0013-7227/78/1036-2240$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 103, No. 6 Printed in U.S.A.
Spontaneous Dissociation of Human Pituitary Luteinizing Hormone in Solution* J. G. LOEBER, J. W. G. M. NABBEN-FLEUREN, L. H. ELVERS,f M. F. G. SEGERS, AND R. M. LEQUINf University Clinic for Obstetrics and Gynecology, Catholic University, Nijmegen, the Netherlands ABSTRACT. Aliquots of a solution of highly purified human pituitary LH (hLH) were incubated with variations in temperature and time. The incubates were chromatographed on Sephadex G-100 as well as on DEAE-Sephadex A25. The column effluents were assayed in radioligand assay and in specific hLH,,- and hLH/?-RIA systems. The results indicate that there is spontaneous dissociation of hLH at elevated temperatures under otherwise "normal" conditions concerning,
T
HE PRESENCE of molecular heterogeneity within highly purified pituitary glycoprotein hormone preparations has been recognized (e.g. Refs. 1-5). Frequently, discrepancies between the potencies of such preparations in different types of assays have been encountered (5-7). One reason for these discrepancies is that in bioassays and in radioligand assays (RLA), only "intact" hormone molecules are measured, i.e. molecules consisting of a human LHa- and a human LH^subunit combined, while in RIAs, free hormone subunits and fragments also may be detected. In addition, the conditions of a particular assay have a direct influence on the assay results, e.g. strain of animals, injection schedules, choice of antiserum, incubation temperature, etc. Previous studies in this laboratory indicated that there is a relationship between the potencies of human pituitary LH (hLH)
Received March 28, 1978. * This work was supported in part by the Foundation for Medical Research (FUNGO), which is subsidized by The Netherlands' Organization for the Advancement of Pure Research (ZWO). Address requests for reprints to: Dr. J. G. Loeber, Rudolf Magnus Institute for Pharmacology, University of Utrecht, Medical Faculty, Vondellaan 6, Utrecht, The Netherlands. f Present address: National Institute of Public Health, P.O. Box 1, Bilthoven, The Netherlands.
for example, pH and ionic strength. The degree of dissociation is both time and temperature dependent. This dissociation is not due to proteolytic enzymes. It is concluded that one should be alert when using hLH subunit RIA systems at elevated temperatures for measuring hLH levels, e.g. in clinical samples, because artificial high levels may be obtained. (Endocrinology 103: 2240, 1978)
preparations, as measured by a specific RIA, and the temperature of the incubation (5). In the present studies we report evidence for the formation of hLH subunits during incubation at elevated temperatures. Materials and Methods Highly purified preparations of hLH and its subunits were prepared from acetone-dried pituitary glands, as described by Closset et al. (8). The potencies, as measured in the (ovarian ascorbic acid depletion OAAD) assay, were 9.55 (6.31-16.27) U NIH-LH-Sl/mg for intact hLH and 1 mg/ml) favoring association. Another aspect concerns the correct interpretation of RIA data of clinical samples. Although from a therapeutic point of view the absolute hormone levels may be less important than the relative levels, e.g. during medical treatment, one tries to measure the "true" value as accurately as possible. Nowadays, in many laboratories, sets of reagents ("kits") are being used for RIA. Some of these have been designed to produce results within a short period of time. Usually the reaction equilibrium is attained in a reduced incubation time
2245
