Role of Carbohydrate in Human Chorionic Gonadotropin

Transcription

1 THX JOURNAL OF BIOLOGICAL CHXMISTRY Vol. 258, No. 1, Issue of Jnury 10, pp , 1983 Printed m US A. Role of Crbohydrte in Humn Chorionic Gondotropin EFFECT OF DEGLYCOSYLATION ON THE SUBUNIT INTERACTION AND ON ITS IN VITRO AND IN VIVO BIOLOGICAL PROPERTIES* (Received for publiction, June 25, 1982) Nrender K. Klyn nd Om P. Bhlt From the Deprtment of Biologicl Sciences, Division of Cell nd Moleculr Biology, Stte University of New York t Bufflo, Amherst Cmpus, Bufflo, New York Previous studies from our lbortory hve found tht prtil removl or modifiction of the crbohydrte of humn chorionic gondotropin (hcg) results in the impirment of the biologicl ctivity in vitro nd in vivo without ffecting its binding to the receptor. In this report, the complete removl of crbohydrte from hcg hs been ttempted by the ppliction of trifluoromethne sulfonic cid to the subunits followed by the reconstitution of the resulting deglycosylted subunits. The tretment with trifluoromethne sulfonic cid removed ll the crbohydrtes except the lst peptidelinked hexosrnines, without ffecting the polypeptide chins s shown by their mino cid compositions, NH2- terminl nlysis, moleculr weights, CD spectroscopy, nd immunorectivity. The reconstitution of the ntive nd deglycosylted subunits resulted in one deglycosylted derivtive, hcg-d+d@ nd two mixed hybrids, hcg-d+@ nd hcg-+d@. All the reconstituted hcg derivtives not only bound to the testiculr receptor with high ffinity of the order of lo1 M-, but lso regined immunorectivity (90 to 105%) comprble to tht of hcg. None of the reconstituted hormones showed binding to the liver membrne receptor. However, plsm clernce rte of rdiolbeled hcg-d+d@ (tl/, 5 min) nd hcg-+d@ (tli, 15 min) ws considerbly fster thn tht of hcg (tli, 48 min). Orgn distribution of the rdioctivity showed tht kidney, not the liver, ws the mjor orgn for the clernce of these derivtives. The dt on the in vivo cellulr responses of camp nd progesterone production in corpus luteum cells nd the in vivo response of depletion of ovrin scorbic cid showed tht the deglycosyltion of one or both subunits led to drstic drop in the biologicl ctivity of the hormone. hile the deglycosylted hcg filed to stimulte hormonl ctivity, the mixed hybrids produced responses intermedite between those of the ntive nd the deglycosylted hcg. In ddition, the deglycosylted hcg ws found to be potent inhibitor of hcg ctivity in the bove hormonl responses. These studies further demonstrte tht the crbohydrte is not involved in the ssocition of the subunits or in the ntibody or receptor binding ctivity of the hormone lthough it is required for the expression of the hormonl ctivity. * This work ws supported by United Sttes Public Helth Grnt R01-HD nd orld Helth Orgnition Grnt H9/181/337. The costs of publiction of this rticle were defryed in prt by the pyment of pge chrges. This rticle must therefore he hereby mrked dvertisement in ccordnce with 18 U.S.C. Section 1734 solely to indicte this fct. * To whom ll correspondence regrding this mnuscript should be sent. 67 Previous investigtions from our lbortory hve exmined in vitro (1-3) nd in vivo (4, 5) the role of crbohydrte in hcg. hile the sequentil removl of crbohydrte from hcg did not impir its receptor binding properties in vitro, its bility to stimulte camp nd steroidogenesis in rt Leydig cells (1) nd grnulos cells (2, 3) ws considerbly reduced. Similr results were obtined in vivo studies crried out with chemiclly modified nlogs of silo-hcg (4, 5). In ll the hcg derivtives thus fr tested, the crbohydrte ws