. Figure 1 shows a typical e x p e r i m e n t of separation of [125I]TSH from [125I!iodide o b t a i n e d by

a. Chromatoelectrophoresis. ~0~

~Chromatoelectrophoresis of i o d i n a t i o n m i x t u r e or p u r i f i e d 125I-labelled TSH was p e r f o r m e d a c c o r d i n g to Berson a n d Yalow [10] on W h a t m a n 3 MC p a p e r strips. After m i g r a t i o n , strips were cut in 1 c m - w i d t h segments a n d counted. b. Polyacrylamide gel eleclrophoresis. Aliquots of [12~I]TSH were s u b m i t t e d to polya c r y l a m i d e gel electrophoresis (10 p. cent acrylamide, 4 p. cent N , N - m e t h y l e n e b i s a c r y l a m i d e ) i n 0.05 M tris-glycine p H 8.5 c o n t a i n i n g 0.1 p. cent s o d i u m dodecylsulfate a c c o r d i n g to W e b e r a n d Osborn [ l l l . At the t e r m i n a t i o n of electrophoresis, gels were frozen, cut into 1 rum slices a n d counted. Native P-TSH was detected by s t a i n i n g w i t h Coomassie Brilliant Blue. Mol. wt. estimations w e r e obtained b y c o m p a r i s o n w i t h the m o b i l i t y of m a r k e r p r o t e i n s of k n o w n Mol. wt. BIOCHIMIE, 1974, 56, n ° 5.

10

i

~52 20

40

60

f r a c t ions ( 0 . s m l )

Fro. 1. - - Sephadex G-50 gel filtration of iodination mixture. Iodination with 2.8 1~5I atoms per mole of TSH. The peak eluting at about 30 ml corresponds to iodide. Bars: immunoreactivily of some fraclions.

Enzymatic

radioiodination

Sephadex G-50 gel filtration. The i m m u n o r e a c t i vity of several fractions of the excluded radioi o d i n a t e d material t o w a r d s anti P-TSH a n t i s e r u m is also indicated. Chromatoelectrophoresis of pooled excluded peak fractions showed that 81.6 _+ 1.9 p. cent of labeled m a t e r i a l r e m a i n e d at origin for i o d i n a t i o n levels of 2.2 to 8.8 i o d i n e atoms added per mole TSH.

4

3

b

The b a l a n c e of r a d i o a c t i v i t y losses d u r i n g the steps of labeling and p u r i f i c a t i o n of the h o r m o n e showed that 81.9 _ 5.1 p. cent (14 experiments) of [125I1TSH was recovered w h a t e v e r the a m o u n t of added :25I b e t w e e n 2.2 a n d 8.8 i o d i n e atoms per mole TSH was. To eliminate the possible overestimation of labeled-TSH specific r a d i o a c t i v i t y that might be related to the i o d i n a t i o n of laetoperoxidase or c o n t a m i n a n t p r o t e i n s p r e s e n t in the c o m m e r c i a l p r e p a r a t i o n of enzyme, i o d i n a t i o n e x p e r i m e n t s were p e r f o r m e d i n the c o n d i t i o n s described omitting TSH. Less t h a n 2 p. cent p r o t e i n i o d i n a t i o n (10 experiments) occured as s h o w n by chromatoelectrophoresis.

2.

,r--

P U R I T Y , CHEMICAL P R O P E R T I E S AND BIOLOGICAL

ACTIVITY OF T H E

x

E o_2 o

771

of thyrotropin.

[~25IITSH

]?REPARATIONS.

Unless otherwise noted, all the e x p e r i m e n t s desc r i b e d below c o n c e r n e d [125I]TSH p r e p a r a t i o n s c o n t a i n i n g between 1.2 to 2.5 i o d i n e a t o m s / m o l e

1

0

J

100

\

FRACTIONS

(Iml)

50

150

FIG. 2. - - S e p h a d e x G-200 gel f i l t r a t i o n of the excluded peak eluted f r o m the S e p h a d e x G-50 column

.k "til

(fig. 1). Void volume, fraction 54.

