165 Horm. Metab. Res. 11 (1979) 165-167
The Carbohydrate Moiety of Human Chorionic Gonadotropin: lack of Competition with HCG for Testicular Receptors and Anti-HCG-Serum D. Puett, R. Benveniste, A. Kenner and D. Rabinowitz Departments of Biochemistry and Medicine, Vanderbilt University, Nashville, Tennessee, U.S.A.
A glycopeptide fraction has been prepared from human chorionic gonadotropin (HCG) by digesting the reduced, Scarboxymethylated hormone with pronase and fractionating the digest by gel exclusion chromatography. The glycopeptide fraction was estimated to contain (w/w) 29% sialic acid, 31% hexose, 23% hexosamine, and 17% amino acids and/or peptides; thus, the glycopeptide mixture is 83% carbohydrate compared to intact HCG which is about 30% carbohydrate. There was no cross-reactivity of the glycopeptide fraction with an antiserum directed against HCG. Also, when corrected for minimal non-specific effects, the fraction failed to displace 125 I-HCG from a rat testicular preparation even when tested at a 10,000-fold (w/w) excess. Thus, any model involving carbohydrate effects in gonadotropin action must include the pro tein moiety as a necessary component. Key-Words: Human Chorionic Gonadotropin - Glycopeptides - Radio-/mmunoassay - Radio-Receptor Assay
Introduction Several studies have appeared on the possible role of the carbohydrate moiety of HCG in the hormonereceptor interaction and subsequent biological responses (Tsuruhara, Dufau, Hickman and Catt 1972; Moyle, Bahl and Marz 1975). In these investigations, HCG derivatives were prepared using various glycosidases on the intact hormone to specifically remove different sugars, although in no case was it possible to eIiminate aIl of the carbohydrate residues. In the present study a totally different approach has been taken in which most of the pro tein moiety was removed from the hormone leaving the various carbohydrate side-chains intact and covalently attached to either amino acids or peptides. The carbohydraterich fraction so obtained was assayed for its binding affinity (via competition with 125I-HCG) to testicuIar receptors and for any cross-reactivity to antiserum against HCG. Material and Methods Crude (pregnancy urine) HCG was purchased from Organon, lnc., and had astated potency of about 3,000 lU/mg. Purification was achieved using anion exchange chromatography Received: 9 March 1978
Accepted: 10 March 1978
001B-5043/79
.03.00
0232-D165
(DEAE-Sephadex A-25) followed by gel exc1usion chromatography (Bahl 1969; Hoiladay and Puett 1975). The fraction used for the preparation of the glycopeptides was eluted from the ion-exchanger with 0.2 M NaCl, 40 mM Tris-phosphate, pH 8.7. The purified HCG was reduced, S-carboxymethylated, and digested three times with pronase as described elsewhere (Hoiladay and Puett 1975; Puett, Nureddin and Hoiladay 1976; Puett, Hoiladay and Robinson 1977). Following the second enzymic digestion, the mixture was chromatographed using Bio-Gel P-4 and the column fractions were monitored for hexose-positive and amino-positive material (Hoiladay and Puett 1975). The hexose-positive fraction was pooled, lyophilized, digested again with pronase, and the mixture was chromatographed on a Bio-Gel P-6 column. For final purification, the HCG glycopeptide fraction was chromatographed on a Bio-Gel P-30 column. This separated the glycopeptide fraction from any residual pron~se, HCG, and oligopeptides. The major hexose-positive fractions were pooled and lyophilized; 11 mg were obtained from 74 mg of starting HCG. A control with no HCG was processed using identical conditions to those employed for the glycopeptides. The glycopeptide fraction was analyzed for total hexose, hexosamine, sialic acid, and amino acid composition of the peptide portions using techniques described elsewhere (Holloday and Puett 1975; Puett, Nureddin and Hoiladay 1976). Hexose was based on the orcinol-sulfuric acid reaction with a galactose standard; sialic acid was determined using the resorcmol method with N-acetylneuraminic acid as standard; and hexosamines and amino acids were measured on 4 N HCl hydrolysates (4 hours, 110°C) using a Beckman 120 amino acid analyzer (appropiate standards were glucosamine, galactosamine, and an amino acid kit). HCG was similarly analyzed except that the amino acid composition was obtained on 6 N HCl hydrolysates (20 hours, 110°C). The amount of each component was referred to the dry weight of the starting material; in all cases the experimentally determined composition accounted for ca. 8590% of the total weight (the 10-15% difference is weil within the overall experimental accuracy). For convenience, the compositions have been normalized to 100%. The activities of HCG and the glycopeptide fraction were tested in an homologous second antibody radioimmunoassay (RIA) and a radio-receptor assay (RRA) which have been decribed (Benveniste, Frohman, Beil, Spitz and Rabinowitz 1975; Schneider, Benveniste, Spitz and Rabinowitz 1973). Briefly, the RRA consisted of using a rat testicular particulate fraction (18,000 xg); incubation with 1251-HCG and HCG or the glycopeptide fraction was for 8 hours at 21°C. High speed centrifugation was used to separate free from bound f251-HCG. In order to determine the nonspecific effect of the glycopeptide preparation, parallel dilutions were performed on the control.
© 1979 Georg Thieme Publishers
Downloaded by: Boston University. Copyrighted material.
Summuy
166
D. Puett, R. Benveniste, A. Kenner and D. P.abinowitz
0.50
@
®
A420 0.25
0 0 25
50
75
100
6OOr---------~~~~~--------,
@ 400 HEXOSE }IIJ Iml 200
50
Figure 1 shows the column chromatograms of the HCG glycopeptide fraction following the second (Fig. lA) and third (Fig. 18) digestion with pronase, and the fmal purification of the 3-times digested material (Fig. 1C). The HCG glycopeptide fraction was by weight: 29% sialic acid, 31% hexose, 23% hexosamine, including 20% glucosamine and 3% galactosamine (all probably N-acetylated), and 17% amino acids or peptides. The overall recovery of total carbohydrate (from HCG) in the glycopeptide fraction was ca. 40%. However, the weight percentages of sialic acid, hexose, and hexosamine in the total carbohydrate component of HCG was 30, 40, and 30, respectively; the corresponding values fot the glycopeptide fraction 6"elated to its total carbohydrate component) are 35, 37, and 28. Thus, there was no preferential removal of sialic acid or other sugar residues during the pro nase digestions. Consequently, the incomplete recovery of total carbahydrate is attributed to normal handling losses during chrornatography and lyophilization. Following acid hydrolysis, which converts asparagine and glutamine to the free ß and 'Y carboxylic acids, respectively, the major amino acids were proline, aspartic acid, serine, threonine, alanine, and leucine, Normalized to aspartic acid, the corresponding molar ratios were l.I, l.O, 0.8, 0.6, 0.4, and 0.3. The amino acids lysine, histidine, arginine, glutamic acid, glycine, and valine, were present in amounts between 0.1 to 0.2 (nonnalized to aspartic acid); the other amino acids, (carboxymethyl) cysteine, methionine, isoleucine, tyrosine, and phenylalanine were either absent or present in trace amounts only.
100
75 VOLUME, ml
70% protein. The amino acid composition was quite similar to that of sequenced HCG (Bahl 1973; Mo,gan, Bi,ken and Can/ield 1975).
Fig. 1 Gel exc1usion chromatograms following digestion of reduced, S-carboxymethylated HCG with pronase. A. 1.5 x 86 cm column of Bio-Gel P-4 following the second pronase digestion. The hexose-positive fractions were pooled as indicated; the arrow denotes the position of the main ninhydrin-positive material. B. 1.5 x 90 ~ column of Bio-Gel P-6 following the third pronase digestion; the dashed line shows ninhydrin-positive material. C. Rechromatography of the pooled material from B on a 1.5 x 52 cm column of Bio-Gel P-30. The ordinate in panels A and B is the absorbance at 420 nm for the orcinol reaction; ninhydrin reactions were monitored at 570 nm. The hexose content (panel C) is based on a mannose standard. In all cases the columns were equilibrated and developed with 0.1 M pyridine-acetate, pH 5.0, at ambient temperature.