either prtilly removed or ws modified. The sequentil tretment with exoglycosidses hydrolyed less thn 60% crbohydrte from intct hcg or subunits, while the modifictions removed no more thn 30% crbohydrte, predominntly silic cid (4). In the present work, the complete removl of crbohydrte hs been ttempted by the ppliction of TFMS to the subunits of hcg. This pproch hs enbled us to investigte the involvement of crbohydrte in the subunit interction, in ddition to its effect on the in vitro nd in vivo biologicl properties of the hormone such s receptor binding, camp stimultion, steroidogenesis, plsm hlf-life, nd bility to cuse ovrin scorbic cid depletion. Furthermore, by prepring the hybrids of the deglycosylted subunits with the corresponding ntive subunits, the role of crbohydrte in the individul subunits hs been investigted. A preliminry report of this work hs previously been mde (6). MATERIALS AND METHODS AND RESULTS2 Immunologicl nd Receptor Binding Activity of Purified Reconstituted Deglycosylted nd Hybrid hcg Derivtiues- Immunorectivities of hcg nd the reconstituted nlogs were determined by homologous rdioimmunossy. As shown in Fig. 8, ll the derivtives exhibited prllel doseresponse curves in lz5i-hcg:nti-hcg rdioimmunossy system. Reltive immunologicl potencies of vrious hormone nlogs were determined from the dosge for 50% inhibition (IDSo) nd re given in Tble IV. ithin experimentl error, ll derivtives hd essentilly identicl immunorectivities The bbrevitions used re: hcg, humn chorionic gondotropin or humn choriogondotropin; hcg-dol nd hcg-dd, deglycosylted - nd 8-subunits of hcg; hcg-dol+dp, reconstituted deglycosylted hcg; hcg-d+p nd hcg-+dp, reconstituted mixed hybrids of hcg; TFMS, trifluoromethne sulfonic cid; PK-CAMP, protein kinseboyd CAMP. Portion of this pper (including Mterils nd Methods, prt of Results, Figs. 1-7, nd Tbles 1-111) re presented in miniprint t the end of this pper. Miniprint is esily red with the id of stndrd mgnifying glss. Full sie photocopies re vilble from the Journl of Biologicl Chemistry, 9650 Rockville Pike, Bethesd, MD Request Document No. 82M-1692, cite the uthors, nd include check or money order for $6.80 per set of photocopies. Full sie photocopies re lso included in the microfh edition of the Journl tht is vilble from verly Press.

2 68 FIG. 8. Immunorectivity (A) nd receptor binding ctivity (B) of the reconstituted hcg derivtives. A, homologous rdioimmunossy ws performed using '251-hCG (30,000 cpm) nd nti-hcg ntiserum (1:30,000 dilution). Zero dose binding determined in the bsence of the hormone ws bout 9,OOO cpm nd ws tken s 100% binding. B, "'I-hCG (100,000 cpm) ws incubted with crude testiculr membrne (50 pg of protein) in the presence of vrying concentrtions of the hormones. In the bsence of hcg, bout 25,000 cpm were bound nd ssumed s 100% binding. Nonspecific binding ws determined in the presence of 24 IU (2 pg) of hcg nd ws usully less thn 2% of the totl counts/min dded. Properties of Deglycosylted hcg n $ 50 0 I- u" 20 (L I I I I o CONCENTRATION OF HORMONE [nm) &- 3 TABLE IV Comprison of immunorectivity nd testiculr receptor binding ffinity.. of hcg nd the reconstituted hcg derivtives ~~~ ~~ ~, Immunorectivity" Testiculr receptor binding ffinityh Hormone IDm x Reltive rc, X 10"' Reltive 10"' M potency' (men f S.E.) potency' hcg k hcg-+p f hcg-d+dp k hcg-d+p f hcg+dp f The immunorectivity of hormone derivtives ws compred on the bsis of ID,, (hormone concentrtion required for hlf-mximl inhibition) determined from the rdioimmunossy inhibition binding curves shown in Fig. 10. ' The pprent ffinity constnts (KO) of hormones