Chemical and immunochemical studies on pregnant mare serum gonadotropin

BIOCI-IIMICAET BIOPHYSICAACTA

I39

BBA 36064 CHEMICAL AND IMMUNOCHEMICAL STUDIES ON P R E G N A N T MARE SERUM GONADOTROPIN

D I E T E R SCHAMS AND HAROLD P A P K O F F

Hormone Research Laboratory, University of California, San Francisco, Calif. (U.S.A.) (Received October 5th, 1971)

SUMMARY

Highly purified pregnant mare serum gonadotropin (PMSG) can be prepared from crude commercial preparations of PMSG by chromatography on sulfoethylSephadex C-5o and gel filtration on Sephadex G-Ioo. The preparation was examined by disc electrophoresis and gel filtration and found to be of high purity. Amino acid analysis shows similarities to pituitary gonadotropins. The PMSG contains a high content of proline and cystine and low amounts of the aromatic amino acids. Phenylalanine is the major amino terminal amino acid. The carbohydrate content totals 45% of which lO% is the content of sialic acid. The PMSG is relatively stable in 8 M urea or 4 M guanidine, but inactivated by performic acid oxidation or treatment with neuraminidase. Antiserum to PMSG was characterized by agar diffusion, immunoelectrophoresis, and quantitative precipitiu reactions; it was specific for PMSG and did not cross react with human or ovine interstitial cell-stimulating hormone and folliclestimulating hormone.

INTRODUCTION

The primary structures of the subunits of ovine interstitial cell-stimulating hormone 1-3 and thyrotropin 4 have been reported. Evidence has been published that follicle-stimulating hormoneS,6 and human chorionic gonadotropinV, s consist of subunits as is the case with interstitial cell-stimulating hormone and thyrotropin. Little, however, is known about the properties of pregnant mare serum gonadotropin (PMSG). Several years ago we described 9 a simple procedure for the isolation of PMSG. The limited quantities of PMSG obtained at the time precluded extensive chemical characterization of the material. Some years prior to this study, LEGAULTDEMARE et a/.l°, n and HARBON-CHABBATet al. TM had also described methodology for the preparation of highly purified PMSG and some of its physicochemical properties. We have now found that commercial preparations of PMSG (15oo-25oo I.U./mg) Abbreviation: PMSG, pregnant mare serum gonadotropin.

Biochim. Biophys. Acta, 263 (1972) 139-148

14o

D. S C H A M S , H. P A P K O F I ;

can be further purified by utilizing the final two steps of our original procedure. In addition, we have extended the chemical as well as immunological characterization of the PMSG isolated in this manner and report the results here. M A T E R I A ] . S A N D METH()I),~

Commercial preparations of PMSG assaying I7OO-25oo I. U./mg were obtained from Organon (Oss) and Schering (Berlin). Preparations were assayed by the nlethod of COLE AND ERWA¥ la in 26-day-old female Holtzman (Madison, Wisc.) rats. Potencies were obtained from the statistical analysis of multiple dose (2 t 2 and 3 i- 3 design) assays in which a commercial preparation (Organon) of 17oo I. U./mg was used as standard. Sephadex G-Ioo and sulfoethyl-Sephadex C-5o (SE-C-5o) chromatography were performed as previously described 9. Disc electrophoresis in columns of polyacrylamide were done by the method of DAVIS14. Samples for amino acid analysis were hydrolyzed in constant boiling HC1 (I mg/ml) in sealed, evacuated tubes at lO5 ° for 2o h. Hydrolysates were analyzed in a Beckman Model i2oB automatic analyzer by the method of SPACKMAN et al. ~5. Tyrosine and tryptophan were also determined spectrophotometrically ~6. Amino terminal determinations were by the dansyl method xT,~s. Dansylated samples were hydrolyzed for 6 and 16 h prior to analysis. Carboxypeptidase and leucineaminopeptidase experiments were performed as described previously 19,20. Carbohydrate components were determined by the following methods: sialic acid by the thiobarbituric acid procedure of WARREN2~; hexose by the orcinol reaction2~; hexosamine by the method of RON DLE ANI) M O R G A N 2a. Antiserum against purified PMSG was obtained from rabbits immunized by standard procedures previously employed in this laboratory for other glycot)rotein hormones aa. The antiserum was employed for double diffusion tests in agar (()uchterhmy), inmmnoelectrot)horesis, and quantitative precipitation tests as en~ployed before "a.

