0013-7227/79/1045-1484$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 104, No. 5 Printed in U.S.A.
Luteinizing Hormone-Releasing Hormone Inactivation by Purified Pituitary Plasma Membranes: Effects on Receptor-Binding Studies* R. N. CLAYTON,f R. A. SHAKESPEAR, J. A. DUNCAN, AND J. C. MARSHALL Department of Medicine, University of Birmingham, Edgbaston, Birmingham, United Kingdom; and the Department of Internal Medicine, Division of Endocrinology and Metabolism, University of Michigan (J. A. D., J. C. M.), Ann Arbor, Michigan 48109
ABSTRACT. Inactivation of LHRH by purified bovine pituitary plasma membranes was studied in vitro. After incubation of [l25I]iodo-LHRH with plasma membranes, the amount of tracer bound to the pellet was measured, and the integrity of the unbound tracer in the supernatant was assessed. Reduction in ability to bind to anti-LHRH serum and to rebind to plasma membranes together with altered electrophoretic mobility on polyacrylamide gels showed that the unbound [125I]iodo-LHRH was inactivated. LHRH inactivation occurred rapidly and was dependent upon membrane concentration and incubation temperature. These results indicate that hormone inactivation must be taken into account in the interpretation of LHRH-receptor interactions. During 37 C incubations, the apparent absence of specific LHRH binding can be explained by inactivation of tracer hormone. Significant LHRH inactivation also occurred at 0 C, which in part explains the insensitivity of LHRH receptor assays. Assessment of LHRH inactivation by different particulate subcellular fractions of pituitary tissue showed that the inactivating enzyme was associated with the plasma membranes; other organelles did not alter LHRH. The enzyme appeared to be an
I
T IS widely recognized that the initiation of peptide hormone action is by binding to specific surface membrane receptors of target cells. However, the process(es) involved in termination of hormone action have received little attention and are poorly understood. Knights et al. (1) have recently drawn attention to hormonal destruction being of equal importance to synthesis and release in controlling hormone concentrations at the receptor site. Several examples of polypeptide hormone degradation by target tissues are recognized. Homogenates of adipose tissue degrade ACTH (2), and oxytocin, insulin, and glucagon are broken down by particle-free supernatants Received April 14, 1978. Address requests for reprints to: Dr. John C. Marshall, Division of Endocrinology, University of Michigan, Ann Arbor, Michigan 48109. * Presented in part at the 59th Meeting of the Endocrine Society, Chicago, IL, June 1977. This work was supported in part by a Medical Research Project grant, a faculty research grant from the Rackham School of Graduate Studies, and the Michigan Memorial Phoenix Project. t Supported by the Medical Research Council.
integral part of the plasma membrane structure, since enzymic activity could not be removed by washing without reducing specific LHRH binding. Additionally, reduction of LHRH inactivation by the inhibitors Bacitracin and Trasylol and by magnesium was also accompanied by reduced LHRH binding. Previous studies have shown that the majority of LHRH binding to pituitary plasma membranes is to the low affinity site (~10~(> M), but the significance of this binding has been uncertain. Our findings indicate that low affinity binding probably represents binding of LHRH to the inactivating enzyme. The LHRH analog, D-Ser6(TBu), des Glyio, ethylamide, has greater biological activity than LHRH and is not inactivated to a significant extent by pituitary plasma membranes. The enhanced biological activity of the analog, therefore, may be due to its resistance to inactivation by enzymes on the pituitary cell surface. The membrane-associated inactivating enzyme could play an important role in vivo in determining the concentration of intact LHRH available at the receptor site which initiates gonadotropin release. (Endocrinology
104: 1484, 1979)
of kidney, liver, and fat cells, respectively (3-5). Particulate pellets derived from kidney tissue have also been shown to degrade parathyroid hormone (6). The hypothalamic hormones, LHRH and TRH, are known to be inactivated by peptidases present in homogenates of hypothalamic and other brain tissues (7-11). Different amounts of peptidase activity have been found in hypothalamic extracts from castrated and intact animals. Thus, it has been suggested that steroid hormones can influence the activity of the degrading enzymes and thereby exert control over the amount of hormones released from the hypothalamus (12). More recently, it has been shown that pituitary cytosol can also degrade LHRH (13, 14). Additionally, Koch et al. (15) have shown that analogs of LHRH, which possess greater activity than the native hormone, are resistant to degradation by pituitary cytosol. This finding has suggested that the increased biological activity of the analog is due to resistance to degradation at the level of the pituitary gland. Current evidence however, indicates that LHRH acts by an initial step of binding to pituitary 1484
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 22:21 For personal use only. No other uses without permission. . All rights reserved.
LHRH INACTIVATION BY PITUITARY MEMBRANES gonadotroph plasma membranes. [ 125 I]Iodo-LHRH has been localized at the cell surface by radioautography, (16), and specific binding sites have been demonstrated on plasma membrane preparations (17-19). Thus, the LHRH may not come into contact with the degrading enzyme present in the pituitary cytosol. Previous studies of LHRH-pituitary membrane binding have shown the presence of two distinct binding sites (17, 19-21). One site, found on pituitary membranes, is of high affinity (Kd = 10~9 M) and constitutes some 25% of the specific binding. The second site is of much lower affinity (K
Vol. 104, No. 5 Printed in U.S.A.