for testiculr membrne receptor were clculted from Sctchrd plots mde from the equilibrium competitive binding ssy shown in Fig. 10. Ech vlue represents the men k S.E. of two to three ssys. Immunorectivity nd receptor binding ctivity of hcg ws ssumed to be 100%. " 15 C w tn c '"I d +db I 0' I I CONCENTRATION HORMONE/ DERIVATIVES ng / ml rnging from 85 to 107% of hcg, suggesting the recovery of ll ntigenic sites on ressocition of the deglycosylted subunits. Hlf-mximl inhibition ws chieved t hormone concentrtion round 10"' M. Similrly, hcg nd its reconstituted derivtives possessed identicl receptor binding ctivities nd gve prllel doseresponse curves in the rdiolignd receptor ssy shown in Fig. 8. The binding ffinity constnts (K,) of hcg nd its nlogs were estimted from Sctchrd plots obtined from the competitive receptor binding dt shown in Fig. 8. As is cler from Tble IV, ll reconstituted derivtives hd identicl binding constnts, with K, vlues rnging from 1.4 to 2 X 10" M-', which re not significntly different from tht of the ntive hcg (IC, 1.6 X 10" "I). The bove dt clerly show tht the crbohydrte of both or the individul subunit is not prt of the ntibody or the receptor binding site. Stimultion of camp Production nd Steroidogenesis by hcg nd Its Ressocited Derivtives in Corpus Luteum Cells-In order to investigte theffect of deglycosyltion of hcg in the post-receptor binding events of two cellulr responses, camp production nd steroidogenesis were studied in luteum cells nd re shown in Fig. 9. In the cse of steroidogenesis, none of the reconstituted deglycosylted derivtives produced mximl response equivlent to tht by 1 ng/ml of hcg. hile the hybrids hcg-d+p nd hcg-+dp produced bout 40% of the mximl response, the deglycosylted reconstituted derivtive hcg-d+dp brely responded bove the bsl levels. In camp production, the mixed P I / Jc. / hcg-do@ hcg-db hcg-do t db 0 1 io CONCENTRATIONHORMONE/DERIVATIVES ng /ml FIG. 9. Stimultion of progesterone (A) nd CAMP (B) production by hcg nd its reconstituted derivtives in corpus luteum cells. A, vrying mounts of hcg nd its derivtives were incubted with 0.4 X lo6 cells/tube t 37 "C for 90 min. Both ssys were done in triplicte nd represent the men f S.E. E, bout 1 X lo6 cells were incubted in the presence of the indicted mounts of hormones t 37 "C for 90 min. The medium lso contined 0.2 mm methyl 1,3-isobutylxnthine.

3 brids of hcg t 1 pg/d produced only bout 15% of the mximl response of hcg. In contrst, hcg-d+dp did not respond significntly bove the bsl level. In order to further investigte whether the inbility of deglycosylted hcg to produce the cellulr responses is lso reflected in the levels of intrcellulr CAMP, PK-CAMP ws estimted by highly sensitive rdioimmunossy s described by Dufu et l. (15). As shown in Tble V, hcg stimulted n increse in the level of PK-CAMP in dose-response fshion t hormone concentrtion of 0.1 to 100 ng/ml s previously reported (15). The deglycosylted hcg filed to produce ny significnt increse in the level of PK-CAMP up to concentrtion of 1 pg/d, gin showing tht the derivtive did not hve ny intrinsic hormonl ctivity. Since hcg-&+dp hd high binding ffinity for the trget tissue receptor without producing ny hormonl response, it ws ssessed for its bility to inhibit hcg ctivity in the bove cellulr responses in luteum cells. The results of these studies re shown in Fig. 10 nd Tble V. The derivtive cusedosedependent inhibition of camp production nd steroidogenesis by hcg. In cse of camp production, the response produced by 10 nd 100 ng of hcg ws inhibited by 80 to 90% by n equl or greter mount of the derivtive. On