THE JOURNAL cm BIOLOGICAL CHEMISTRY

Vol. 246, No. 21, Issue of November 10, pp. 6570-6575, 1971

Printed in U.S.A.

Relationship of Thyrotropin to Exophthalmos-producing Substance

FORMATION OF AN EXOPHTHALMOS-PRODUCING SUBSTANCE BY PEPSIN DIGESTION OF PITUITARY GLYCOPROTEINS CONTAINING BOTH THYROTROPIC AND EXOPHTHALMOGENIC ACTIVITY

(Received for publication, July 7, 1971)

LEONARD D. KOHN* AND ROGER J. WINAND$

From the Laboratory of Biochemical Pharmacology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland %‘0014, and The Institute of Medicine, Department of Clinical Medicine and Semiology, University of LGge, Belgium

SUMMARY

Homogeneous bovine pituitary glycoproteins with both thyrotropic and exophthalmogenic activity have been sub- jected to limited proteolysis by pepsin. One fraction lost 90 % of its thyrotropin action, but only 15 % of its exophthal- mogenic activity, when incubated with a 1% weight ratio of pepsin at pH 2.2 and at 3’7”. Analytical disc gel analyses showed that the decrease in thyrotropin activity could be cor- related with the destruction of the glycoprotein originally in this fraction and with the appearance of several new protein- staining bands. One of the digestion products was an oligo- saccharide-containing derivative with terminal galactose on at least one of its carbohydrate moieties. After isolation by electrofocusing, this derivative was shown to have exophthal- mogenic activity but to be without significant thyrotropin action; the residual thyrotropin activity of limited digests re- mained associated with nondigested glycoproteins containing both activities. The exophthalmogenic derivative had an isoelectric point at pH 6.9 and exhibited a single component on analytical disc gels. It had a molecular weight of 20,000 to 22,000 when evaluated by electrophoresis on gels contain- ing sodium dodecyl sulfate, but appeared to be composed of two polypeptide units, one with a molecular weight of ap- proximately 14,000 and the other with a molecular weight of approximately 6,000. The results indicate that limited pro- teolysis of a homogeneous pituitary glycoprotein with both thyrotropic and exophthahnogenic activity can yield a deriva- tive uniquely active as an exophthalmos-producing substance.

In 1931, Schokaert (1) demonstrated that pituitary extracts produced exophthalmos in ducks. Although initial experiments

* Temporary address, Institut de Medecine, Departement de Clinique et de Semiologie medicales, University of Liege, Belgium.

$ Chercheur qualifie au Fonds National de la Recherche Scien- tifique (Belgium).

indicated that this activity was associated with thyrotropin preparations purified from pituitary extracts (2-6), Dobyns suggested that the exophthalmos-producing substance was a factor distinct from TSHi (7) since he could partially purify the TSH activity without concomitant increases in EPS action. Subsequent experiments supported this suggestion. TSH and EPS were shown to have different solubilities in 8% trichloro- acetic acid (8) ; TSH was more rapidly destroyed than EPS by either iodination (9) or partial pepsin digestion of partially purified extracts (10) ; partial separation of the two activities was obtained by ion exchange chromatography (11-14) ; and no EPS activity was measurable in transplantable mouse tumors which secreted TSH (15). Nevertheless, isolation of an EPS fraction without TSH activity could not be accomplished. Ion excha,nge procedures which claimed to separate the activities were not reproducible (ll-14), extensive purification by two laboratories (16-20) yielded fractions with both activities only, and, most recently, homogeneous glycoproteins were isolated from bovine pituitary extracts and shown to contain both ac- tivities within the same macromolecular structure (21).

To reconcile these data (&21), it was suggested that the TSH and EPS determinants resided on the same glycoprotein mole- cule, that the partial structure responsible for each determinant could react differently with selective reagents, and that destruc- tion of the TSH determinant could result in a residual structure having only EPS action (21). In the present report we have evaluated the action of pepsin on crude and highly purified glycoprotein fractions containing both TSH and EPS activity. These experiments were performed with tritium-labeled (21) glycoproteins in order to facilitate the subsequent characteriza- tion of the proteolytic products. Conditions of digestion are described, and a preliminary evaluation of the digestion products is presented. The results demonstrate that an exophthalmos- producing substance with no significant TSH action can be obtained from a pituitary glycoprotein which originally had both determinants on the same macromolecular structure.