RESULTS

Purification of crude P M S G It was found that tile final two steps of the previously published procedure :~ were adequate to prepare highly purified PMSG from crude commercial preparations. In the present series of experiments, however, the chromatography step on sulfoethylSephadex C-5o was performed prior to the gel filtration on Sephadex G-ioo. Fig. I a shows a typical pattern obtained when 178 mg of Schering PMSG (25oo I. U./mg) was chromatographed on a 2o-ml column of sulfoethyl-Sephadex C-5o under the conditions shown in the legend. The fraction which is eluted with o.2 M ammonium acetate, p H 8.5, was taken for final purification and amounted to 12 rag. Fig. Ib shows the pattern obtained when 77 mg of this type of material (derived from i g of crude PMSG) was gel filtrated on a 4 cm × 84 cm column of Sephadex G-IOO. The PMSG activity was found to be associated with the second major peak to emerge from the column. The material from this area (tubes 6o-77), Fig. Ib, amounted to 36 mg and when re-filtered on a smaller colunm of Sephadex G-ioo (1.6 cm ~ 8o cm) eluted as a homogeneous peak (Fig. IC). Following dialysis against distilled water Biochi**~. Biophys. Acta, " 0 3 (1972) ~3 c) ~48

STUDIES ON P M S G

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Fig. I. (a) Sulfoethyl-Sephadex C5o chromatography of crude PMSG. 178 mg in pFI 4.5 buffer applied to column equilibrated to the same pH ; buffer changes as indicated. Peak C was taken for further purification as in (b). (b) Gel filtration of 77 mg sulfoethyl-Sephadex C5 o purified PMSG on Sephadex G-Ioo in phosphate-NaC1 buffer, pH 6.9; Peak B taken for re-run as in (c). (c) Rerun on Sephadex G-Ioo of purified PMSG. Fig. 2. Disc electrophoresis of purified PMSG in pH 8.3 buffer, 7.5% gel. Migration from top to bottom. Stained with Coomassie Blue. 60 min electrophoresis at 3 mA.

Biochim. Biophys. Acta, 263 (1972) 139-148

142

D. 8CHAMS, H. P A P K ( } H ;

and lyophilization, 32 nag was obtained. In a second experiment, the final yield was 34 mg. Bioassay of the purified PMSG showed it to have a biological activity ~}f IO 5oo I.U./mg (95% confidence limits: 8ooo 13 ooo' 2 ..... o.o9), a value {'on]t)arable t that previously reported ~ for the material isolated from sermn. Characterization studies Electrophoresis in columns of t)olyacrylamide (Fig. 2) showed that the purified PMSG migrated as a single, broad and diffuse band, which does not readily stain. The absence of other bands in the column suggests the PMSG free {}f any appreciable quantity of other components. Table I tabulates the results of the amino acid analysis of the PMSG and compares the amino acid content with that of ovine interstitial cell-stinmlating hormone and ovine follicle-stimulating hormone as well as the PMSG studied by HARBON-CHABBOTet al. 1". PMSG is characterized by a low content of pheiwlalanine, tyrosine, and methionine, and a high content of proline and cystine. It is also seen that the composition of PMSG more nearly resembles that of ovine interstitial cellstimulating hormone, although similarities to ovine follicle-stimulating hormone are evident as well. Spectrophotometric determinations suggested the presence of o. 7 residue of tryptophan per roo residues which is similar to the content lkmnd in ovine follicle-stimulating hormone (Table I). TABLE