Luteinizing Hormone-Releasing Hormone Inactivation by Purified Pituitary Plasma Membranes: Effects on Receptor-Binding Studies* R. N. CLAYTON,f R. A. SHAKESPEAR, J. A. DUNCAN, AND J. C. MARSHALL Department of Medicine, University of Birmingham, Edgbaston, Birmingham, United Kingdom; and the Department of Internal Medicine, Division of Endocrinology and Metabolism, University of Michigan (J. A. D., J. C. M.), Ann Arbor, Michigan 48109
ABSTRACT. Inactivation of LHRH by purified bovine pituitary plasma membranes was studied in vitro. After incubation of [l25I]iodo-LHRH with plasma membranes, the amount of tracer bound to the pellet was measured, and the integrity of the unbound tracer in the supernatant was assessed. Reduction in ability to bind to anti-LHRH serum and to rebind to plasma membranes together with altered electrophoretic mobility on polyacrylamide gels showed that the unbound [125I]iodo-LHRH was inactivated. LHRH inactivation occurred rapidly and was dependent upon membrane concentration and incubation temperature. These results indicate that hormone inactivation must be taken into account in the interpretation of LHRH-receptor interactions. During 37 C incubations, the apparent absence of specific LHRH binding can be explained by inactivation of tracer hormone. Significant LHRH inactivation also occurred at 0 C, which in part explains the insensitivity of LHRH receptor assays. Assessment of LHRH inactivation by different particulate subcellular fractions of pituitary tissue showed that the inactivating enzyme was associated with the plasma membranes; other organelles did not alter LHRH. The enzyme appeared to be an
I
T IS widely recognized that the initiation of peptide hormone action is by binding to specific surface membrane receptors of target cells. However, the process(es) involved in termination of hormone action have received little attention and are poorly understood. Knights et al. (1) have recently drawn attention to hormonal destruction being of equal importance to synthesis and release in controlling hormone concentrations at the receptor site. Several examples of polypeptide hormone degradation by target tissues are recognized. Homogenates of adipose tissue degrade ACTH (2), and oxytocin, insulin, and glucagon are broken down by particle-free supernatants Received April 14, 1978. Address requests for reprints to: Dr. John C. Marshall, Division of Endocrinology, University of Michigan, Ann Arbor, Michigan 48109. * Presented in part at the 59th Meeting of the Endocrine Society, Chicago, IL, June 1977. This work was supported in part by a Medical Research Project grant, a faculty research grant from the Rackham School of Graduate Studies, and the Michigan Memorial Phoenix Project. t Supported by the Medical Research Council.
integral part of the plasma membrane structure, since enzymic activity could not be removed by washing without reducing specific LHRH binding. Additionally, reduction of LHRH inactivation by the inhibitors Bacitracin and Trasylol and by magnesium was also accompanied by reduced LHRH binding. Previous studies have shown that the majority of LHRH binding to pituitary plasma membranes is to the low affinity site (~10~(> M), but the significance of this binding has been uncertain. Our findings indicate that low affinity binding probably represents binding of LHRH to the inactivating enzyme. The LHRH analog, D-Ser6(TBu), des Glyio, ethylamide, has greater biological activity than LHRH and is not inactivated to a significant extent by pituitary plasma membranes. The enhanced biological activity of the analog, therefore, may be due to its resistance to inactivation by enzymes on the pituitary cell surface. The membrane-associated inactivating enzyme could play an important role in vivo in determining the concentration of intact LHRH available at the receptor site which initiates gonadotropin release. (Endocrinology
104: 1484, 1979)
of kidney, liver, and fat cells, respectively (3-5). Particulate pellets derived from kidney tissue have also been shown to degrade parathyroid hormone (6). The hypothalamic hormones, LHRH and TRH, are known to be inactivated by peptidases present in homogenates of hypothalamic and other brain tissues (7-11). Different amounts of peptidase activity have been found in hypothalamic extracts from castrated and intact animals. Thus, it has been suggested that steroid hormones can influence the activity of the degrading enzymes and thereby exert control over the amount of hormones released from the hypothalamus (12). More recently, it has been shown that pituitary cytosol can also degrade LHRH (13, 14). Additionally, Koch et al. (15) have shown that analogs of LHRH, which possess greater activity than the native hormone, are resistant to degradation by pituitary cytosol. This finding has suggested that the increased biological activity of the analog is due to resistance to degradation at the level of the pituitary gland. Current evidence however, indicates that LHRH acts by an initial step of binding to pituitary 1484
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 22:21 For personal use only. No other uses without permission. . All rights reserved.
LHRH INACTIVATION BY PITUITARY MEMBRANES gonadotroph plasma membranes. [ 125 I]Iodo-LHRH has been localized at the cell surface by radioautography, (16), and specific binding sites have been demonstrated on plasma membrane preparations (17-19). Thus, the LHRH may not come into contact with the degrading enzyme present in the pituitary cytosol. Previous studies of LHRH-pituitary membrane binding have shown the presence of two distinct binding sites (17, 19-21). One site, found on pituitary membranes, is of high affinity (Kd = 10~9 M) and constitutes some 25% of the specific binding. The second site is of much lower affinity (K