by raising the incubation temperature to 37 C. When these kits employ an assay system based upon the a- or /?-subunit of hLH (or hCG), there is a fair chance that artefacts are measured, unless it has been proven that the antiserum employed binds the free subunit and the intact hormone in equimolar quantities. In general, one should be cautious when interpreting the results of such experiments carried out at elevated temperatures. References 1. Reichert, L. E., Electrophoretic properties of pituitary gonadotropins as studied by electrofocusing, Endocrinology 88: 1029,1971. 2. Canfield, R. E., F. J. Morgan, S. Kammerman, J. J. Bell, and G. M. Agosto, Studies of human chorionic gonadotropin, Recent Prog Horm Res 27: 121, 1971. 3. Pierce, J. G., T. Liao, S. M. Howard, B. Shome, and ' J. S. Cornell, Studies on the structure of thyrotropin: its relationship to luteinizing hormone, Recent Prog Horm Res 27: 165, 1971. 4. Rathnam, P. and B. B. Saxena, Subunits of FSH from human pituitary glands, In Saxena, B. B., C. G. Beling, and H. M. Gandy (eds.), Gonadotropins, Wiley-Interscience, New York, 1970, p. 120. 5. Loeber, J. G., Human luteinizing hormone; structure and function of some preparations, Acta Endocrinol [Suppl. 210] 85: 1, 1977. 6. Sharpe, R. M., M. Shamanesh, M. G. Elwood, M. Hartog, and P. S. Brown, Discrepancy between radioimmunoassay and radioligand-receptorassay of luteinizing hormone released in vitro by pituitary tissue from male rats of different ages, J Endocrinol 65: 265, 1975. 7. Schlamowitz, M., J. Cronquist, M. Esfahami, and D. N. Ward, A comparative study of preparations of ovarian luteinizing hormone by bioassay, immuno double diffusion, radioimmunoassay, radioreceptorassay and polyacrylamide gel electrophoresis, Acta Endocrinol (Kbh) 81: 270, 1976. 8. Closset, J., J. L. Vandalem, G. Hennen, and R. M. Lequin, Human luteinizing hormone; isolation and characterization of the native hormone and its a- and /3-subunits, Eur J Biochem 57: 325, 1975. 9. Lequin, R. M., M. F. G. Segers, J. Closset, and G. Hennen, Immunochemical characterization of antisera to human luteinizing hormone and its subunits, Horm Metab Res (Suppl) 5: 49, 1974. 10. Jerne, N. K., and W. L. M. Perry, The stability of biological standards, Bull WHO 14: 167, 1956. 11. Adams, E., and E. L. Smith, Proteolytic activity of pituitary extracts, J Biol Chem 191: 651, 1952. 12. Lewis, U. J., Enzymatic transformations of growth hormone and prolactin, J Biol Chem 237: 3141,1962. 13. Reichert, L. E., and A. F. Parlow, Proteinase contam-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 22:31 For personal use only. No other uses without permission. . All rights reserved.
2246
LOEBER ET AL.
ination of purified pituitary gonadotrophins, Endocrinology 74: 809, 1964. 14. De la Llosa, P., and M. Jutisz, Reversible dissociation into subunits and biological activity of ovine luteinizing hormone, Biochim Biophys Ada 181: 426, 1969. 15. Reichert, L. E., A. R. Midgley, G. D. Niswender, and D. N. Ward, Formation of a hybrid molecule from subunits of human and bovine luteinizing hormone, Endocrinology 87: 534, 1970. 16. Papkoff, H., M. R. Sairam, S. Walker Farmer, and C. H. Li, Studies on the structure and function of interstitial cell-stimulating hormone, Recent Prog Horm Res 29: 563, 1973.
Endo 1978 Vol 103 , No 6
17. Bewley, T. A., M. R. Sairam, and C. H. Li, The kinetics of dissociation and reassociation of ovine pituitary interstitial cell-stimulating hormone, Arch Biochem Biophys 163: 625, 1974. 18. Liu, W. K., K. P. Yang, Y. Nakagawa, and D. N. Ward, The role of the aminogroup in subunits association and receptor site interaction for ovine luteinizing hormone as studied by acylation, J Biol Chem 249: 5544, 1974. 19. De la Llosa, P., C. Courte, and M. Jutisz, On the mechanism of reversible inactivation of luteinizing hormone by urea, Biochem Biophys Res Commun 26: 411, 1967.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 22:31 For personal use only. No other uses without permission. . All rights reserved.