I

AMINO ACID CONTENT

OF P~/I.~G,

FOLLICLE-STIMULATING

HORMONE

OVINE INTERSTITIAL

CELL-STIMULATING

H O R M O N 1 ~2, A N D O V I N E

F o l l i c l e - s t i m u l a t i n g h o r n l o n e d a t a t a k e n f r o m PAPKOFF AND EKBLAD 5. A m i n o acid

PMSG* Harbon-Chabbot et al. l"a

Lysine Histidine Argininc Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine T r y p t o p h a n *'"

Schams and Papkoff

,t.5 -,. 2 7.8 4.5 {}.7

5.4 -'-5 (}. 1 5-5 8. ()

,1. I

8.O

()vine inlerstilial cellstimulating

()vine follicle-stim~tlating hormone

hoYmone** 5.0 z.8 5.1 7.4 0. 5

8.{} 3.1 4.2 q.7 7.3 ~}. I

5. l

S.9

8.4

i i. |

I 2..5

0. 5 ~3.o

I x. 3 5-9

5.o

5.

.5. t

5.2

7.8

7.5

7.o

7.8

s.9 3.3 2.2 3.3 5.0 2.2 3.3 o.6

7.5 4-6 1-7 4.8 6.3 2-4 (3.5"**) 4.o o.7

[0.2

(),I

6.0 3.2 3.2 6.5 3.2 .3.7

0.7 1.[ 3.I 7-3 3-3 4 .t

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2o h h y d r o l y s i s ; r e s u l t s a r e c a l c u l a t e d a s r e s i d u e s per i o o a m i n o a c i d r e s i d u e s ; d a t a o t HARBON-CHABBOT et al. 12 r e c a l c u l a t e d for c o m p a r i s o n . ** C a l c u l a t e d f r o m s t r u c t u r a l f o r m u l a t o f i n t e r s t i t i a l c e l l - s t i m u l a t i n g t m r m o n e . *** S p e c t r o p h o t o m e t r i c d e t e r n l i n a t i o I L Bio chim. Biophys. Acta, 2(}3 (1972) I 3 9 - 1 4 8

STUDIES ON P M S G

143

Two samples of PMSG, one derived from crude Schering PMSG and the other from crude Organon PMSG, were analyzed for the presence of amino terminal acids by the dansyl technique. The dansylated preparations were hydrolyzed for 6 and 16 h and the dansyl amino acids identified by polyamine thin-layer chromatography TM. Identical results were obtained with both preparations. Phenylalanine was the major amino acid identified. In each case, traces of glycine and serine were also seen. Treatment of performic acid-oxidized PMSG with leucineaminopeptidase and ana!ysis of the digestion mixture did not reveal the release of any amino acids. Carboxypeptidase A was also without effect on both the purified PMSG and the performic acid oxidized PMSG. PMSG was oxidized with performic acid as previously described ze and the oxidized preparation digested with both trypsin and chymotrypsin, respectively (o.i M ammonium acetate, p H S . I ; enzyme/substrate = 1:5o; 8h, 37 °) and the resultant digests analyzed by paper chromatography and high-voltage electrophoresis (fingerprint technique, Whatman 3 M paper, details in legend of Fig. 3). Peptides were stained with ninhydrin spray and arginine-containing peptides were located by means of the Sakaguchi reaction. Tracings of the results are seen in Fig. 3. The number of peptides obtained, both total and arginine-containing, are consistent with the amino acid composition of the PMSG molecule (see Table I), assuming the presence

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Fig. 3 Peptide maps of tryptic and chymotryptic digests of performic acid-oxidized PMSG. Chromatography in butanol-acetic acid-water (4 :I :5, b y vol.) ; electrophoresis in p H 2.1 (formic acid-acetic acid), 2ooo V, .z h; spots with + are those which stained positive with Sakaguchi reagent (arginine detection) in addition to ninhydrin spray.

