Binding proteins for insulin-like growth factors in adult rat serum. Comparison with other human and rat binding proteins

Vol. 147, No. 1,1987 August31,1987

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BINDING PROTEINS FOR INSULIN-LIKE GROWTH FACTORS IN ADULT RAT SERUM. COMPARISON WlTH OTHER HUMAN AND RAT BINDING PROTEINS Robert C. Baxter and Janet L. Martin Department of Endocrinology, Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia

Received July 8, 1987

S U M M A R Y : Insulin-like growth factor (IGF) binding protein has been purified from adult rat serum by affinity chromatography on agarose-IGF-II and high performance reverse-phase chromatography. The final preparation contains two components, of apparent molecular mass 50 and 56 kDa nonreduced, or 44 and 48 kDa reduced, both of which specifically bind IGF-I and IGF-II. Competitive binding data indicate association constants of 5-10 x 10 l° l]mol for both IGFs, with a slightly higher affinity for IGF-II than IGF-I. Amino-terminal sequence analysis yields a unique sequence, identical in 11 of the first 15 amino acids with that of a human plasma IGF binding protein (Martin, J. L., and Baxter, R. C. (1986) J. Biol Chem. 261, 8754-8760), and with slight homology to other human and rat IGF binding proteins characterized to date. By analogy with the binding protein from human plasma, it is likely that the rat protein is part of the growth-hormone dependent complex which appears to carry most or all of the circulating IGFs. o 1987 Academic P. . . . . Inc.

In recent years, binding proteins for peptides of the insulin-like growth factor (IGF) family have been purified from a variety of sources.

A BP preparation isolated in this laboratory from human

plasma was found to contain two protein species: a major component of molecular mass 53 kDa and a minor component of 47 kDa, nonreduced, or 43 kDa and 40 kDa respectively, reduced (1). Both species had IGF binding activity, and the mixture yielded a single amino-terminal sequence, suggesting identity in this region between the two components (2). An antibody raised against this preparation reacts predominantly with a GH-dependent species of 125-150 kDa in human plasma (3,4). A smaller BP, with an apparent molecular mass of about 34 kDa reduced and 28 kDa nonreduced, and an aminoterminal sequence different from that of the plasma protein, has been isolated from human amniotic fluid (5-7) and from culture medium conditioned by the human hepatoma line, HEP-G2 (8). Human placenta contains a similar protein, termed placental protein 12, with IGF binding activity and an identical amino-terminal decapeptide (9). RIAs developed against these BP types detect a protein of similar size in human plasma, which is present in much higher concentrations in fetal and cord blood than in the adult circulation (6, 10,11).

A b b r e v i a t i o n s : IGF, insulin-like growth factor; BP, binding protein; GH, growth hormone; RIA, radioimmunoassay; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography. 0006-291X/87 $1.50 Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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The first n o n - h u m a n BP to be purified was a protein from the r a t liver-derived cell line BRL-3A. Originally isolated by K n a u e r et al. (12), this protein h a s been characterized extensively in three laboratories (13-15). It has an a p p a r e n t molecular mass of 33-36 kDa reduced and 31.5 kDa nonreduced (13-15), and approximately equal affinity for IGF-I and IGF-II (14). RIAs for this protein fail to detect activity in adult r a t serum, whereas fetal and neonatal sera contain h i g h concentrations (13,15). Thus this BP bears a strong resemblance to the h u m a n protein of about 30 kDa, both in its physical properties a n d in the age-dependence of its circulating levels. Although adult r a t s e r u m contains undetectable levels of the ~30 kDa BP, significant IGF binding activity is present in the adult r a t circulation, in a v a r i e t y of molecular forms (16-21). This s t u d y aimed to isolate a n d c h a r a c t e r i z e the proteins responsible for this activity, and in this report we describe the physical and binding properties of a r a t s e r u m BP preparation, a n d compare its amino-terminal sequence w i t h t h a t of the other known BP species.