Refiltration of the Sephadex G-50 excluded peak on Sephadex G-200 (fig. 2) gave a m a j o r symmetrical peak w i t h one or occasionally two shoulders at the foot of the peak. Top peak fractions c o n t a i n e d 94 to 96 p. cent of material r e m a i n i n g at origin i n chromatoelectrophoresis. I o d i n a t i o n e x p e r i m e n t s were p e r f o r m e d u s i n g 0.2 nmole TSH a n d i n c r e a s i n g a m o u n t s of c a r r i e r free 125I- (0.4 to 1.6 nmoles) c o r r e s p o n d i n g to 2.2 to 8.8 i o d i n e atoms per mole TSH. The n u m b e r of iodine-125 atoms i n c o r p o r a t e d per mole TSH was d e t e r m i n e d by c h r o m a t o e l e c t r o p h o r e t i c analysis of the r a d i o a c t i v i t y i n c o r p o r a t e d into TSH v e r s u s u n r e a c t e d iodide. I n these c o n d i t i o n s specific radioactivities from 54 to 240 ~Ci/~g were obtained c o r r e s p o n d i n g to 1.2 to 5.6 i o d i n e atoms i n c o r p o r a t e d per mole TSH. A l i n e a r r e l a t i o n s h i p betw e e n the a m o u n t of iodide added a n d i o d i n e i n c o r p o r a t e d was s h o w n ,(fig. 3). BIOCHIMIE, 1974, 56, n ° 5.

ATOMS"Sl I N C O R P O R A ~

TSH ~o~

Fro. 3. - - Relation between the a m o u n t of [I~5I] iodide reacted and iodine content of radioiodinated TSH ( 0 ) and its i m m u n o r e a c t i v i t y (o ).

TSH. P o l y a c r y l a m i d e gel electrophoresis i n the presence of 0.1 p. cent sodium dodecylsulfate performed w i t h ten different [:25I]TStt p r e p a r a t i o n s c o n s i s t e n t l y showed the p r e s e n c e of a m a j o r peak of mol. wt. 41-43.00.0 c o n t a i n i n g 95 p. cent or more of the total r a d i o a c t i v i t y applied to the gel (fig. 4). That this c o m p o n e n t c o r r e s p o n d s to TSH is b e y o n d doubt on the basis of i m m u n o r e a c t i v i t y a n d biological activity (see below). P o l y a c r y l a m i d e gel electrophoresis i n s o d i u m d o d e c y l s u l fate of the native P-TSH w h i c h served as substrate for i o d i n a t i o n disclosed a mol. wt. of 60.000 w h i c h is about twice that of native m o n o m e r i c 51

P. Jaquet, G. Hennen and S. Lissitzky.

772

TSH. The n a t u r e of the a m i n o acid residues i n v o l v e d i n TSH i o d i n a t i o n w a s investigated b y i o n - e x c h a n g e c h r o m a t o g r a p h y after e n z y m a t i c digestion. As s h o w n in figure 5 two r a d i o a c t i v e peaks exactly c o r r e s p o n d i n g to t h e : e l u t i o n volume

r a d i o a c t i v i t y recovery after c h r o m a t o g r a p h y was quantitative. Up to 4.7 i o d i n e atoms i n c o r p o r a t e d per mole of TSH, no definite r e l a t i o n b e t w e e n DIT c o n t e n t a n d the degree of i o d i n a t i o n was observed. W i t h excess of homospecific a n t i b o d y a n d a ratio of i o d i n e atoms i n c o r p o r a t e d per mole of TSH --~ 2, 74.3 _ 2.2 p. cent of [125IlTSH were i m m u n o p r e c i p i t a b l e . An i n v e r s e r e l a t i o n s h i p betw e e n i o d i n e c o n t e n t a n d i m m u n o r e a c t i v i t y was o b s e r v e d (fig. 3). Using 3 p r e p a r a t i o n s c o n t a i n i n g 1.5, 2.0 and 3.0 i o d i n e atoms p e r mole of TSH, essentially i d e n t i c a l dose-response curves were o b t a i n e d (fig. 6). The starting B / T were 44, 41 a n d 29 p. cent, respectively. The d i s p l a c e m e n t of the labelled h o r m o n e expressed by B/B o w i t h

4 ? o3 u m

10

20

30

40

100

SLICE N U M B E R

FI6. 4. - - P o l y a c r y l a m i d e gel electrophoresis in the presence of s o d i u m dodecylsulfate of [1~5I] TSH (2.$ iodine atoms~mole TSH).