Biochim. Biophys. Acta, 263 (1972) 139-148

144

D. SCHAMS, H. PAPKOFF

TABLE II PMSG Data in g/ioo g glycoprotein; uncorrected for moisture and ash. C A R B O H Y D R A T E C O N T E N T OF

SugaF

HARBON-CtIABBOT

~CHAMS AND P A P K O F F

el al? ~ Hexose

24.o

E4-3

Hexosamine Sialic acid

15.2

2o.6

12.0

10.2

of a similar n u m b e r of residues as in ovine 1 i n t e r s t i t i a l cell-stimulating hormone or ovine follicle-stimulating hormone. The c a r b o h y d r a t e c o n t e n t of PMSG is s u m m a r i z e d in Table I I a n d compared with the d a t a of HARBON-CHABBOT et al.12; triplicate analysis of two preparations were performed. It is seen t h a t the c a r b o h y d r a t e c o n t e n t totals 45%, a high value which is in accord with values reported earlier 21. N o t e w o r t h y is the high content, lO.2%, of sialic acid. A n u m b e r of s t a b i l i t y studies have been performed a n d are s u m m a r i z e d in T a b l e I I I . T r e a t m e n t with either 6 M or 8 M urea (pH 7.o, o.I I phosphate), or 4 M g u a n i d i n e effected only a small degree of i n a c t i v a t i o n (6o-65% as active as native PMSG). T r e a t m e n t with n e u r a m i n i d a s e (enzyme/suhstrate = I:~OO, p H 5.o, o.I I sodium acetate -!- 5 mM CaC12) abolished more t h a n 95% of the biological activity. Performic acid-oxidized PMSG was also i n a c t i v a t e d to an e x t e n t greater t h a n (-o,,)5/,,. Immunological studies

Wlfite New Z e a l a n d r a b b i t s (3-4 kg) were i m m u n i z e d with purified PMSG. The r a b b i t s were injected with 2oo-/~g doses of PMSG in saline mixed with an equal volume of F r e u n d ' s complete a d j u v e n t at 8-day intervals. IO days after the fourth injection, animals were given a booster dose of PMSG in saline only (intraperitoneal) a n d IO days later t h e y were bled. E x a m i n a t i o n of the a n t i s e r u m by the O u c h t e r l o n y agar diffusion technique showed t h a t the a n t i s e r u m formed a single line with PMSG, b u t in a d d i t i o n c o n t a i n e d TABLE [ [ I E F F E C T OF VARIOUS T R E A T M E N T S * ON T I I E BIOLOGICAL A C T I V I T Y OF

Conditions

Activity (%)**

pH 7.o (phosphate buffer) 6 M urea 8 M urea 4 M guanidine Neuraminidase Performic acid oxidized

8i.o (57.5-1o2) 6o.o (49.5-74-5) 65.0 (48.o-97.o) 61.5 (46.5-85.5) < 5-° <5.0

PMSG

* I mg/ml treated as indicated for 20 h at 21 °. ** Treated PMSG compared in bioassays with untreated PMSG and expressed as percentage of untreated PMSG activity; 95% confidence limits in parentheses. Biochim. Biophy~. A~;'a, 263 (1972) 139-148

STUDIES ON PMSG

145

Fig. 4- Photographs of Ouchterlony plates showing precipitin reactions; 12/*1 PMSG antiserum in central well in each case; 12/zg of antigens used. (a) I, 2, and 3: PMSG; 4, 5, and 6: normal mare serum. (b) i and 4 : PMSG; 2 : human interstitial cell-stimulating hormone ; 3 : ovine folliclestimulating hormone ; 5 : ovine interstitial cell-stimulating hormone ; 6 : human follicle-stimulating hormone. (c) I and 4: PMSG; 2: neuraminidase-treated PMSG; 3 : 4 M guanidine-treated PMSG; 5 : 6 M urea treated PMSG; 6 : 8 M urea-treated PMSG.