the other hnd, inhibition of the 50% steroidogenic response produced by 0.1 nd 0.5 ng required 2 to 3-fold greter mount of the derivtive. As is cler from Tble V, the intrcellulr level of PK-CAMP mesured in the bove responses showed corresponding inhibition by the hcg-d+dp. 2 pg of the deglyco- sylted derivtive cused decrese in the level of PK-CAMP from 305 f 11 fmol/tube, stimulted by 0.5 ng of hcg, to 220 rt 11 fmol/tube. The bsl level of PK-CAMP in these studies ws fmol/tube. Binding of 1251-lbeled Deriutives to Liver Plsm Membrne-In order to investigte the effect of deglycosyltion of hcg on its uptke by the liver, direct binding studies using 125 I-lbeled hcg derivtives nd crude liver plsm membrnes were performed s shown in Fig. 11. None of the deglycosylted nd hybrid hcg derivtives bound to the liver membrnes pprecibly. As reference, silo-hcg ws shown to bind specificlly to the glctose receptor on liver membrnes. Plsm Clernce of 1251-lbeled hcg, Deglycosylted hcg, nd Mixed Hybrids in Mture Femle Rts-Fig. 12 shows the plsm clernce curves of I5I-lbeled hcg derivtives in rts, nd Tble VI lists the orgn distribution of rdioctivity 30 min fter injection of the hormone. The TABLE V Inhibition of hcg-induced intrcellulr PK-CAMP by the reconstituted deglycosylted hcg, hcg-d+dp The results re expressed s men f S.D. of three vlues. Hormone Pk-CAMP fmol/loh cells Control 188 f 7 hcg 0.1 ng 240 f ng 305 f ng 415 f ng 430 f 15 hcg-d+dp 100 ng 180 & 8 lo00 ng 205 f 10 hcg (0.5 ng) + hcg-d+dp 0.5 ng 240 f ng HCG (IO ng) + hcg-d+dp 220 f 4 10 ng 210 f ng 180 f 4 Properties of Deglycosylted hcg t- 0 (L w 75 n to.lng hcg I 1 I I I I I CONCENTRATION OF hcg-do+d (ng/ml) FIG. 10. Inhibition of hcg stimulted progesterone (A) nd CAMP (B) production in luteum cells by hcg-d+d/?. The derivtive ws mixed with hcg before ddition to the cells. No hormone ws dded to the control tube. The tubes for camp studies lso contined 0.2 m~ methyl 1,3-isobutylxnthine. The results re expressed s men f S.E LIVER MEMBRANE PROTEIN (mg) FIG. 11. Binding of hcg nd its reconstituted derivtives to liver plsm membrnes. To 5 ng (l00,ooo cpm) of 1Z51-lbeled hormone were dded vrying mounts of crude liver membrnes, nd these were incubted t 37 "C for 1 h s described under "Mterils nd Methods."!- u '9 A hcg. I I I TIME (rnin) FIG. 12. Plsm clernce curves of hcg nd its reconstituted derivtives in femle rts. About 0.5 X 10' cpm of I5Ilbeled nd 100 ng of unlbeled hormone were injected vi til vein, nd blood smples were tken from the opposite til vein t indicted time intervls. Rdioctivity in 100 p1 of plsm ws counted nd expressed s per cent of the injected dosge/ml of plsm. Echpoint represents the verge from two nimls. plsm clernce rte of mixed hybrid contining ntive p- subunit, hcg-d+p (til2, 50 min), ws found to be comprble to tht of hcg (til2, 48 min). However, the plsm hlf-lives of hcg-d+db nd hcg-+dp were considerbly lower, with tllr of 5 nd 13 min respectively, thn tht predicted from the in vitro liver receptor binding dt. Distribution of the rdioctivity in vrious orgns showed tht kidney contined bout 56 nd 39% rdioctivity. As reference, silo-hcg with terminl glctosyl residue ws found to be concentrted in