MATERIALS AND M E T H O D S Reagents - - IGF-I and IGF-II were isolated from Cohn fraction IV of h u m a n plasma, as in our previous studies (22). Both peptides were i o d i n a t e d u s i n g c h l o r a m i n e T a n d purified by gel c h r o m a t o g r a p h y at n e u t r a l pH. Iodinated IGF-I was f u r t h e r purified by hydrophobic i n t e r a c t i o n chromatography (23). The specific activities of the iodinated peptides were estimated to be 150 Ci/g for IGF-I and 100 Ci/g for IGF-II. The IGF-II affinity column was prepared using Affi-Gel 15 (Bio-Rad, Richmond, CA), as previously described (1). Bovine a l b u m i n (RIA grade) was from Sigma; most electrophoresis r e a g e n t s and t h e reverse p h a s e HPLC column from Bio-Rad. Molecular weight m a r k e r s for electrophoresis were purchased from P h a r m a c i a (Sydney, NSW), the Coomassie blue s t a i n (Gradipure) from Gradipore, Sydney, NSW, amido black 10B from Corning, Palo Alto, CA, and nitrocellulose m e m b r a n e (BA 85) from Schleicher and Schuell, Dassell, West Germany. HyperfilmMP for autoradiography was obtained from Amersham, Bucks, England. P u r i f i c a t i o n o f R a t B P - - The starting material for this preparation was a pool of 240 ml of normal adult r a t serum, and 100 ml of serum from adult rats bearing the GH-secreting tumor MtT/W15. We have shown previously t h a t the hepatic production rate of IGF BP in adult r a t s bearing this t u m o r is approximately double t h a t seen in control r a t s (24). The pool was mixed with an equal volume of 2 M acetic acid, and the pH adjusted to 3.0. After standing for 2 h at 22 ° C, the acidified serum was pumped at 24 ml/h onto a 2.5 x 19 cm column of SP-Sephadex, previously equilibrated with 1 M acetic acid, 75 mM NaCl, pH 3.0. S e r u m remaining on the column was washed t h r o u g h with equilibration buffer, and the entire flow-through volume (690 ml) was adjusted to pH 6.5 with 7 M NaOH, and centrifuged to remove precipitated material. The s u p e r n a t a n t was pumped at 18 ml/h onto a 1 x 12 cm column of agarose-IGF-II, the column was washed with 100 ml of 0.25 M NaC1 at 75 ml/h, and BP was eluted with 0.5 M acetic acid, pH 3.0, at 60 ml/h. Fractions of 1 ml were collected and assayed for IGF-II binding activity, as previously described (25). Active fractions were pooled, lyophilized, and dissolved in 15% acetenitrile in 0.1% trifluoroacetic acid, t h e n subjected to reverse-phase HPLC on a Bio-Rad Hi-Pore RP-318 column, exactly as previously described for the h u m a n protein (1). Active fractions were pooled a n d freeze-dried, a n d the dry powder weighed a n d dissolved i n 1 M acetic acid a t a n o m i n a l concentration of 1 mg/ml. S D S - P A G E a n d E l e c t r o b l o t t i n g - - SDS-PAGE was performed according to the method of Laemmli (26) on 7-18% gradient slab gels. Standards and lyophilized samples were reconstituted in 10 mM TrisC1, 1% SDS, pH 8.0 before being applied to the gel. Electrophoresis at 50 V was continued until the sample had r u n into the stacking gel, t h e n at 100 V until the dye front h a d reached the bottom of the gel. For protein staining, gels were fixed in methanol-acetic acid-water (40 : 10 : 50), t h e n s t a i n e d u s i n g Gradipure Coomassie blue. For blotting, gels were equilibrated after electrophoresis for 1 h i n 25 mM Tris-C1, 192 mM glycine, 20% m e t h a n o l , pH 8.3. P r o t e i n s were t r a n s f e r r e d from t h e gel to