1 The abbreviations used are: TSH, thyrotropin; EPS, exoph- thalmos-producing substance; CM-cellulose, carboxymethyl- cellulose; SDS, sodium dodecyl sulfate.

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MATERIALS

Crude bovine pituitary thyrotropin was a commercial prepara- tion (Ambinon Organon, Oss, Holland) with a specific activity of 0.6 i.u. per mg. Galactose oxidase, 45 units per mg, was purchased from Worthington, and pepsin was a 3X-recrystal- lized preparation from Pentex. Sephadex G-25 and Sephadex G-100 were products of Pharmacia; CM-cellulose and DEAE- cellulose were products of the Reeve Angel Company, New York, New York. The sodium [*H]borohydride (200 mCi per mmole) was obtained from Amersham-Searle, Arlington Heights, Illinois.

METHODS

TSH was assayed by the method of McKenzie (22). Ex- ophthalmogenic activity was detected by the method of Brouhon- Massillon (23) a.fter a screening test described by Dedman, Fawcett, and Morris (20) which eliminated “nonresponder” fish. TSH specific activity has been defined in terms of international units per mg of protein and was assayed against the bovine international standard. EPS specific activity has been defined in terms of equivalent TSH units (23) in a crude commercial preparation (Ambinon) . Protein was determined colori- metrically with the use of recrystallized bovine serum albumin as the standard (24).

Analytical disc gel electrophoresis was performed with a pH 9.5 system (25). Gels were stained for protein with Amido black or Coomassie blue (26) and for carbohydrate with a periodic acid-Schiff reagent (27).2 Radioactivity on the gels was measured by the method of Tishler and Epstein (28). Electrofocusing was performed in an LKB 8102 column and used an ampholyte gradient (pH 3 to 10) in sucrose. The anode was at the bottom of the column, and cooling was effected by circulating ice water. Electrofocusing was continued until the current had decreased to a constant value at a constant voltage, usually 24 to 48 hours.

Terminal gala&se residues on the glycoproteins of bovine TSH (Ambinon) were tritiated by the method of Morell et al. (29), as applied by Winand and Kohn (21). Galactose oxidase, 675 units, and 6.0 g of crude TSH were dissolved in 40 ml of 0.05 M sodium phosphate, pH 7.0, which was 0.05 M in sodium chloride. Toluene, 0.04 ml, was added to the mixture, and it was incubated at 37” for 24 hours. The reactants were then diluted 5-fold by 0.05 M sodium phosphate, pH 7.8, containing 0.05 M sodium chloride; sodium [aH]borohydride, 100 mCi, was added; and the solution was allowed to react for 30 min at room temperature. After an equivalent period of reduction by non- labeled sodium borohydride, 1 InM, the solution was concen- trated to 30 ml by evaporation in a vacuum.

Glycoproteins containing both TSH and EPS activity were purified by means of the series of sequential column chroma- tographic procedures previously described (21) : Sephadex G-100 chromatography, CM-cellulose chromatography, and DEAE- cellulose chromatography. The pooled concentrate of the final DEAE-cellulose fractionation procedure contained four glyco- protein components with both TSH and EPS activity, two major (RF 0.25 and 0.13 on analytical gels) and two minor (RF 0.4 and 0.35 on analytical gels); these four components were

2 We are indebted to Dr. V. Marchesi, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland, for this technique.

separated and individually isolated by preparative disc gel electrophoresis (21). The specific activities of the DEAE- purified glycoprotein fraction and of the individual preparative gel fractions are listed in Table I. The amino acid compositions of all four of the preparative gel fractions were effectively identical and have previously been reported (21). The carbohydrate composition of each fraction is noted in Table II; only the galactose moiety was labeled with tritium.