4 70 Properties of Deglycosylted hcg liver (73%). However, when silo-fetuin (2 mg) ws co-injected with '2sI-silo-hCG, the liver uptke decresed to 27% with concomitnt increse in uptke by the kidney (4). In order to determine the ovrin uptke, '251-lbeled hcg derivtives were dministered to superovulted femle rts. hcg nd hcg-d+p both showed substntil ovrin uptke of 15 to 20% rdioctivity t 1 h fter injection. In the cse of hcg-d+dfi nd hcg-+db, only bout 2.5 nd 5%of the TABLE VI Tissue distribution of '"I-lbeled hcg nd the reconstituted ntive nd deglycosylted hcg hybrids The group of rts used for plsm clernce studies shown in Fig. 14 were killed 30 min fter injection of rdiolbeled hormone. The rdioctivity in the indicted orgns ws determined nd expressed s per cent of totl dose injected per orgn. Vlues in prentheses re counts/min X 1O"/g of wet tissue. Ech vlue represents the men of dt obtined from two rts. Per cent of '"1 lbeled hormone injected per orgn Orgn hcg hcg-d+dp hcg-d+p hcg-+d@ Blood Liver 13.3 (7.1) 8.6 (5.2) 16.5 (9.8) 18.5 (14.2) Kidney 8.1 (19.6) 56.2 (180.8) 6.9 (12.7) 39.0 (120.8) Hert 0.2 (0.8) 0.36 (1.9) 0.58 (1.9) 0.30 (1.4) Spleen 0.34 (1.5) 0.27 (2.0) 0.43 (2.4) 0.34 (2.1) Uterus Ovries 0.05 (0.9) 0.54 (23.4) 0.39 (3.8) 0.1 (3.8) 0.15 (1.3) 0.30 (14.8) 0.32 (2.8) 0.36 (9.6) t 0 I E 0 il I t? I AMOUNT OF hcg OR DERIVATIVES INJECTED (pg) '/ ; AMOUNT OF hcg -dd + dfi ( pg) FIG. 13. Top, in uiw biologicl ctivity of the reconstituted hcg nlogs determined by mesuring the depletion of ovrin scorbic cid in pseudopregnnt rts. The rts in groups of three were dministered the indicted dosge of either hcg (12,000 IU/mg) or the derivtives in 0.9% NCl contining 1% bovine serum lbumin. The control groups received vehicle only. The results re expressed s per cent depletion of scorbic cid reltive to the control vlues. Bottom, inhibition of hcg ctivity by hcg-d+dp in n ovrin scorbic cid ssy. The derivtive ws co-injected with hcg into pseudopregnnt rts, nd the rest of procedure ws exctly the sme s described bove. I I rdioctivity ws found in the ovries. This is consistent with their shorter plsm hlf-lives. Kidney ws gin found to be the mjor orgn responsible for the clernce of these derivtives. In Vivo Biologicl Activity of hcg nd its Reconstituted Derivtives-The effect of deglycosyltion of hcg on its in vivo biologicl properties ws studied by mesuring the depletion of scorbic cid in pseudopregnnt rt ovries nd is shown in Fig. 13. Consistent with the in vitro observtions, the mixed hybrids hd reduced potencies rnging from 1.5 to 7% ctivity. Bsed upon the specific ctivity of hcg s 12,000 IU/mg, the corresponding vlues for hcg-d+p nd hcg+dp were found to be 650 IU/mg (520 to 780,95% confidence limit) nd 170 IU/mg (110 to 230, 95% confidence limit), respectively. The lower potency estimte of hcg-+db my prtly be due to its shorter plsm hlf-life. On the other hnd, the deglycosylted derivtive, hcg-d+dp, did not produce ny response up to dosge of 100 pg. Consequently, it ws ssessed for its bility to neutrlie the hcg ctivity in the bove ssy system. As shown in Fig. 13, the derivtive ws ble to inhibit the ctivity of 0.5 nd 2 pg of hcg in dose-dependent fshion. About 20-fold excess of the derivtive ws needed to chieve 50% inhibition of the response. This ws probbly due to its shorter plsm hlf-life thn hcg (Fig. 12). DISCUSSION hcg hs 30 to 33% crbohydrte distributed in 4 sprginelinked "complex"-type crbohydrtes, 2 in ech - nd p- subunit nd 4 serine-linked tetrscchride chins locted exclusively in the COOH terminus of the P-subunit (22, 23). The tretment with TFMS selectively removed lmost ll but the linkge hexosmine residues from both subunits with good recovery of the products. Detiled physicochemicl nd immunologicl properties such s mino cid composition, NH,-terminl mino cid nlysis, moleculr weight determintion, CD spectr, s well s immunorectivity of the deglycosylted subunits, filed to revel ny significnt ltertion in the polypeptide chin. The dt on the ressocition of the deglycosylted