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nitrocellulose m e m b r a n e at 45 V for 6 h in the Bio-Rad T r a n s - B l o t cell. After blotting, the lanes containing molecular weight markers were cut off, stained in 0.1% amido black in 45% methanol, 7% acetic acid, a n d d e s t a i n e d in 7% m e t h a n o l , 5% acetic acid. The nitrocellulose sheets containing binding proteins were incubated overnight at 37 °C in a 3% bovine albumin solution in 50 mM sodium phosphate, pH 6.5, t h e n sealed in plastic bags with 35 ml of a solution containing 2 x 106 cpm ofiodinated IGF-I (10 ng) or IGF-II (15 ng), with or without 10 p.g of unlabeled IGF-I or IGF-II respectively, in 50 mM sodium p h o s p h a t e , 3 % bovine albumin, pH 6.5. After i n c u b a t i o n at 22 °C for 2 h, non-bound radioactivity was removed with three washes in cold phosphate buffer, the sheets were dried in air, and exposed to Hyperfilm-MP autoradiography film for 24 h at -80 °C. Competitive Binding Studies - - Binding was performed in 0.3 ml of 0.05 M sodium phosphate buffer, 0.25 % bovine albumin, pH 6.5, for 2 h at 22 °C. Incubations contained r a t BP (routinely 0.5 ng, based on dry weight), iodinated IGF-I or IGF-II (30 pg), and various concentrations of unlabeled IGF-I or IGF-II ranging from 0.01 to 5 ng/tube. Nonspecific binding was measured in the presence o f l 0 0 ng of either peptide. At the end of the incubation period, bound radioactivity was separated from free by precipitation with 200 ~g/tube concanavalin A, as previously described for the h u m a n binding protein (1). This method was chosen after preliminary experiments had established t h a t precipitation of bound counts with concanavalin A gave slightly higher binding t h a n adsorption of free tracer with charcoal. Determination - - Approximately 40 ~g (0.8 nmol) of unreduced r a t binding protein was by Dr. M. I. Tyler, School of Chemistry, Macquarie U n i v e r s i t y , N o r t h Ryde, NSW, by automatic E d m a n degradation on a n Applied Biosystems 470 A protein sequencer, as described (2). Sequences of h u m a n plasma and amniotic fluid proteins were determined as described (2).

Sequence

sequenced Australia, previously previously

RESULTS When HPLC-purified r a t serum IGF BP was subjected to SDS-PAGE and stained with Coomassie blue, two protein components were evident, of apparent molecular mass 50 kDa and 56 kDa. Figure l(a) compares these two b a n d s with those seen i n preparations of h u m a n plasma BP, to which we have previously assigned molecular masses of 53 and 47 kDa (1). Unlike the h u m a n BP preparation, in which the higher molecular mass species was always the more heavily Coomassie stained, in the rat BP preparation the lower molecular mass (50 kDa) band generally appeared somewhat darker t h a n the 56 kDa band. After reduction with 2-mercaptoethanol, the two b a n d s corresponded to a p p a r e n t molecular masses of 44 and 48 kDa (data not shown), showing a comparable decrease to t h a t previously reported for the h u m a n BP preparation (1). Following electroblotting onto nitrocellulose after SDS-PAGE, and incubation with IGF-I or IGF-II tracer, both protein components were found to bind both IGFs. As shown in Figure l(b), the 50 kDa component of r a t BP showed greater binding of both tracers t h a n the 56 kDa component, in c o n t r a s t to the h u m a n BP p r e p a r a t i o n which showed higher binding to the higher molecular mass (53 kDa) band. Tracer binding was specific, as indicated by the elimination of bound radioactivity w h e n incubations were performed in the presence of excess unlabeled IGF-I or IGF-II. The specificity of IGF binding was examined in more detail by performing competitive binding experiments in liquid phase. As preliminary experiments had indicated t h a t r a t BP was glycosylated, we separated bound from free ligand in these experiments by precipitating the BP-IGF complex with concanavalin-A plus polyethylene glycol. As shown in Figure 2, r a t BP bound IGF-II tracer slightly 410

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(a) C o o m a s s i e

(b) IGF Binding 94),

671~

431

IGF-I

IGF-II

.

431~

3 0 i~lllm 301~

20.1~

20.11, Excess IGF

~"

"~-

-~-

-I-

Figure 1. Nonreduced SDS-PAGE of human and rat serum IGF binding proteins. (a) 10 ~tg of nonreduced rat and human BP, as indicated, were electrophoresed on 7-18% gradient gels as described in the Methods. Binding proteins and molecular weight markers (left lane, values in kDa) were stained with Coomassie blue. (b) 1.25 ~g per lane of nonreduced rat or human BP were electrophoresed as described above, blotted onto nitrocellulose, and probed with IGF-I tracer in the absence (-) or presence (+) of excess unlabeled IGF-I, or IGF-II tracer in the absence (-) or presence (+) of excess unlabeled IGF-II, as indicated in the figure. The nitrocellulose sheets were then autoradiographed as described in the Methods. IGF-I binding to the minor 47 kDa band of the human preparation, not visible in this experiment, has been demonstrated previously (2).

better t h a n IGF-I tracer, a n d IGF-II w a s more potent t h a n IGF-I in displacing either tracer. Scatchard a n a l y s i s of b i n d i n g d a t a indicated a h i g h affinity b i n d i n g component (Ka = 5-10 x 1010 1/mol) plus a nonspecific binding component (Ka < 107 ]/mol) for each tracer (Figure 3). Based on a molecular m a s s of 50 kDa, the b i n d i n g capacity for either ligand w a s approximately 0.5 mol/mol, s u g g e s t i n g a single binding site for either ligand per 50 kDa protein.