TABLE I

TSH and EPS activities of pituitary glycoproteins isolated by preparative gel electrophoresis

Speciiic activityb Fraction Active glycoprotein

components= TSH EPS

RF i.u./?ng i.u./mg

PurifiedDEAEfraction.. 0.4, 0.35, 0.25, 0.13 5.4 4.8 Preparative disc fraction

I.. . . . . . . . . . . . . . . . . . . . 0.4 0.04 0.05 II.. . . . . . . . . . . . . . . . . . . 0.4,0.35 0.05 0.045 III.. 0.25 5.0 4.5 IV .._................. 0.13 6.0 5.5

a The components present in each fraction are indicated by their migration (RF) on 7% analytical gel at pH 9.5 (25). Preparative disc fractions I, III, and IV remained single banded when 10, 12, or 15% running gels were also used.

b TSH and EPS activities were in all cases measured with the use of lyophilized concentrates. As noted previously (21), the low TSH activity can in part, be explained on this basis; however, the tritiation procedure (which now includes reduction with ex- cess nonlabeled borohydride) appears to increase the TSH la- bility as well as decrease the specific activity of purified fractions. TSH activity is defined in terms of bovine international units; EPS activity is defined in equivalent TSH unite (23) in reference to the Ambinon preparation.

TABLE II

Carbohydrate composition” of TSH- and EPS-active glycoprotein fractions

Purified DEAE glycoprotein

fractionb

Galactose. 2.3 (0.5)d Mannose.. _. . 6.7 (7.3) Fucose... _. 1.1 (1.1) Glucosamine.. 6.8 (7.5) Galactosamine 4.3 (3.1)

-

Preparative disc gl %

coprotein fractions

I I I

II III

moles/mole )r&id

2.5 2.4 2.4 7.0 6.7 6.8 1.1 1.1 1.0 6.5 6.7 6.8 4.1 4.2 4.1

- I

L-

Iv

2.0 6.1 1.0 6.7 4.2

a Sugars were measured by two different methods (30, 31) ; the average of all results is presented.

b The TSH- and EPS-active glycoprotein components present in each fraction are noted in Table I.

c These data are based on a molecular weight of 28,000 for each component (21).

d The numbers in parentheses are the average values for each sugar as measured by Kim et al. (32) on similarly purified bovine material. Differences in galactose measurements appear to re- flect, a difference in methods since gas chromatography yields a value of less than 1 on the present samples.

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6572 Th yrotropin and Exopktkalmos-producing Xubstarze Vol. 246, x0. 21

CRUDE TSH

T,ME OF PEPSIN DIGESTION (MINUTES)

FIG. 1. Action of pepsin on TSH and EPS activities of pituitary extracts. Samples containing 5 mg of a crude pituitary TSH preparation (Ambinon) (A) or 5 mg of the DEAE-purified glyco- protein fraction (B) of Table I were incubated with 50 pg of pepsin. Incubations were at 37” in 0.2 M sodium citrate, pH 2.2; final volumes were 0.2 ml. At the time indicated, the reaction mixtures were neutralized with 0.2 M NaOH, diluted 5-fold with normal NaCl solution, quick frozen in a dry ice-acetone mixture, and stored at -10” until assayed. Multiple aliquots were evaluated for both TSH (0) and EPS (0) activity. Bars represent the range of all experimental values; points designate the average.

RESULTS

Optimal Conditions of Proteolytic Digestion--The sensitivity of

the TSH and EPS determinants to pepsin digestion was ex- amined with the use of a tritiated crude TSH preparation (Ambinon) as well as the DEAE-purified glycoprotein fraction containing only the four tr,tiated glycoproteins with TSH and EPS activity. In both preparations (Fig. 1, A and B), there was a loss of TSH activity without a coincident decrease m EPS activity when fractions were incubated with pepsin (1% by

weight) in 0.2 M sodium citrate, pH 2.2. After 15 min of diges- tion at 37”, residual TSH activity was 20% of its initial level, whereas resdual EPS activity remained at 80 to 90%. Con- trol incubations, maintained under identical conditions without pepsin, had no measurable loss of TSH or EPS activity in the first hour of reaction. Aliquots from control incubations con- taining a boiled, inactivated crude extract (Ambinon) plus pepsin had no activity in either the TSH or EPS bioassay and did not affect the activity of standards used to calibrate the TSH and EPS response curves (19-21). Ambinon preparations and DEAE-purified glycoprotein fractions which had not been subjected to the galactose tritiation procedure gave analogous results, i.e. the tritiation procedure did not affect the proteolytic sensitivity of these glycoproteins.

Digestions at pH 3.8 and 4.5 did not result in as great a preferential destruction of TSH activity as noted at pH 2.2 (Fig. 2A). Increases or decreases in the ratio of pepsin to glycoprotein caused corresponding changes in the rate of diges- tion as well as changes in the differential sensitivity of the two

determinants during the first portion of the reaction (Fig. 2B).