of MIT a n d DIT came out of the column. Two m i n o r c o m p o n e n t s eluting before a n d after the DIT peak likely c o r r e s p o n d to u n d i g e s t e d material. Calculated on 1i experiments, MIT a n d DIT

80

Q 0

60

x 0 en

40 MIT

20 30

? o

I

E o

0.5

20-

I

1.0

P-TSH

I

1.5

I

2.0

I

2.5

ing~

FIG. 6. - - Dose r e s p o n s e curves using P - T S H and [1~5I] P - T S H of iodine content 1.5 (/5_), 2.0 ( © ) and

I ,o

DIT

I

I 2O

I

fractions [ 0.5 ml ]

Fro. 5. - - S e p a r a t i o n of radioiodinated c o m p o u n d s

by ion-exchange c h r o m a t o g r a p h y (Dowex 50 × 4) of the pronase and leueylaminopeptidase digest of [1~5I] TSH (2.$ iodine atoms~mole TSH).

r e p r e s e n t e d 80.4 ± 1.1 p. cent a n d 12.4 ___ 1 p. cent of the total r a d i o a c t i v i t y eluted from the column, respectively, a n d u n d i g e s t e d m a t e r i a l 10.3 _ 2.2 p. cent. W i t h i n e x p e r i m e n t a l errors, BIOCHIMIE, 1974, 56, n ° 5.

3.0 ( e ) iodine a t o m s per mole of TSH. A n t i s e r u m to P - T S H used at 1:50 000 solution.

i n c r e a s i n g a m o u n t s of native P-TSH b e t w e e n 0.075 a n d 2.5 ng was very s i m i l a r for all three preparations. Bioassay of [125I]T,SH p r e p a r a t i o n s u s i n g the p r o p e r t y of TSH to specifically stimulate the reorganization into follicles of isolated c u l t u r e d thyroid cells [15], showed r a d i o i o d i n a t e d TSH to have a p o t e n c y of 0.8 to 1.2 relative to the P-TSH solution w h i c h served for r a d i o i o d i n a t i o n . I n seven experiments, the m i n i m u m a m o u n t of labelled-TSH s t i m u l a t i n g cell r e o r g a n i z a t i o n was 100 to 150 p g p e r 1 X 105 cells i n a volume of 0.2 ml.

Enzymatic radioiodination of thyrotropin. DISCUSSION. R a d i o i o d i n a t i o n of TSH u s i n g lactoperoxidase oxidation of iodide resulted in p r e p a r a t i o n s of [125I]TSH of very high specific r a d i o a c t i v i t y w i t h preserved biological potency. As r e c e n t l y r e p o r t e d [5], these p r e p a r a t i o n s were suitable for investigating the b i n d i n g of TSH to specific t h y r o i d cell receptor sites. After sequential p u r i f i c a t i o n on Sephadex G-50 and G-20,0 the level of c o n t a m i n a n t s r e p r e s e n t e d 4 to 6 p. cent of total i o d i n e for p r e p a r a t i o n s cont a i n i n g b e t w e e n 1.2 a n d 4.7 i o d i n e a t o m s / m o l e TSH as s h o w n by chromatoelectrophoresis. Highly p u r i f i e d p r e p a r a t i o n s of TSH show poiym o r p h i s m in a c r y l a m i d e gel electrophoresis, the different c o m p o n e n t s b e i n g closely related to each other c h e m i c a l l y a n d i m n m n o l o g i c a l l y a n d all e x h i b i t i n g h o r m o n a l activity [17]. It is therefore not excluded that the small a m o u n t of labelled material m i g r a t i n g b e t w e e n the zone of deposition and i o d i n e in p a p e r electrophoresis might represent biologically active r a d i o i o d i n a t e d material and not h o r m o n e damages. I n p o l y a c r y l a m i d e gel electrophoresis i n s o d i u m dodecylsulfate r a d i o i o d i n a t e d P-TSH migrates as a sharp homogenous peak but some special features have been noted. The a p p a r e n t nmlecular weight of the native P-TSH used i n our investigalions was controlled by approach-to-equil i b r i u i n u l t r a c e n t r i f u g a t i o n a n d was s h o w n to be 2,8-3.0,0,0,0 in agreement w i t h the size of TSH mon o m e r d e m o n s t r a t e d by s t r u c t u r a l studies [17]. However, a tool. wt. of about 60.0,0~0 c o r r e s p o n d ing to the TSH d i m e r was f o u n d by p o l y a c r y l amide gel electrophoresis in sodium dodecylsulfate w h e r e a s [125I]'PSH analyzed i n the same conditions disclosed an a p p a r e n t mol. wt. of 42,000 w h i c h a p p r o x i m a t e l y could c o r r e s p o n d to the association of three about 14,00,0 subunits. These results suggest that molecular r e a r r a n g e m e n t s occur consecutive to r a d i o i o d i n a t i o n a n d / o r electrophoresis i n sodium dodecylsulfate. I o d i n e 125-labelling of P-TSH can be a t t r i b u t e d essentially to m o n o i o d i n a t i o n of t y r o s i n e ; the [~25I]DIT c o n t e n t does not i n c r e a s e significantly w i t h i n c r e a s i n g i o d i n e content of the h o r m o n e suggesting that a d d i t i o n a l t y r o s i n e residues were m o n o i o d i n a t e d w i t h i n c r e a s i n g i o d i n e content. It is therefore likely that p r e p a r a t i o n s of [125I]TSH c o n t a i n i n g 2 atoms of i o d i n e p e r mole of TSH were almost d e p r i v e d of u n l a b e l l e d h o r m o n e . As judged b y c h r o m a t o e l e c t r o p h o r e s i s a n d i m m u n o r e a c t i v i t y , t h e labelled P-TSH p r e p a r a -