Vol. 103, No. 6 Printed in U.S.A.
Spontaneous Dissociation of Human Pituitary Luteinizing Hormone in Solution* J. G. LOEBER, J. W. G. M. NABBEN-FLEUREN, L. H. ELVERS,f M. F. G. SEGERS, AND R. M. LEQUINf University Clinic for Obstetrics and Gynecology, Catholic University, Nijmegen, the Netherlands ABSTRACT. Aliquots of a solution of highly purified human pituitary LH (hLH) were incubated with variations in temperature and time. The incubates were chromatographed on Sephadex G-100 as well as on DEAE-Sephadex A25. The column effluents were assayed in radioligand assay and in specific hLH,,- and hLH/?-RIA systems. The results indicate that there is spontaneous dissociation of hLH at elevated temperatures under otherwise "normal" conditions concerning,
T
HE PRESENCE of molecular heterogeneity within highly purified pituitary glycoprotein hormone preparations has been recognized (e.g. Refs. 1-5). Frequently, discrepancies between the potencies of such preparations in different types of assays have been encountered (5-7). One reason for these discrepancies is that in bioassays and in radioligand assays (RLA), only "intact" hormone molecules are measured, i.e. molecules consisting of a human LHa- and a human LH^subunit combined, while in RIAs, free hormone subunits and fragments also may be detected. In addition, the conditions of a particular assay have a direct influence on the assay results, e.g. strain of animals, injection schedules, choice of antiserum, incubation temperature, etc. Previous studies in this laboratory indicated that there is a relationship between the potencies of human pituitary LH (hLH)
Received March 28, 1978. * This work was supported in part by the Foundation for Medical Research (FUNGO), which is subsidized by The Netherlands' Organization for the Advancement of Pure Research (ZWO). Address requests for reprints to: Dr. J. G. Loeber, Rudolf Magnus Institute for Pharmacology, University of Utrecht, Medical Faculty, Vondellaan 6, Utrecht, The Netherlands. f Present address: National Institute of Public Health, P.O. Box 1, Bilthoven, The Netherlands.
for example, pH and ionic strength. The degree of dissociation is both time and temperature dependent. This dissociation is not due to proteolytic enzymes. It is concluded that one should be alert when using hLH subunit RIA systems at elevated temperatures for measuring hLH levels, e.g. in clinical samples, because artificial high levels may be obtained. (Endocrinology 103: 2240, 1978)
preparations, as measured by a specific RIA, and the temperature of the incubation (5). In the present studies we report evidence for the formation of hLH subunits during incubation at elevated temperatures. Materials and Methods Highly purified preparations of hLH and its subunits were prepared from acetone-dried pituitary glands, as described by Closset et al. (8). The potencies, as measured in the (ovarian ascorbic acid depletion OAAD) assay, were 9.55 (6.31-16.27) U NIH-LH-Sl/mg for intact hLH and 1 mg/ml) favoring association. Another aspect concerns the correct interpretation of RIA data of clinical samples. Although from a therapeutic point of view the absolute hormone levels may be less important than the relative levels, e.g. during medical treatment, one tries to measure the "true" value as accurately as possible. Nowadays, in many laboratories, sets of reagents ("kits") are being used for RIA. Some of these have been designed to produce results within a short period of time. Usually the reaction equilibrium is attained in a reduced incubation time
2245