a n t i b o d i e s which r e a c t e d with n o r m a l horse serum. Following a d s o r p t i o n of the a n t i s e r u m with l y o p h i l i z e d horse serum, only a single line of p r e c i p i t a t i o n was observed, t h a t with P M S G (Fig. 4a). F i g u r e 4b shows t h a t the a n t i s e r u m d i d not cross-react w i t h i n t e r s t i t i a l c e l l - s t i m u l a t i n g h o r m o n e (human or ovine) or follicle-stimulating h o r m o n e ( h u m a n or ovine); in addition, no cross reaction was o b s e r v e d with h u m a n chorionic g o n a d o t r o p i n or, m o s t interestingly, with e x t r a c t s of horse pituitaries. Performic acid o x i d i z a t i o n of the PMSG results in a loss of a b i l i t y to form a line of p r e c i p i t a t i o n w i t h t h e antiserum, b u t t r e a t m e n t of PMSG with n e u r a m i n i d a s e d i d n o t affect t h e r e a c t i v i t y of t h e m a t e r i a l with t h e a n t i s e r u m (Fig. 4c). The same figure suggests t h a t u r e a a n d g u a n i d i n e t r e a t m e n t i n h i b i t e d the a n t i b o d y - a n t i g e n r e a c t i o n insofar as no p r e c i p i t a t i o n occurred. I t seems likely t h a t this was a result of t h e high c o n c e n t r a t i o n o f u r e a a n d g u a n i d i n e in the solutions as q u a n t i t a t i v e precipitin a n a lysis (Fig. 5), where the reagents were c o n s i d e r a b l y diluted, showed s t r o n g reactions. Biochim. Biophys. Acla, 263 (1972) 139-148

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I

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ANTIGEN (~g) Fig. 5. Q u a n t i t a t i v e precipitin curves obtained w i t h PMSG a n t i s e r u m and its reaction with PMSG and various treated PMSG preparations. Details in text.

Ouchterlony experiments employing anti-ovine interstitial cell-stimulating hormone antiserum specific for ovine interstitial cell-stimulating hormone 25, did not indicate any reaction with the purified PMSG. Immunoelectrophoresis experiments were performed in i.j:°/..o agar in Tris, p H 7.4. Electrophoresis was for 2 h in the cold room and diffusion with antiserum for 24 h. The results, in general were confirmatory of the Ouchterlony immunodiffusion experiments described above. With the adsorbed antiserum only a single arc of precipitation was obtained with the purified PMSG. The quantitative precipitin reaction was studied with adsorbed antiserum which was diluted 1:4 with o.9~ ~ saline. The antigen in o. 5 ml o.9~, saline was added to o.5 ml of the diluted antiserum. The mixture was incubated for I 1/at 37 ° and 72 h at 4 °. The precipitate which formed was collected by centrifugation, washed twice with cold o.9% NaC1, and the protein determined by the method of LOWRY et al74. Fig. 5 shows the precipitin curves obtained with PMSG and several treated PMSG preparations. The PMSG presents a typical precipitin curve with an equivalence point located at IO fig of antigen. PMSG treated with 8 M urea was identical to untreated PMSG, however PMSG which was treated with 4 M guanidine, while qualitatively similar in its precipitin reaction, showed a tendency to plateau in the antigen excess region (i.e. > IO #g). Neuraminidase-treated PMSG showed a similar pattern, and performic acid-oxidized PMSG, as expected, was totallv unreactive in the system. DISCUSSION

The studies described here show that highly purified PMSG can be prepared from crude commercially available preparations by a very simple two-step procedure derived from the method previously developed 9 for serum. This is advantageous in that the initial steps of the serum procedure involve large volumes and a great deal of centrifugation; not only time-consuming processes but not amenable to m a n y Biochim. Biophys. Acta, 263 (1972) 139-148