BIOCHIMIE, 1974, 56, n ° 5.

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tions r e m a i n e d stable for at least 1,6 days w h e n stored at 0 ° or - - 1 9 6 ° C . I n the presence of an excess of specific anti TS,H a n t i s e r u m , the p e r c e n t of i m m u n o p r e c i p i t able [12~I]TSH never exceeded 80 p. cent for the lowest studied i o d i n a t i o n levels (1.2 i o d i n e a t o m s / mole TSH). This c a n n o t be e x p l a i n e d by contam i n a n t s of the p r e p a r a t i o n s , since r a d i o i o d i n a t e d TSH 94 to 96 p. cent homogenous as s h o w n b y electrophoretic controls b e h a v e d similarly. Since i m m u n o r e a c t i v i t y of [12~I]TSH decreased w i t h i n c r e a s i n g i o d i n e i n c o r p o r a t i o n , it is highly probable that loss of i m m u n o r e a c t i v i t y is related to i o d i n a t i o n even for the less i o d i n a t e d p r e p a r a tions. E x t r a p o l a t i o n of the line r e p r e s e n t i n g i m n m n o r e a c t i v i t y versus i o d i n e c o n t e n t crosses the y axis at about 9f2 p. cent (fig. 3) w h i c h is close to the p u r i t y of the labelled p r e p a r a t i o n s analyzed (94-9,6 p. cent). An i n t e r e s t i n g feature of P-TSH i o d i n a t i o n was that w h e r e a s i m m u n o r e a c t i v i t y of freshly prepared [a25I]TSH decreased with i n c r e a s i n g i o d i n e content, biological activity, as s h o w n by the specific p r o p e r t y of TSH to stimulate the reassociation of isolated t h y r o i d cells into follicle a n d to b i n d to t h y r o i d cell receptor sites [5] r e m a i n e d almost unaffected. No e x p l a n a t i o n of this p h e n o m e n o n is yet available. However, it is p r o b a b l e that discrete d e t e r m i n a n t s for b i n d i n g to cell receptors a n d to a n t i b o d y are p r e s e n t i n the TSH molecule. After completion of the e x p e r i m e n t s described in this paper, we observed that the use of 1:6 the a m o u n t of LPO (0.8 ~g) a n d a stoechiometric a m o u n t of H20 ~ (0.34 ng) to i o d i n a t e 5 ~g P-TSH gave r e p r o d u c i b l y i d e n t i c a l results. This procedure is n o w adopted to p r e p a r e [12~I] TSH of high specific radioactivity.