subunits nd in vitro receptor binding ctivity of the reconstituted hormone further support the integrity of the polypeptide chins. TFMS tretment of intct hcg cused its deglycosyltion s extensively s the individul subunits, lthough the hormone underwent dissocition during the tretment s shown by the loss of its recept,or binding ctivity. Under suitble conditions, however, the dissocited hormone could be ressocited with the complete restortion of the receptor binding ctivity of the hormone:' HF hs lso been used for the deglycosyltion of glycoproteins; when pplied to olh subunits, it removed only 60 to 70% of the crbohydrte (24) while TFMS removed bout 90% of the crbohydrte from hcg. Thus, TFMS is significntly more effective deglycosylting gent thn HF. Moreover, it is convenient to use since it does not wrrnt ny specific hndling s is required for HF (6, 18, 24). The vilbility of the deglycosylted subunits of hcg for the fist time hs enbled us to investigte the role of crbohydrte in individul subunits. Severl conclusions cn be drwn from the present work. Importntly, the crbohydrte in both subunits is not involved in the subunit interction nd in the receptor binding ctivity of the hormone. Thus, the deglycosyltion of the subunits does not ffect their bility to ressocite with ech other or with the corresponding ntive subunit (Figs. 5 nd 6). In fct, the deglycosylted subunits recombine t rte twice tht of the ntive subunits. This N. Klyn nd 0. Bhl, unpublished dt

5 probbly is due to the elimintion of electronegtive chrge by the loss of silic cid during deglycosyltion. It is worth noting tht ll the reconstituted hcg derivtives bind to the receptor with high ffinity of the order of 10" M-', comprble to tht of hcg (Fig. 8 nd Tble IV). In ddition, the deglycosyltion of one or both subunits leds drstic to drop in the biologicl ctivity of the hormone. Unlike the ntive hormone, the deglycosylted hcg fils to stimulte the in uitro cellulr responses such s camp nd progesterone production in rt luteum cells (Fig. 9). In uiuo, in rts, the biologicl response of ovrin scorbic cid depletion is lso impired (Fig. 13). The mixed hybrids of deglycosylted nd ntive subunits produce hormonl responses intermedite between those of the ntive nd the deglycosylted hcg. The deglycosylted hcg is potent inhibitor of camp nd progesterone stimultion (Fig. 10). The inhibitory effect ws much more pronounced in the in uitro system thn in uiuo. This probbly ws due to its rpid clernce from plsm by kidney nd not by liver (Figs. 11 nd 12 nd Tble VI). The resons for the rpid clernce by kidney re not cler t present. It my be due prtly to the reduced sie nd chrge. It is interesting to note tht the deglycosylted hcg does not bind in uitro or in vivo to the liver which hs receptors for N- cetylhexosmines (25). It ppers tht the linkge hexosmines re not ccessible to the cellulr receptors due to steric effects. Since the deglycosylted hormone binds to the receptor s well s hcg or better, the loss of crbohydrte does not seem to chnge the conformtion of the receptor binding site of the hormone. This, however, does not rule out the possibility of the deglycosylted hormone-receptor complex undergoing conformtionl chnge which my led to the loss of its bility to form microclusters in the membrnes nd/or its bility to stimulte the subsequent steps in the hormone ction s the ctivtion of GTP or nucleotide binding protein nd denylte cyclse (26,27). Obviously, the precise site of the crbohydrte ction in the currently vilble model of polypeptide hormone ction is not known. Our preliminry dt, however, indicte tht the removl of crbohydrte from hcg ffects n event(s) prior to the ctivtion of nucleotide binding pro- Properties of Deglycosylted hcg 71 moleculr level, it hs the potentil of ppliction to fertility regultion. Acknowledgments-e wish to express our pprecition to Jmes Stmos for prepring the figures nd to Ursul Brunn for typing the mnuscript. REFERENCES 1. Moyle,. R., Bhl, 0. P., nd Mr, L. (1975) J. Biol. Chem. 250, Chnning, C. P., nd Bhl, 0. P. (1978) Biol. Reprod. 