STUDIES ON PMSG

147

laboratory operations. The final yield of purified PMSG (32-34 mg/g of crude PMSG) represents about a 35% yield on the basis of biological activity. The side fractions obtained from the sulfoethyl-Sephadex C-5o column and Sephadex G-Ioo column have not as yet been examined with respect to the possibility of re-working them to obtain further amounts of material. The material prepared by this procedure appears to be identical to that isolated from serum, and the chemical studies reported here further characterize the material. Thus, the amino acid analysis and peptide maps suggest a composition similar to interstitial cell-stimulating hormone and follicle-stimulating hormone. More definitive structural analyses, however, would be necessary to establish this point. I f one assumes a peptide content equal to that of ovine 1 interstitial cell-stimulating hormone and a moisture content of 2O~/o, one can calculate from the carbohydrate value (45%) that the molecular weight of PMSG is about 52 ooo; previous reports have suggested a molecular weight of either 28 ooo or 68 ooo depending on the experimental conditions ~9. Clearly further definitive studies are needed in this area. Amino terminal group analyses show that the major end group in PMSG is phenylalanine, but traces of glycine and serine are present. The inability of carboxypeptidase to release any amino acids from the carboxyl terminus in all probability reflects the nature of the terminal amino acids rather than the lack of a free terminal residue. The stability studies indicate that the hormonal activity is largely unaffected by treatment with concentrated urea or guanidine solutions. These results are in contrast to those of VISUTAKUL et al. 3° who showed that crude PMSG was inactivated to a much greater extent in high concentrations of urea. The stability in urea, as observed here, is much like that observed with purified ovine follicle-stimulating hormone31m. Inactivation of PMSG by neuraminidase has been reported earlier 29. The immunological data show that it is possible to prepare an antiserum against PMSG which displays a high degree of specificity for the hormone and does not cross react with related gonadotropins of pituitary origin. The extensive cross reaction seen with neuraminidase-treated PMSG indicates that the sialic acid present is not a factor contributing to the antigenicity of the molecule. Finally, mention should be made of the possibility that PMSG consists of chemically dissimilar subunits. This has been shown to be true for the pituitary gonadotropins, interstitial cell-stimulating hormone 34-~6 and follicle-stimulating hormoneS, 6 as well as thyrotropin s6. In addition, human chorionic gonadotropin has been shown to consist of subunitsT, 8. Although no evidence is presented in this paper supporting this concept, it is reasonable to expect that such will be the case, and the chemical data herein presented will provide a basis from which further studies can now be undertaken. ACKNOWLEDGMENTS

We wish to thank Professor C. H. Li for suggesting and encouraging these studies. The work was supported in part by grants from the National Institute of Arthritis and Metabolic Diseases (AM-6o97) and the National Institute of Child Health and Development (HD-o5722), National Institutes of Health. One of us (H.P.) is a Career Development Awardee of the National Institute of General Biochim. Biophys. Acta, 263 (1972) 139-148