by raising the incubation temperature to 37 C. When these kits employ an assay system based upon the a- or /?-subunit of hLH (or hCG), there is a fair chance that artefacts are measured, unless it has been proven that the antiserum employed binds the free subunit and the intact hormone in equimolar quantities. In general, one should be cautious when interpreting the results of such experiments carried out at elevated temperatures. References 1. Reichert, L. E., Electrophoretic properties of pituitary gonadotropins as studied by electrofocusing, Endocrinology 88: 1029,1971. 2. Canfield, R. E., F. J. Morgan, S. Kammerman, J. J. Bell, and G. M. Agosto, Studies of human chorionic gonadotropin, Recent Prog Horm Res 27: 121, 1971. 3. Pierce, J. G., T. Liao, S. M. Howard, B. Shome, and ' J. S. Cornell, Studies on the structure of thyrotropin: its relationship to luteinizing hormone, Recent Prog Horm Res 27: 165, 1971. 4. Rathnam, P. and B. B. Saxena, Subunits of FSH from human pituitary glands, In Saxena, B. B., C. G. Beling, and H. M. Gandy (eds.), Gonadotropins, Wiley-Interscience, New York, 1970, p. 120. 5. Loeber, J. G., Human luteinizing hormone; structure and function of some preparations, Acta Endocrinol [Suppl. 210] 85: 1, 1977. 6. Sharpe, R. M., M. Shamanesh, M. G. Elwood, M. Hartog, and P. S. Brown, Discrepancy between radioimmunoassay and radioligand-receptorassay of luteinizing hormone released in vitro by pituitary tissue from male rats of different ages, J Endocrinol 65: 265, 1975. 7. Schlamowitz, M., J. Cronquist, M. Esfahami, and D. N. Ward, A comparative study of preparations of ovarian luteinizing hormone by bioassay, immuno double diffusion, radioimmunoassay, radioreceptorassay and polyacrylamide gel electrophoresis, Acta Endocrinol (Kbh) 81: 270, 1976. 8. Closset, J., J. L. Vandalem, G. Hennen, and R. M. Lequin, Human luteinizing hormone; isolation and characterization of the native hormone and its a- and /3-subunits, Eur J Biochem 57: 325, 1975. 9. Lequin, R. M., M. F. G. Segers, J. Closset, and G. Hennen, Immunochemical characterization of antisera to human luteinizing hormone and its subunits, Horm Metab Res (Suppl) 5: 49, 1974. 10. Jerne, N. K., and W. L. M. Perry, The stability of biological standards, Bull WHO 14: 167, 1956. 11. Adams, E., and E. L. Smith, Proteolytic activity of pituitary extracts, J Biol Chem 191: 651, 1952. 12. Lewis, U. J., Enzymatic transformations of growth hormone and prolactin, J Biol Chem 237: 3141,1962. 13. Reichert, L. E., and A. F. Parlow, Proteinase contam-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 22:31 For personal use only. No other uses without permission. . All rights reserved.
2246
LOEBER ET AL.
ination of purified pituitary gonadotrophins, Endocrinology 74: 809, 1964. 14. De la Llosa, P., and M. Jutisz, Reversible dissociation into subunits and biological activity of ovine luteinizing hormone, Biochim Biophys Ada 181: 426, 1969. 15. Reichert, L. E., A. R. Midgley, G. D. Niswender, and D. N. Ward, Formation of a hybrid molecule from subunits of human and bovine luteinizing hormone, Endocrinology 87: 534, 1970. 16. Papkoff, H., M. R. Sairam, S. Walker Farmer, and C. H. Li, Studies on the structure and function of interstitial cell-stimulating hormone, Recent Prog Horm Res 29: 563, 1973.
Endo 1978 Vol 103 , No 6
17. Bewley, T. A., M. R. Sairam, and C. H. Li, The kinetics of dissociation and reassociation of ovine pituitary interstitial cell-stimulating hormone, Arch Biochem Biophys 163: 625, 1974. 18. Liu, W. K., K. P. Yang, Y. Nakagawa, and D. N. Ward, The role of the aminogroup in subunits association and receptor site interaction for ovine luteinizing hormone as studied by acylation, J Biol Chem 249: 5544, 1974. 19. De la Llosa, P., C. Courte, and M. Jutisz, On the mechanism of reversible inactivation of luteinizing hormone by urea, Biochem Biophys Res Commun 26: 411, 1967.
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