17, Chnning, C. P., nd Bhl, 0. P. (1978) Endocrinology 103, Mdnick, H.., Klyn, N. K., Segl, H. L., nd Bhl, 0. P. (1981) Arch. Biochem. Biophys. 212, Klyn, N. K., Lippes, H. A., nd Bhl, 0. P. (1982) J. Biol. Chem. in press 6. Klyn, N.K., nd Bhl, 0. P. (1981) Biochem. Biophys. Res. Commun. 102, Crlsen, R. B., Bhl, 0. P., nd Swminthn, N. (1973) J. Bwl. Chem. 248, Lehnhrdt,. F., nd inler, R. J. (1968) J. Chromtogr. 34, rren, L. (1959) J. Biol. Chem. 234, Swminthn, N., nd Bhl, 0. P. (1970) Biochem. Biophys. Res. Commun. 40, Pndin, M. R., nd Bhl, 0. P. (1977) Arch. Biochem. Biophys. 182, Ashwell, G., nd Morell, A. G. (1974) Adu. Enymol. Rel. Ares Md. Biol Achord, D. T., Brot, F. E., nd Sly,. S. (1977) Biochem. Biophys. Res. Commun. 77, Ferguson, K. A. (1964) Metb. Clin. Ep. 13, Dufu, M. L., Tsuruhr, T., Horner, K. A., Podest, E., nd Ctt, K. J. (1977) Proc. Ntl. Acd. Sci. U. S. A. 74, Hrper, J. F., nd Brooker, G. (1975) J. Cyclic Nucleotide Res. 1, Mindlin, R. L., nd Butler, A. M. (1933) J. Biol. Chem. 122, Edge, A. S. B., Fltynek, C. R., Hof, L., Reichert, L. E., Jr., nd eber, P. (1981) Anl. Biochem. 118, Bellisrio, R., Crlsen, R. B., nd Bhl, 0. P. (1973) J. Biol. Chem. 248, Holldy, L. A., nd Puett, D. (1975) Arch. Biochem. Biophys. 171, Bewley, T. A., Sirm, M. R., nd Li, C. H. (1972) Biochemistry tein nd denylte cyclse but on or fter the hormone- 11, receptor complex formtion (28). 22. Kessler, M. J., Reddy, M. S., Shh, R. H., nd Bhl, 0. P. (1979) In summry, the present work clerly estblishes the effi- J. Biol. Chem. 254, ccy of TFMS in the selective removl of crbohydrte from 23. Kessler, M. J., Mise, T., Ghi, R. D., nd Bhl, 0. P. (1979) J. Biol. Chem. 254, hcg or hcg subunits without ny detectble dverse effect 24. Sirm, M. R. (1980) Arch. Biochem. Biophys. 204, on the polypeptide chins. It further demonstrtes tht the 25. Kwski, T., nd Ashwell, G. (1977) J. Biol. Chem. 252, crbohydrte is not involved in the ssocition of the subunits 6543 or in the ntibody or receptor binding ctivity of the hormone. 26. Amsterdm, A., Berkowit, A,, Nimrod, A., nd Kohen, F. (1980) Finlly, it is clerly shown tht the deglycosyltion of hcg Proc. Ntl. Acd. Sci. U. S. A. 77, results not only in the loss of biologicl ctivity, but it lso 27. Abrmowit, J., Iyengr, R., nd Birnbumer, L. (1980) in Funcimprts ntgonistic properties to the hormone. The deglytionl Correltes of Hormone Receptors in Reproduction (Mhesh, V. B., Muldoon, T. G., Sxen, B. B., nd Sdler, w. A,, cosylted hcg thus is importnt from both the fundmentl eds) pp , Elsevier/North-Hollnd, New York nd clinicl point of view. hile the derivtive cn serve s 28. Thotkur, N. R., nd Bhl, 0. P. (1982) Biochem. Biophys. Res. probe in the study of the hormone ction t cellulr nd Commun., 108,

6 72 Properties of Deglycosylted hcg

7 Properties of Deglycosylted hcg 73 A - " Y E P Y CI, s 1.7- hcg-d 1.6 I I I I I PERCENT GEL CONCENTRATION r 1.o 0.5 hcg-dfiysozyme FRACTION NUMBER 1.o I I I ( M ) 'I3

8 74 Properties of Deglycosylted hcg AVELENGTH (nml, o FRACTION NUMBER Cl hcg- d +B Dl hcg-n +d/ io 100 CONCENTRATION OF HORMONE (nm) 100 P c 80 & m 5 60 w v 40 Y I hcc-ihcg-b hcgdcllhcg-db hcc-dcl+hcg-b hcg+hcg-db