I4~

D. SCHAMS, H. PAPKOFF

Medical Sciences, National Institutes of Health. D.S. was supported by a fellowship from the Deutsche Forschungsgemeinschaft, German Research Foundation. We also thank Mrs. Inge Schams for invaluable assistance in these studies. It is also a pleasure to thank Dr. H. Gibian of Schering (Berlin) and Dr. J. D. H. Homan of Organon (Oss) for their generous supply of PMSG preparations. REFERENCES i U. 1)APKOFF, M. R. SAIRAM AND C. H. LI, J. Am. Chem. Soc., 93 (1971) 1531. 2 W.-K. Lltr, C. M. SWEENEY, H. S. NAHM, G. N. HOLCOMB AND D. N. WARD, Res. Commun. Chem. Palhol. Phatm., i (197 o) 463 . 3 W . - K . Lltl, H. S. NAHM, C. M. SWEENEY, It. N. BAKER, W. M. LAMKIN AND D. N. WARD, Res. Commun. Chem. Pathol. Pharm., 2 (I97 I) 168. 4 T.-H. LIAO AND J. G. PIERCE, J. Biol. Chem., 246 (1971 ) 850. 5 H. PAPKOFF AND M. EKBLAD, Bioehem. Biophys. Res. Commun., 4 ° (197 o) 614. 6 B. B. SAXENA AND 1). ]{ATHNAM, ./r. Biol. Chem., 246 (1971) 3549. 7 N. S\VAMINATHAN AND O. P. BAHL, Biochem. Biophys. Res. Commun., 4 ° (t97 o) 422. 8 F. J. MORGAN AND H. E. CANFIELD, Endocrinology, 88 (1971) lO45. 9 D. GOSPODAROWlCZ AND H. PAPKOFF, Endocrinology, 80 (1967) 699. IO J. LEGAULT-DEMARE, H. CLAUSER AND M. JOTISZ, Biochim. Biophys. Acta, 3 ° (1958) 109. i i J. LEGAULT-DEMARE, H. CLAUSER AND M. JUTISZ, Bull. Soc. Chim. Biol., 43 (1961) 897. 12 S. HARBON-CHABBOT, J. LEGAULT-DEMARE, P. JOLLES AND H. CLAUSER, Bull. Soc (?him. Biol., 43 (1961) 1339. 13 H. H. COLE AND J. ERWAY, Endocrinology, 29 (1941) 514 . 14 B. J. DAVIS, Ann. N . Y . Acad. Sci., 12I (1964) 404 . 15 D. H. SPACKMAN, W. U. STEIN AND S. MOORE, Anal. Chem., 3° (1958) 119o. 16 G. H. BEAVEN AND E. R. HOLIDAY, Adv. Protein Chem., 7 (1952 ) 319 . 17 B. S. HARTLEY AND V. MASSEY, Biochim. Biophys. Acta, 21 (1956) 58. 18 K. H. WOODS AND K. T. WANG, Biochim. Biophys. Acta, 133 (1967) 369. 19 T. S. A. SAMY, H. PAPROFF AND C. H. LI, Arch. Biochem. Biophys., 13 ° (1969) (>74. 20 C. H. LI, J. S. DixoN, T.-B. Lo, K. D. SCHMIDT AND Y. A. PANKOV, Arch. Biochem. Biophvs , 141 (197 ° ) 705 . 21 L. "WARREN, J. Biol. Chem., 234 (1959) 1971. 22 t . J. WINZLER, ~Ieth. Biochem. Anal., 2 (1955) 279. 23 C. J. M. HONDLE AND W. T. J. MORGAN, Biochem. J., 61 (1955) 586. 24 N. R. MOUDGAL AND C .H. LI, Arch. Biochem. Biophys., 95 (1961) 93. 2.5 H. PAPKOFF, J. SOLIS-WALLCKERMANN, M. MARTIN AND C. I-I. LI, Arch. Biochem. Biophys., 143 (1971 ) 226. 26 C. H. LI, J. Biol. Chem., 229 (1957) 157. 27 H. BOtrRRILLON AND R. GOT, Acta Endocrinol., 24 (1957) 82. 28 O. H. LowRY, N. J. ROSEBROUGH, A. L. FARR AND H. J. HANDLE, ,]. Biol. Chem., 193 (1951) 265. 29 R. BOURRILLON AND R. GOT, Acta Endocrinol., 31 (1959) 5593° P. VISUTAKUL, F. T. BELL, J. A. LORAINE AND R. B. FISHER, J. Endocrinol., 36 (i966) 15 31 H. PAPKOEF, Acla Endoerinol., 48 (1965) 439. 32 C. HERMIER, P. DE LA LLOSA AND M. JUTISZ, Endocrinology, 87 (197o) 136433 M. E. RAFELSON, H. CLAUSER AND J. LEGAULT=DEMARE, Biochim. Biophys. Acta, 47 (196I) 406. 34 H. PAPKOFF AND T. S. A. SAMY, Biochim. Biophys. Aeta, 147 (1967) 17.5. 35 L. E. HEICHERT, M. A. HASCO, D. N. WARD, G. D. NISWENDER AND A. R. MIDGLEY, ./r. Biol. Chem., 244 (1969) 511o. 36 T.-H. LIAO AND J. G. PIERCE, ,[. Biol. Chem., 245 (197 o) 3275 •

Biochim. Biophys. Acta, 263 (1972) 139-148