A m i n o - t e r m i n a l sequence a n a l y s i s on 40 ~tg of r a t BP yielded a u n i q u e sequence, s u g g e s t i n g t h a t the two protein components were identical in their a m i n o - t e r m i n a l region (or t h a t one component w a s terminally blocked). Figure 4 compares this sequence to t h a t obtained previously for the h u m a n p l a s m a BP preparation (2), now extended to 15 residues, and to those reported by Mottola et al. (14) a n d Lyons and S m i t h (15) for the BP from r a t BRL-3A cells. Six of the t e n r e s i d u e s previously reported for the h u m a n BP (2) are identical in the r a t protein, and positions 11 - 15 are entirely homologous, a s s u m i n g residue 13 (not determined) to be cysteine. In contrast, t h e homology w i t h the BRL-3A protein is m u c h weaker in this region, a l t h o u g h residues 5, 6 and 8 in the sequence of Lyons and S m i t h (corresponding

411

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1.00 i a : IGF-I

4O

0.75 ~

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~'~-~=o

0.01

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Q

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b : IGF-I[ Tracer 0 , , O.~o_..=-",,M 0.00 0.01 0.10 1.00 Peptide Added (ng/tube)

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i

....

.4.

,

0.1 0.2 0.3 IGF-II Bound (ng/ng rBP)

0.4

Figure 2. Competitive binding curves showing the displacement of (a) IGF-I tracer and (b) IGF-II tracer from purified rat IGF BP (0.5 ng) by increasing concentrations of unlabeled IGF-I (solid s y m b o l s ) or IGF-II (open symbols). The reaction volume was 0.3 ml. Bound radioactivity was separated from free by precipitating the BP-IGF complex with concanavalin A plus polyethylene glycol (~). B/T is the ratio of protein-bound radioactivity to total radioactivity. Figure 3. Scatchard plots of (a) IGF-I and (b) IGF-II binding to rat serum IGF BP (rBP). P o i n t s represent experimental data, solid lines are computer-assisted fits to a two-site model (high-affinity binding plus nonspecific binding), and dotted lines represent the resolved components. B/F is the ratio of bound to free ligand.

to r e s i d u e s 2,3 a n d 5 in t h e s e q u e n c e o f M o t t o l a et a].) a p p e a r to correspond to r e s i d u e s 12, 13 a n d 15 of t h e h u m a n a n d r a t s e r u m proteins. Also s h o w n for c o m p a r i s o n a r e t h e first 15 r e s i d u e s of h u m a n amniotic fluid I G F B P i s o l a t e d in t h i s l a b o r a t o r y (2,7), initially reported up to r e s i d u e 10 b y £ 6 v o a et al. (5). T h e s e q u e n c e differs from t h a t r e p o r t e d for t h e r e l a t e d or identical protein, p l a c e n t a l p r o t e i n 12, w h i c h h a s A s p - G l u - at r e s i d u e s 11 a n d 12 (9). A m n i o t i c fluid I G F BP, a l t h o u g h only w e a k l y h o m o l o g o u s to the h u m a n a n d r a t s e r u m p r o t e i n s at t h e a m i n o - t e r m i n u s , is i d e n t i c a l in 6 of i t s first 15 r e s i d u e s w i t h the r a t B R L - 3 A protein.