In RIA, 1 mg/mI of the HCG glycopeptide fraction did not affect the binding of 125 I-HCG to rabbit antiHCG serum, while 50% displacement of the tracer was observed with 5 ng/mI of HCG. The results of RRA are shown in Figure 2 which compares the
3Or-----------------------------------------,
10'
10·
10' nQ/ml
10'
10·
10"
Fig. 2 RRA dilution curves of native HCG (e) and the glycopeptide fraction (0). BIT refers to the bound to total ratio of 125I-HCG in the presence of various concentrations (ng/ml) of HCG or g1ycopeptides.
Downloaded by: Boston University. Copyrighted material.
Results The purified HCG preparation had a potency (relative to CR1l9) of II ,304 lU/mg (with 95% confidence limits of 10,466-12,142 lU/mg) by RIA. It was estimated to contain (by weight), 9% sialic acid, 12% hexose, 9% hexosamine (mainly glucosamine), and
The Carbohydrate Moiety of Human Chorionic Gonadotropin
Discussion These results indicate that the HCG glycopeptide fraction has less than 1: 10,000 (w/w) the binding affmity of HCG; on a molar basis the difference would be even greater. This suggests that any biological role of the HCG carbohydrate moiety must be in concert with the pro tein component. The lack of cross-reactivity with an antiserum directed against HCG suggests that the carbohydrate side-chains do not represent an antigenic site; other anti sera need to be investigated to confirm this suggestion.
four glycosidases was capable of stimulating steroidogenesis, but not cAMP, and the apparent binding was approximately 10-fold less than that of HCG. Since our results suggest that the isolated carbohydrate components have limited, if any, binding affmity, the lO-fold reduction in binding may result from a conformational change in the protein moiety upon removal of a portion of the carbohydrate. Also, it is possible that the carbohydrate moieties may undergo conformational changes when removed from the polypeptide domain of HCG. However, with this possible caveat, our results suggest that under physiological conditions where the criteria of high specificity and binding affinity are presumably met, the pro tein component dictates the initial receptor recognition and interaction, and this may be influenced slightly by the carbohydrate components.
Thus, the carbohydrate moiety of HCG may regulate secretion and metabolism of the hormone, but its role, if any, in stimulating biological events, on ce binding has occurred, remains to be clarified. It appears, however, not to be responsible for the initial These findings, based on a different approach capable receptor recognition. of recognition independent from the protein moiety of glycoprotein hormones, confirm in general the results of others (Tsuruhara et al. 1972; Moyle et al. Acknowleclgements 1975). Moyle et al. (1975) studied the binding, It is a pleasure to than Ms. Betty Kay Wasserman for ex· pert technical assistance. This research was supported in testosterone production, and cAMP production of large part by the National Institutes of Health: AM 15838, HCG and derivatives prepared by sequential treatHO 10128, H005797, and HO 07043. O.P. is a Research ment with neuraminidase, galactosidase, N-acetylCarear Oevelopment Awardee (AM 00055) and thanks the glucosaminidase, and mannosidase. It is noteworthy Oreyfus Foundation and the VU Research Council for partial support. that the derivative obtained by treatment with the