Acknowledgements. T h e skilful technical assistance of Mrs L. Vinet and Miss M. Leone is gratefully acknowledged. These studies were supported in part by the D~lfigation h la R e c h e r c h e Scientifiquc et Technique and the Commissariat h l'Energie Atomiquc. R~SUMI~.

Le marquage de la thyrotropine de pore hautement purifi6e (37 U1/mg) par l'iode radioactif a 6t6 r6alis6 en utilisant la l a c t o p e r o x y d a s e , l'eau o x y g d n ~ e et Na125I. Des pr6parations de l'hormone radioioddc de radioactivitds sp6eifiques ~lev6es (54 h 240 ,IxCi/,~g, soit 1,2 h 5,6 atomes d'iode par mole de thyrotropine) out 6t$ obtcnues. L'activitfi biologiquc de ces prdparations mcsurfic par la propri~t~ de l'hormone de stimuler la r6organisation cn follicules de cellulcs thyroidiennes

P . J a q u e t , G. H e n n e n a n d S. L i s s i t z k y .

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isol~es en culture est eonserv~e (80, h 100 p. cent d'aetivit6). Par contre, leur immunor~aetivit~ d~eroit en fonctiou de la quantit~ d'iode ineorpor~ dans la moldeule. La puret~ des p r e p a r a t i o n s a 6t6 eontrol6e p a r ~leetrophor~se en gel de p o l y a e r y l a m i d e - d o d ~ e y l s u l fate de sodium. Dans ce syst~me analytique, la t h y r o tropine radioiod~e a une masse mol6eulaire a p p a r e n t e d ' e n v i r o n 42 000 alors qu'elle est d'environ 60 000 p o u r l ' h o r m o n e non iod~e, sugg~rant que des r~arrangements mol6eulaires eons~eutifs h la r a d i o i o d a t i o n e t / o u h l'61eetrophor~se en presence de dod~eylsulfate de sodium ont lieu. Mesur~s p a r c h r o m a t o g r a p h i c d'6ehange d'ions des h y d r o l y s a t s e n z y m a t i q u e s de F h o r m o n e radioiod~e, les taux de 1251 mono- et diiodotyrosines repr~sentent respeetivement environ 80 p. cent et 12 p. cent de la radioaetivit6 h o r m o n M e totale quelque soit la quantit~ d'iode incorpor~ j u s qu'h 4,7 atomes p a r mole d'hormone.

3. Miyaehi, Y., Vaitutakis, J. L., Nieschlag, E. & Lipsett, M. B. (1972) J. Clin. Endocr., 34, 23-28. 4. Marchalonis, J. J. (1969) Biochem. J., 113, 299-305. 5. Lissitzky, S., Fayet, G., Verrier, B., Hennen, G. ,¢ Jaquet, P. (1973) FEBS Letters, 29, 20-24. 6. Hennen, G., Prusik, Z. a Maghuin-Rogister, G. (1971) Eur. J. Biochem., 18, 376-383. 7. Liao, T. H., Hennen, G., Howard, S. M., Shome, B. & Pierce, J. G. (1969) J. Biol. Chem., 24~1, 6458-6464. 8. McKenzie, J. M. (1958) Endocrinology, 63, 372-382. 9. Jaquet, P., Ketelslegers, J. M., Jakubowski, H. F r a n c h i m o n t , P. (1917) Ann. Endocrinol. (Paris), 32, 483-494. 10. Berson, S. A. ~ Yalow, R. S. (1958) Ann. N.Y. Acad. Sci., 35, 56. 11. Weber, K. ,~ Osborn, M. (1969) J. Biol. Chem., 244, 4406-4412. 12. Rolland, M., Aquaron, R. ~ Lissitzky, S. (1970) Analyt. Biochem., 33, 307-317. 13. Fayet, G. & Lissitzky, S. (1970) FEBS Letters, 11, 185-188.

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14. Lissitzky, S., Fayet, G., Giraud, A., Verrier, B. & Torresani, J. (1971) Eur. J. Biochem., 24, 88-99. 15. Planells, R., Fayet, G., Hennen, G. a Lissitzky, S., in preparation. 16. Yang, K. P. ~ Ward, D. N. (1972) Endocrinology, 91, 317-320. 17. Pierce, J. G. (1971) Endocrinology, 89, 1331-1334.