1

2

3 4

5

6

7

8

9 10 11 12 13 14 15

-~-AlafGly-Pro-VaI-Val-Arg-(Cys)~-Glu~-Pro t er-Ser-Ala GL~Leulr, Gly-Pro-VaI-VaI-ArgC y s , Rat BRL-3A Cells (Lyons& Smith) Rat BRL-3A Cells (Mottola st ah) Human Amniotic Fluid

Glu-VaI-LeutPhetArg-Cys

~ t , G lPro u

Rat Serum Human Serum

ro-Cys-Thr~

/Phe~-Arg-Cys~_~Pro-Cys~T~Oro~---~Arg~'-~AlaAla-Pro-Trp- GinCy~la~Pro-Cys~ Ser-Ala 1~.~4-ys~-Leu-Ala~Leu1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Figure 4. Amino-terminal sequence of rat serum IGF binding protein, compared to sequences of IGF BPs from h u m a n serum, rat BRL-3A cell culture medium, and h u m a n amniotic fluid. Residue 13 in the rat serum BP sequence, not identified, is assumed to be Cys. The BRL-3A BP sequences are those of Lyons and Smith (15) and Mottola et al. (14); the latter sequence was reported to 31 residues. Enclosed areas represent regions of homology between different sequences. 412

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DISCUSSION

In this study we have demonstrated t h a t adult r a t serum contains a glycosylated binding protein for IGF-I and IGF-II which is different in size and amino-terminal sequence from t h a t isolated from culture medium conditioned by r a t BRL-3A cells, b u t homologous with a protein which we previously isolated from h u m a n plasma. The p r e p a r a t i o n c o n t a i n s two b i n d i n g components, of a p p a r e n t molecular mass 50 a n d 56 kDa nonreduced, or 44 and 48 kDa reduced. It thus resembles a n IGF BP p r e p a r a t i o n isolated from h u m a n plasma which also contains two binding components, b o t h of which decrease significantly in a p p a r e n t molecular m a s s on reduction (1,2). In contrast, t h e r a t BRL-3A b i n d i n g protein, a n d the b i n d i n g protein from h u m a n amniotic fluid, have nonreduced molecular masses around 30 kDa, which show a significant increase on reduction (5,7,14,15).

The r a t BP preparation described in this report showed slightly higher affinity for IGF-II t h a n for IGF-I. The relatively close affinity of this BP for the two IGFs resembles t h a t of the BP i n acidchromatographed r a t serum, and in medium conditioned by primary cultures of adult r a t hepatocytes (25). IGF-I has been reported to show twice the potency of IGF-II in displacing IGF-I tracer from a BP preparation from cultured r a t liver explants (27), but, given slight differences between the potencies of IGF preparations from different laboratories, this may not be inconsistent with our results. Binoux et al. (28) have also suggested t h a t rat liver may produce a BP with selective affinity for IGF-II; however, this still remains to be conclusively demonstrated.

The exact relationship between the two IGF binding components of our BP preparation, and r a t s e r u m IGF BPs described in other studies, is not clear. In r a t serum, much of the endogenous IGF activity and IGF binding sites have a n a p p a r e n t molecular mass, determined by gel chromatography at n e u t r a l pH, of about 150 kDa, whereas after acidification the binding activity corresponds to a n acidstable protein somewhat smaller t h a n a l b u m i n (17). This observation parallels t h a t seen in h u m a n p l a s m a (29), where antibodies raised against the acid-stable 53 kDa BP have b e e n shown to react predominantly with the plasma BP-IGF complex of approximately 150 kDa (3). This complex, which carries most of the endogenous IGFs (30,31), contains sufficient binding s u b u n i t s (as determined by RIA) to account fully for the total circulating concentrations of IGF-I and IGF-II (4), a s s u m i n g one binding site per 53 kDa protein, as previously demonstrated by direct binding studies (1).

By analogy with the binding proteins of h u m a n plasma, the purified r a t binding species of 50 and 56 kDa described in this paper may be structurally related to the major circulating BP-IGF complex in 413

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rat serum. The two proteins may also be related to some of the binding species demonstrated by D'Ercole and Wilkins using affinity labeling (20). In that study the major GH-dependent BP in rat serum appeared to form a complex with IGF-I of 95 kDa, with a fainter 49 kDa band also present. These might correspond to a monomer and dimer of one or both of the binding proteins we have isolated, which had reduced molecular masses of 44 and 48 kDa. Rat hepatocytes in primary culture also produce binding activity of 50 kDa after acidification (25), consistent with the size of the purified rat serum BP. Future structural and immunological studies may explain how these proteins aggregate to form complexes of 95 kDa or 150 kDa with the IGFs, and how they regulate the delivery of IGFs to target cell receptors.