References Bahl, O.P.: Human chorionic gonadotropin. I. Purification and physicochemical properties. J. Biol. Chem. 244: 567-574 (1969) Bahl, O.P.: Chemistry of human chorionic gonadotropin. In: Hormonal proteins and peptides, Vol. I, Li, C. H., Ed., p. 171-199, Academic Press, New York 1973 Benveniste, R., L.A. Frohman, 1. Bell, I. Spitz, D. Rabinowitz: Subunit of g1ycoprotein hormone: presence in peripheral serum after LHRH. Europ. J. Clin. Invest. 5: 123-131 (1975) Holladay, L.A., D. Puett: Gonadotropin and subunit conformation. Arch. Biochem. Biophys. 171: 708-720 (1975) Morgan, F./., S. Birken, R.E. Canfield: The amino acid sequence of human chorionic gonadotropin. J. Biol. Chem. 250: 5247-5258 (1975)
Moyle, W.R., O.P. Bahl, L. Marz: Role of the carbohydrate of human chorionic gonadotropin in the mechanism of hormone action. J. Biol. Chem. 250: 9163- 9169 (1975) Puett, D., A. Nureddin, L.A. Holladay: Circular dichroism of human pituitary luteinizing hormone and its glycopeptides. Int. J. Peptide Protein Res. 8: 183-191 (1976) Puett, D., L.A. Holladay, I.P. Robinson: Circular dichroism of human urinary Tamm-Horsfall glycoprotein. Molecular Cellular Biochemistry 15: 109-116 (1977) Schneider, B., R. Benveniste, I. Spitz, D. Rabinowitz: Bind· ing of human luteinizing hormone to particulate frac· tion of rat testis. Metabolism 22: 1225-1233 (1973) Tmruhara, T., M.L. Du/au, I. Hickman, K./. Catt: Biological properties of HC'G after rem oval of terminal sialic acid and galactose residues. Endocrinology 91: 296- 30 I (1972)
Requests for reprints should be addressed to: Or. O. Puett, Oepartment of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232 (U.S.A.)
Downloaded by: Boston University. Copyrighted material.
displacement of m I-HCG by the HCG glycopeptide fraction and intact HCG. The displacement of 125I-HCG from the rat testicular preparation was unaffected up to concentrations of 50 J,lg/ml of the HCG glycopeptide fraction. The slight displacement noted at 100 J,lg/ml and above is non-specific since comparable displacement was observed with the HCGfree control. Similar results were obtained with the minor glycopeptide fraction present in the leading edge to the major fraction (cf. Fig. IB).
167
The Carbohydrate Moiety of Human Chorionic Gonadotropin: lack of Competition with HCG for Testicular Receptors and Anti-HCG-Serum D. Puett, R. Benveniste, A. Kenner and D. Rabinowitz Departments of Biochemistry and Medicine, Vanderbilt University, Nashville, Tennessee, U.S.A.
A glycopeptide fraction has been prepared from human chorionic gonadotropin (HCG) by digesting the reduced, Scarboxymethylated hormone with pronase and fractionating the digest by gel exclusion chromatography. The glycopeptide fraction was estimated to contain (w/w) 29% sialic acid, 31% hexose, 23% hexosamine, and 17% amino acids and/or peptides; thus, the glycopeptide mixture is 83% carbohydrate compared to intact HCG which is about 30% carbohydrate. There was no cross-reactivity of the glycopeptide fraction with an antiserum directed against HCG. Also, when corrected for minimal non-specific effects, the fraction failed to displace 125 I-HCG from a rat testicular preparation even when tested at a 10,000-fold (w/w) excess. Thus, any model involving carbohydrate effects in gonadotropin action must include the pro tein moiety as a necessary component. Key-Words: Human Chorionic Gonadotropin - Glycopeptides - Radio-/mmunoassay - Radio-Receptor Assay