The optimal conditions for pepsin digestion, i.e. those condi- tions under which TSH activit,y was maximally destroyed while EPS activity was maximally preserved, were thus: 37”, pH 2.2, 0.2 M sodium citrate, and a 1% weight ratio of pepsin.

Products of Proteolytic Digestion-Aliquots of the partially digested DEAE-purified glycoprotein fraction were subjected to

I I TSH I 60 120 60 i;O

TlME OF PEPSlN DfGESTiON (MINUTESI

FIG. 2. Effect of the pH (A) and the pepsin:protein ratio (B) on the TSH and EPS activity of pituitary extracts. Conditions of incubation were the same as in Fig. 1A with the following varia- tions. In A the incubation pH was at 3.8, 4.5, or 7.0. A Tris- chloride buffer was used for the pH 7.0 incubation; sodium citrate was used at pH 2.2, 3.8, and 4.5. In B the pH was 2.2, but the pepsin added was, respectively, 1, 0.5, and 2y0 by weight of the pituitary extract. Nontritiated crude preparations (Ambinon) and both tritiated and nontritiated DEAE-purified glycoprotein fractions yielded similar data.

analytical disc electrophoresis at different times after the ini- tiation of pepsin digestion (Fig. 3). During the first 30 min of digestion, the TSH- and EPS-active glycoprotein having an RF of 0.25 (Fig. 3, Peak 111) was almost completely destroyed without marked changes in the other active components, RF 0.45, 0.35, and 0.13 (Fig. 3, Peaks I, 11, and IV, respectively). In the same period of time, several new protein-staining bands appeared at RF values of 0.64, 0.60, 0.21, and 0.18 (Fig. 3, Peaks A, B, C, and D, respectively). Two of these new species, RF 0.64 and 0.60, stained positively for the presence of carbo- hydrate when gels were exposed to the periodic acid-Schiff re- agent. The same two species were also correlated with peaks of tritium counts, i.e. they had a carbohydrate moiety containing a terminal galactose residue. At 1 hour after the addition of pepsin, when TSH activity was only 20% of controls (Fig. 1, A and B), the glycoproteins having both TSH and EPS activity (Fig. 3, Peaks Z through IV) were markedly diminished in quantity, i.e. their disappearance could be correlated with the loss in TSH activity.

By similarly digesting glycoprotein fractions isolated by preparative gel electrophoresis (Table I), the differential pro- teolytic sensitivity of the RF 0.25 glycoprotein was confirmed (Table III). Analytical disc gels showed the formation of the same major digestion products, and, in a parallel experiment in which nonstained gels were laterally sliced and the slices eluted with 0.05 M ammonium bicarbonate, pH 7.4, bioassays of the eluates indicated that the major digestion product (Fig. 3, Peak B) had EPS activity. The preservation of EPS activity in partial pepsin digests thus appeared to be related to the formation of an EPS-active derivative having a terminal galac- tose moiety and little or no thyrotropin action.

Isolation of EPS-active Digestion Derivative-The DEAE- purified glycoprotein fraction which contained all four of the TSH- and EPS-active glycoproteins was digested with pepsin for 15 to 30 min under the optimal conditions described. Di- gests were neutralized, cooled to 2’, and desalted by either di- alysis or Sephadex G-25 chromatography with the use of O.lyc

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10 20 30 40 GEL SLICE

FIG. 3. Snalytical disc gel analyses of the DEAE-purified glycoprotein fraction (Table I) which was subjected to pepsin digestion (Fig. 1B). Samples were exposed to pepsin under conditions identical with those described (Fig. 1). After diges- tion had been initiated, individual samples were neutralized with 0.2 M NaOH at the indicated times and stored frozen until all the timed samples had been obtained. Duplicate loo-fig aliquots of each sample were then run simultaneously on analytical disc gels. One gel from each sample was stained with Amido black, and the protein components were evaluated by scanning the gel in a Joyce-Lobe1 densitometer. A second gel from each sample was sliced laterally into 44 equal pieces (1.5 mm), and the tritium content of each piece was determined. The tritium present in each gel slice (o- - -0) was correlated with the protein-staining species located by densitometry (--). Peaks I, ZZ, ZZZ, and IV are the original doubly active glycoproteins listed in Table I, RF 0.45, 0.35, 0.25, and 0.13, respectively. The blackened peak in the center of each frame is a glycoprotein standard included in each sample; it was used to correlate the locations of the different peaks on different gels and to validate the carbohydrate-staining procedure (see under “Methods”). The cross-hatched peaks locate the new protein-staining bands appearing during the pepsin digestion, i. e. Peaks A, B, C, and D.

ampholine solution (pH 3 to 10) which was 1 mM in Tris-chloride at pH 7.4. The desalted digest was electrofocused as described under “Methods.”