ACKNOWLEDGEMENTS This study was supported by the National Health and Medical Research Council, Australia. The assistance of Dr. M. I. Tyler, School of Chemistry, Macquarie University, NSW, who performed the amino acid sequence analyses, is gratefully acknowledged. Donor rats bearing the MtT/W15 tumor were originally obtained from the Mason Research Institute, Worcester, MA.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Martin, J. L., and Baxter, R. C. (1986) J. Biol. Chem. 261, 8754-8760 Baxter, R. C., Martin, J. L., Tyler, M. I., and Howden, M. E. H. (1986) Biochem. Biophys. Res. Commun. 139,1256-1261 Martin, J. L., and Baxter, R. C. (1985) J. Clin. Endocrinol. Metab. 61, 799-801 Baxter, R. C., and Martin, J. L. (1986) J. Clin. Invest. 78, 1504-1512 P6voa, G., Enberg, G., JSrnvall, H., and Hall, K. (1984) Eur. J. Biochem. 144, 199-204 Drop, S. L. S., Kortleve, D. J., and Guyda, H. J. (1984) J. Clin. Endocrinol. Metab. 59, 899-907 Baxter, R. C., Martin, J. L., and Wood, M. H. (1987) J. Clin. Endocrinol. Metab. 65 (in press) P6voa, G., Isaksson, M., J6rnvall, H., and Hall, K. (1985) Biochem. Biophys. Res. Commun. 128, 1071-1078 Koistinen, R., Kalkkinen, N., Huhtala, M.- L., Sepp~l~i, M., Bohn, H., and Rutanen, E.- M. (1986) Endocrinology 118, 1375-1378 Rutanen, E. - M., Bohn, H., and Sepp~il~i, M. (1982) Am. J. Obstet. Gynecol. 144, 460-463 P6voa, G., Roovete, A., and Hall, K. (1984) Acta Endocrinol. (Copenh.) 107, 563-570 Knauer, D. J., Wagner, F. W., and Smith, G. L. (1981) J. Supramol. Struct. Cell. Biochem. 15, 177191 Romanus, J. A., Terrell, J. E., Yang, Y. W.- H., Nissley, S. P., and Rechler, M. M. (1986) Endocrinology 118, 1743-1758 Mottola, C., MacDonald, R. G., Brackett, J. L., Mole, J. E., Anderson, J. K., and Czech, M. P. (1986) J. Biol. Chem. 261,11180-11188 Lyons, R. M., and Smith, G. L. (1986) Mol. Cell. Endocrinol. 45, 263-270 Moses, A. C., Nissley, S. P., Cohen, K. L., and Rechler, M. M. (1976) Nature 263, 137-140 Moses, A. C., Nissley, S. P., Passamani, J., and White, R. W. (1979) Endocrinology 104, 536-546. Takano, K., and Shizume, K. (1978) Proceedings, VI Asia & Oceania Congr. Endocrinol., Singapore, 1, 349-357 Baxter, R. C., Brown, A.S., and Turtle, J. R. (1979) Horm. Metab. Res. 11, 216-220 D'Ercole, A. J., and Wilkins, J. R. (1984) Endocrinology 114, 1141-1144 Kaufmann, U., Zapf, J., and Froesch, E. R. (1978) Acta Endocrinol (Copenh) 87, 716-727 Baxter, R. C., and De Mellow, J. S. M. (1986) Clin. Endocrinol. 24, 267-278 Baxter, R. C., and Brown, A. S. (1982) Clin. Chem. 28, 485-487 414

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Scott, C. D., Martin, J. L., and Baxter, R. C. (1985) Endocrinology 116, 1102-1107 Scott, C. D., Martin, J. L., and Baxter, R. C. (1985) Endocrinology 116, 1094-1101 Laemm]i, U. K. (1970) Nature 227, 680-685 Binoux, M., Seurin, D., Lassarre, C., and Gourmelen, M. (1984) J Clin Endocrinol Metab 59, 462 Binoux, M., Hardouin, S., Lassarre, C., and Hossenlopp, P. (1982) J Clin Endocrinol Metab 55, 602 Hintz, R. L. (1984) Clinics Endocrinol Metab 13, 31-42 White, R. M., Nissley, S. P., Moses, A. C., Rechler, M. M., and Johnsonbaugh, R. E. (1981) J Endocrinol Metab 53, 49-57 Daughaday, W. H., Ward, A. P., Goldberg, A. C., Trivedi, B., and Kapadia, M. (1982) J Endocrinol Metab 55, 916-921

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