Introduction Several studies have appeared on the possible role of the carbohydrate moiety of HCG in the hormonereceptor interaction and subsequent biological responses (Tsuruhara, Dufau, Hickman and Catt 1972; Moyle, Bahl and Marz 1975). In these investigations, HCG derivatives were prepared using various glycosidases on the intact hormone to specifically remove different sugars, although in no case was it possible to eIiminate aIl of the carbohydrate residues. In the present study a totally different approach has been taken in which most of the pro tein moiety was removed from the hormone leaving the various carbohydrate side-chains intact and covalently attached to either amino acids or peptides. The carbohydraterich fraction so obtained was assayed for its binding affinity (via competition with 125I-HCG) to testicuIar receptors and for any cross-reactivity to antiserum against HCG. Material and Methods Crude (pregnancy urine) HCG was purchased from Organon, lnc., and had astated potency of about 3,000 lU/mg. Purification was achieved using anion exchange chromatography Received: 9 March 1978
Accepted: 10 March 1978
001B-5043/79
.03.00
0232-D165
(DEAE-Sephadex A-25) followed by gel exc1usion chromatography (Bahl 1969; Hoiladay and Puett 1975). The fraction used for the preparation of the glycopeptides was eluted from the ion-exchanger with 0.2 M NaCl, 40 mM Tris-phosphate, pH 8.7. The purified HCG was reduced, S-carboxymethylated, and digested three times with pronase as described elsewhere (Hoiladay and Puett 1975; Puett, Nureddin and Hoiladay 1976; Puett, Hoiladay and Robinson 1977). Following the second enzymic digestion, the mixture was chromatographed using Bio-Gel P-4 and the column fractions were monitored for hexose-positive and amino-positive material (Hoiladay and Puett 1975). The hexose-positive fraction was pooled, lyophilized, digested again with pronase, and the mixture was chromatographed on a Bio-Gel P-6 column. For final purification, the HCG glycopeptide fraction was chromatographed on a Bio-Gel P-30 column. This separated the glycopeptide fraction from any residual pron~se, HCG, and oligopeptides. The major hexose-positive fractions were pooled and lyophilized; 11 mg were obtained from 74 mg of starting HCG. A control with no HCG was processed using identical conditions to those employed for the glycopeptides. The glycopeptide fraction was analyzed for total hexose, hexosamine, sialic acid, and amino acid composition of the peptide portions using techniques described elsewhere (Holloday and Puett 1975; Puett, Nureddin and Hoiladay 1976). Hexose was based on the orcinol-sulfuric acid reaction with a galactose standard; sialic acid was determined using the resorcmol method with N-acetylneuraminic acid as standard; and hexosamines and amino acids were measured on 4 N HCl hydrolysates (4 hours, 110°C) using a Beckman 120 amino acid analyzer (appropiate standards were glucosamine, galactosamine, and an amino acid kit). HCG was similarly analyzed except that the amino acid composition was obtained on 6 N HCl hydrolysates (20 hours, 110°C). The amount of each component was referred to the dry weight of the starting material; in all cases the experimentally determined composition accounted for ca. 8590% of the total weight (the 10-15% difference is weil within the overall experimental accuracy). For convenience, the compositions have been normalized to 100%. The activities of HCG and the glycopeptide fraction were tested in an homologous second antibody radioimmunoassay (RIA) and a radio-receptor assay (RRA) which have been decribed (Benveniste, Frohman, Beil, Spitz and Rabinowitz 1975; Schneider, Benveniste, Spitz and Rabinowitz 1973). Briefly, the RRA consisted of using a rat testicular particulate fraction (18,000 xg); incubation with 1251-HCG and HCG or the glycopeptide fraction was for 8 hours at 21°C. High speed centrifugation was used to separate free from bound f251-HCG. In order to determine the nonspecific effect of the glycopeptide preparation, parallel dilutions were performed on the control.
© 1979 Georg Thieme Publishers
Downloaded by: Boston University. Copyrighted material.