The tritiated derivative with EPS activity (Fig. 3, Peak III)

had a p1 of 6.9 (Fig. 4, Peak I) and was easily separated from the residual nondigested glycoproteins with p1 values between 8.9 and 9.9 (Fig. 4, Peak II). The fractions denoted by the

shaded bars (Fig. 4) were pooled, dialyzed against 0.05 M am- monium bicarbonate, pH 7.4, and lyophilized. The yield and

biological activity of each fraction were measured (Table IV). The glycoprotein derivative in Peak I contained tritiated (ter-

TABLE III

TSH and EPS activities of glycoprotein fractions puGfLed by preparative gel electrophoresis and exposed to pepsin

digestion for 25 min

Preparative disc fractions

I II III IV

I Specific activityb

Glycoprotein compcnentsQ Before pepsin digestion

After pepsin digesti&

RF units/mg units1mg 0.4 0.04 0.05 - - 0.4, 0.35 0.05 0.045 - - 0.25 5.0 4.5 0.5 3.8 0.13 6.0 5.5 4.0 3.8

TSH 1 EPS 1 TSH / EPS

Q The components present in each fraction are indicated by their migration on 7% analytical gels at pH 9.5 (25).

b TSH activity is defined in terms of bovine international units; EPS activity is defined in equivalent TSH units (23) in reference to the Ambinon preparation.

0 Dashes designate the absence of detectable activity.

FRACTlON

FIG. 4. Electrofocusing of TSH- and EPS-active glycoproteins after partial pepsin digestion. Tritiated glycoproteins in the “DEAE-purified” fraction (Table I), 20 mg, were digested with pepsin for 15 min at pH 2.2 and 37”. The digestion was stopped by neutralization, salts were removed by dialysis, and the sample was loaded onto a LKB 8102 electrofocusing column by mixing it with the light solution. An ampholine gradient (pH 3 to 10) in sucrose was used; the anode was at the bottom. After 24 hours at 250 volts, with constant current for the last 10 hours, 3-ml fractions were collected at a flow rate of 2 to 3 ml per min. Frac- tions were evaluated for their protein (O-O) and tritium (O---O) content. Assayed directly, Fraction 84 was positive for EPS activity (5 i.u. per mg) and negative for TSH (0.001 i.u. per mg). Fractions 78 through 88 and Fractions 115 through 140 (shaded bars) were pooled, dialyzed against 0.05 M ammonium bicarbonate, lyophilized, and evaluated for TSH and EPS activity (Table IV) as well as homogeneity (Fig. 5). Fractions 40 to 70, 71 to 77,89 to 95, and 96 to 114 were similarly evaluated and served as controls in the bioassays (Table IV, see footnotes).

minal) galactose and had an EPS:TSH activity ratio of at least 5000; the nondigested glycoprotein material in Peak II had an EPS:TSH activity ratio of approximately 1. Since the thy- rotropin activity of the Peak I glycoprotein derivative was less than 0.1% of the preparation before digestion and at a level beyond the limits of detectable contamination by residual non- digested material, this derivative has been considered an “ex-

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6574 Thyrotropin and Exophthalmos-producing Substance Vol. 246, No. 21

TABLE IV

TSH and EPS activities of fractions obtained after electrofocusing pepsin digest of purijIed DEAE-glycc

1 I Fraction Fracjiog: :,m Total

protein

Applied Purified DEAE

fraction Recovered0

Peak I (tritiated 78 to 88 glycopeptide)

Peak II (non- 115 to 140 digested gly- coproteins)

mi?