Summuy
166
D. Puett, R. Benveniste, A. Kenner and D. P.abinowitz
0.50
@
®
A420 0.25
0 0 25
50
75
100
6OOr---------~~~~~--------,
@ 400 HEXOSE }IIJ Iml 200
50
Figure 1 shows the column chromatograms of the HCG glycopeptide fraction following the second (Fig. lA) and third (Fig. 18) digestion with pronase, and the fmal purification of the 3-times digested material (Fig. 1C). The HCG glycopeptide fraction was by weight: 29% sialic acid, 31% hexose, 23% hexosamine, including 20% glucosamine and 3% galactosamine (all probably N-acetylated), and 17% amino acids or peptides. The overall recovery of total carbohydrate (from HCG) in the glycopeptide fraction was ca. 40%. However, the weight percentages of sialic acid, hexose, and hexosamine in the total carbohydrate component of HCG was 30, 40, and 30, respectively; the corresponding values fot the glycopeptide fraction 6"elated to its total carbohydrate component) are 35, 37, and 28. Thus, there was no preferential removal of sialic acid or other sugar residues during the pro nase digestions. Consequently, the incomplete recovery of total carbahydrate is attributed to normal handling losses during chrornatography and lyophilization. Following acid hydrolysis, which converts asparagine and glutamine to the free ß and 'Y carboxylic acids, respectively, the major amino acids were proline, aspartic acid, serine, threonine, alanine, and leucine, Normalized to aspartic acid, the corresponding molar ratios were l.I, l.O, 0.8, 0.6, 0.4, and 0.3. The amino acids lysine, histidine, arginine, glutamic acid, glycine, and valine, were present in amounts between 0.1 to 0.2 (nonnalized to aspartic acid); the other amino acids, (carboxymethyl) cysteine, methionine, isoleucine, tyrosine, and phenylalanine were either absent or present in trace amounts only.
100
75 VOLUME, ml
70% protein. The amino acid composition was quite similar to that of sequenced HCG (Bahl 1973; Mo,gan, Bi,ken and Can/ield 1975).
Fig. 1 Gel exc1usion chromatograms following digestion of reduced, S-carboxymethylated HCG with pronase. A. 1.5 x 86 cm column of Bio-Gel P-4 following the second pronase digestion. The hexose-positive fractions were pooled as indicated; the arrow denotes the position of the main ninhydrin-positive material. B. 1.5 x 90 ~ column of Bio-Gel P-6 following the third pronase digestion; the dashed line shows ninhydrin-positive material. C. Rechromatography of the pooled material from B on a 1.5 x 52 cm column of Bio-Gel P-30. The ordinate in panels A and B is the absorbance at 420 nm for the orcinol reaction; ninhydrin reactions were monitored at 570 nm. The hexose content (panel C) is based on a mannose standard. In all cases the columns were equilibrated and developed with 0.1 M pyridine-acetate, pH 5.0, at ambient temperature.
In RIA, 1 mg/mI of the HCG glycopeptide fraction did not affect the binding of 125 I-HCG to rabbit antiHCG serum, while 50% displacement of the tracer was observed with 5 ng/mI of HCG. The results of RRA are shown in Figure 2 which compares the
3Or-----------------------------------------,
10'
10·
10' nQ/ml
10'
10·
10"
Fig. 2 RRA dilution curves of native HCG (e) and the glycopeptide fraction (0). BIT refers to the bound to total ratio of 125I-HCG in the presence of various concentrations (ng/ml) of HCG or g1ycopeptides.
Downloaded by: Boston University. Copyrighted material.
Results The purified HCG preparation had a potency (relative to CR1l9) of II ,304 lU/mg (with 95% confidence limits of 10,466-12,142 lU/mg) by RIA. It was estimated to contain (by weight), 9% sialic acid, 12% hexose, 9% hexosamine (mainly glucosamine), and
The Carbohydrate Moiety of Human Chorionic Gonadotropin
Discussion These results indicate that the HCG glycopeptide fraction has less than 1: 10,000 (w/w) the binding affmity of HCG; on a molar basis the difference would be even greater. This suggests that any biological role of the HCG carbohydrate moiety must be in concert with the pro tein component. The lack of cross-reactivity with an antiserum directed against HCG suggests that the carbohydrate side-chains do not represent an antigenic site; other anti sera need to be investigated to confirm this suggestion.
four glycosidases was capable of stimulating steroidogenesis, but not cAMP, and the apparent binding was approximately 10-fold less than that of HCG. Since our results suggest that the isolated carbohydrate components have limited, if any, binding affmity, the lO-fold reduction in binding may result from a conformational change in the protein moiety upon removal of a portion of the carbohydrate. Also, it is possible that the carbohydrate moieties may undergo conformational changes when removed from the polypeptide domain of HCG. However, with this possible caveat, our results suggest that under physiological conditions where the criteria of high specificity and binding affinity are presumably met, the pro tein component dictates the initial receptor recognition and interaction, and this may be influenced slightly by the carbohydrate components.
Thus, the carbohydrate moiety of HCG may regulate secretion and metabolism of the hormone, but its role, if any, in stimulating biological events, on ce binding has occurred, remains to be clarified. It appears, however, not to be responsible for the initial These findings, based on a different approach capable receptor recognition. of recognition independent from the protein moiety of glycoprotein hormones, confirm in general the results of others (Tsuruhara et al. 1972; Moyle et al. Acknowleclgements 1975). Moyle et al. (1975) studied the binding, It is a pleasure to than Ms. Betty Kay Wasserman for ex· pert technical assistance. This research was supported in testosterone production, and cAMP production of large part by the National Institutes of Health: AM 15838, HCG and derivatives prepared by sequential treatHO 10128, H005797, and HO 07043. O.P. is a Research ment with neuraminidase, galactosidase, N-acetylCarear Oevelopment Awardee (AM 00055) and thanks the glucosaminidase, and mannosidase. It is noteworthy Oreyfus Foundation and the VU Research Council for partial support. that the derivative obtained by treatment with the
References Bahl, O.P.: Human chorionic gonadotropin. I. Purification and physicochemical properties. J. Biol. Chem. 244: 567-574 (1969) Bahl, O.P.: Chemistry of human chorionic gonadotropin. In: Hormonal proteins and peptides, Vol. I, Li, C. H., Ed., p. 171-199, Academic Press, New York 1973 Benveniste, R., L.A. Frohman, 1. Bell, I. Spitz, D. Rabinowitz: Subunit of g1ycoprotein hormone: presence in peripheral serum after LHRH. Europ. J. Clin. Invest. 5: 123-131 (1975) Holladay, L.A., D. Puett: Gonadotropin and subunit conformation. Arch. Biochem. Biophys. 171: 708-720 (1975) Morgan, F./., S. Birken, R.E. Canfield: The amino acid sequence of human chorionic gonadotropin. J. Biol. Chem. 250: 5247-5258 (1975)
Moyle, W.R., O.P. Bahl, L. Marz: Role of the carbohydrate of human chorionic gonadotropin in the mechanism of hormone action. J. Biol. Chem. 250: 9163- 9169 (1975) Puett, D., A. Nureddin, L.A. Holladay: Circular dichroism of human pituitary luteinizing hormone and its glycopeptides. Int. J. Peptide Protein Res. 8: 183-191 (1976) Puett, D., L.A. Holladay, I.P. Robinson: Circular dichroism of human urinary Tamm-Horsfall glycoprotein. Molecular Cellular Biochemistry 15: 109-116 (1977) Schneider, B., R. Benveniste, I. Spitz, D. Rabinowitz: Bind· ing of human luteinizing hormone to particulate frac· tion of rat testis. Metabolism 22: 1225-1233 (1973) Tmruhara, T., M.L. Du/au, I. Hickman, K./. Catt: Biological properties of HC'G after rem oval of terminal sialic acid and galactose residues. Endocrinology 91: 296- 30 I (1972)
Requests for reprints should be addressed to: Or. O. Puett, Oepartment of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232 (U.S.A.)
Downloaded by: Boston University. Copyrighted material.
displacement of m I-HCG by the HCG glycopeptide fraction and intact HCG. The displacement of 125I-HCG from the rat testicular preparation was unaffected up to concentrations of 50 J,lg/ml of the HCG glycopeptide fraction. The slight displacement noted at 100 J,lg/ml and above is non-specific since comparable displacement was observed with the HCGfree control. Similar results were obtained with the minor glycopeptide fraction present in the leading edge to the major fraction (cf. Fig. IB).
167