20

5.7

2.4

protein fraction (Fig. 4)

Specific activity

TSH EPS

i.u./mg

5.4

< O025b

2.1

units/mg

4.8 f 1

7.5 i 2.5

2.9 * 1

Q Fractions 45 to 70, 70 to 77, 89 to 95, and 96 to 114 were also pooled, concentrated, and assayed. The protein recovered in each of the concentrates was 1.0,1.6,2.1, and 1.6 mg, respectively. The TSH specific activities were 0, <.0025, <.0025, and 0.25 i.u. per mg, respectively; the EPS specific activities were 0, 1.5 i 0.5, 1.5 f 0.5, and 0.25, respectively.

t TSH activity was not detected in assays sensitive to this level of hormonal activity. Peak I gave a borderline positive response at a sensitivity level of 0.001 i.u. per mg.

Y

y, I I I I 10 20 30 40 20 no 60 80 OEL SLlCE RELATIYE MOalLlTY

FIQ. 5. Analytical disc gels of EPS-active fractions isolated after electrofocusing of pepsin digests (Fig. 4, Table IV). A, analytical gels with a pH 9.5 system (25) and a 12% running gel. The upper frame is a gel run with a 5C+g aliquot of the TSH- and EPS-active nondigested glycoprotein fraction from Peak ZZ of Fig. 4. The lower frame is a gel run with a 50-pg aliquot of the EPS-active derivative from Peak Z of Fig. 4. The arrows (V) denote the tracking dye; the data above (O-O) record the tritium content in each slice after the gels were divided into 44 equal pieces. B, a semilog plot correlating the mobility of the TSH- and EPS-active glycoprotein fraction from Peak ZZ (Fig. 4) and the EPS-active digestion derivative from Peak Z (Fig. 4) with the mobility of various standards after analytical disc gel electrophoresis on 15% SDS gels (33). Before running, samples were previously incubated with 1% SDS or 1% SDS and 0.1 M

mercaptoethanol for 4 hours at 37”. The inserts at the top are pictures of the EPS-active digestion derivative previously incu- bated with SDS only (left) or SDS plus mercaptoethanol (right). The arrows denote the center of the protein bands stained with Coomassie blue. The top protein band in each gel is a bovine albumin standard applied to each gel immediately before electro- phoresis started; it was used to correlate the mobility of these bands with the mobility of standards.

ophthalmos-producing substance” with no significant TSH action.

On regular and SDS disc gels, the Peak I EPS-active deriva- tive exhibited a single component (Fig. 5) ; from the SDS gels a molecular weight of 20,000 to 22,000 was calculated when its migration was compared to trypsin, pepsin, albumin, insulin, and lysozyme standards. When incubated with 0.1 M mercapto- ethanol as well as SDS, smaller units of approximately 14,000 and 6,000 molecular weight were formed. Nondigested Peak II glycoproteins with both TSH and EPS activity had a molecular weight of 28,000 to 30,000 on SDS gels; nondigested glycopro- teins, previously incubated with 0.1 $X0 mercaptoethanol and SDS, exhibited a second component with a molecular weight of 14,000 to 15,000. In addition to having EPS activity in the fish assay, the Peak I glycoprotein derivative isolated from electrofocusing experiments stimulated mucopolysaccharide synthesis and sulfate incorporation in mouse and guinea pig Harderian glands.a

DISCUSSION

In 1959, Wegeluis, Naumann, and Brunish (10) showed that TSH activity was destroyed faster than EPS activity in pituitary preparations which were exposed to pepsin. They, as well as others, used this data to support the view that the determinants for TSH and EPS resided on 2 entirely different protein or glyco- protein molecules. Since more purified bovine pituitary ex- tracts have subsequently been shown to contain glycoproteins with the determinants for both activities on the same molecule (21), an alternative explanation was possible: the pepsin di- gestion (lo), iodination (9), or acid solubilization (8) had dissociated the two activities by destroying a portion of the molecule carrying the TSH determinant while leaving intact a substructure responsible for EPS action.

In the present experiments, this alternative possibility has been tested by exposing glycoproteins containing both TSH and EPS activity to partial pepsin digestion. Optimal conditions were defined for the dissociation of the two activities, i.e. diges- tion in sodium citrate, pH 2.2, for 15 to 30 min with an amount of pepsin 1% that of the glycoprotein added. In Fig. 3 and Table III, it was shown that one of the glycoproteins containing both activities (Rp 0.25 on analytical gels) was almost completely destroyed under these conditions and that coincident with this destruction there was only a 150/, loss in EPS action. The suggestion that one of the fragments produced during the digestion was responsible for the residual EPS action was con- firmed when a glycoprotein derivative containing tritiated galactose was isolated by electrofocusing (Fig. 4) and shown to be EPS-active but TSH-negative (Table IV). Partial pro- teolysis had thus converted a pituitary glycoprotein having both determinants to a molecular derivative having only EPS action.

TSH is known to be composed of two subunits (34, 35), and it is possible that one subunit or a portion of only one subunit contains the EPS determinant. On SDS gels, the EPS-active digestion product (Peak I) had a molecular weight of 20,000 to 22,000, whereas the nondigested TSH-EPS glycoprotein had a molecular weight of 28,000 to 30,000 (Fig. 5). After prior incubation with 1% mercaptoethanol and SDS, the EPS-active digestion product formed units of approximately 14,000 and 6,000 molecular weight, whereas the nondigested glycoproteins with both activities formed subunit-sized species ‘with molecular

3 R. Winand and L. D. Kohn, manuscript, in preparation.

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weights of 14,000 to 15,000. These data suggest that the EPS- active digestion product is composed of a major portion of one subunit and a smaller piece of the second. The presence of at least one completely intact carbohydrate unit in the EPS-active digestion product, as indicated by the terminal (tritiated) galactose, should be helpful in evaluating the molecular sub- structure which remains in this derivative.

Of the several glycoproteins containing both determinants, one, RF 0.25, was more sensitive to the selective action of pepsin than the others. Since previous data (21) suggested that all four of the active glycoproteins in the DEAE-purified fraction had a similar amino acid composition and molecular weight and since present data (Table II) suggest a similar carbohydrate content, no clear explanation exists for the higher proteolytic sensitivity of the TSH determinant in this one species. One possibility is that there exists in the pituitary a glycoprotein with only TSH action and that during fractionation procedures, amide changes, conformational changes, or proteolytic events it yields a multiplicity of forms with EPS activity, one species of which is unusually sensitive to pepsin. This speculation could explain the existence of a “pure” TSH glycoprotein (34-37) and could account for the conflicting past reports of EPS isolation by ion exchange chromatography (1 l-14), i.e. subsequent changes in extraction procedures (38) aimed at improving the TSH preparation could have prevented the formation of an EPS- active derivative similar to the one presently described.

This speculation is interesting experimentally since it sug- gests that limited pepsin proteolysis of “pure” TSH preparations such as tumor TSH might expose an EPS determinant and that there might exist in viva a more specific proteolytic enzyme than pepsin for this action. Such an enzyme might be a con- sequence of a TSH degradative pathway existent in pituitary tissues, a pathway, for example, which functioned in the feed- back control of TSH levels by thyroid hormones. The low levels of TSH in patients with Graves’ disease (39, 40) and exophthalmos could reflect such an active degradative system rather than low TSH production. Such a hypothesis does not exclude the possibility that the proteolytic products were not directly active but instead functioned as EPS through an im- mune mechanism analogous to that proposed for long acting thyroid-stimulating hormone or a foreign protein (41, 42). Such a mechanism would account for the finding that fractions of y-globulin from patients with exophthalmos can yield a positive EPS response in the fish assay.4, 6 It is hoped that the present studies will allow the development of an EPS-specific immune assay and an experimental means to evaluate these questions.

Acknowledgments-We are indebted to Dr. J. E. Rail, Na- tional Institute of Arthritis and Metabolic Diseases and to Dr. A. Nizet, Institute of Medicine, University of Lisge, for their encouragement and support in pursuing this collaborative proj- ect. We also thank Mr. J. Langenaeken, whose technical skills allowed the project to be accomplished.

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1. SCHOKAERT, J. A., Proc. Sot. Ezp. Biol. Med., 29, 306 (1931). 2. SCHOKAERT, J. A., Amer. J. Anat., 49, 379 (1932).

4 R. Winand, J. Salmon, and P. H. Lambert, The Sixth Inter- national Thyroid Conference, 1970.

5 3. M. McKenzie, personal communication.

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Leonard D. Kohn and Roger J. WinandBOTH THYROTROPIC AND EXOPHTHALMOGENIC ACTIVITY

PEPSIN DIGESTION OF PITUITARY GLYCOPROTEINS CONTAININGFORMATION OF AN EXOPHTHALMOS-PRODUCING SUBSTANCE BY

Relationship of Thyrotropin to Exophthalmos-producing Substance:

1971, 246:6570-6575.J. Biol. Chem. 

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