0013-7227/79/1055-1221$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 105, No. 5
Printed in U.S.A.
The Role of Sulfhydryl Groups in Thyrotropin Binding and Adenylate Cyclase Activities of Thyroid Plasma Membranes* YASUNORI OZAWA, INDER J. CHOPRA,f DAVID H. SOLOMON, AND FRANCHISE SMITH Department of Medicine, University of California at Los Angeles, Center for the Health Sciences, Los Angeles, California 90024
ABSTRACT. The effects of sulfhydryl (SH) reagents on the binding of TSH to thyroid membranes and on membrane adenylate cyclase activity were studied. Diamide, a SH-oxidizing agent, enhanced [125I]iodo-TSH binding to thyroid membranes in a dose-dependent manner. On the contrary, dithiothreitol (DTT), a disulfide-reducing reagent, markedly inhibited this activity in a dose-dependent manner. These effects could not be attributed to a direct interaction between the reagents and the radioactive TSH. Scatchard plot analysis of the data was limited by curvilinear plots but suggested that diamide increases while DTT decreases the affinity of the receptors for TSH in thyroid
I
N THE PAST two decades, data on the important role of sulfhydryl groups (SH) in many physiological and biochemical processes have accumulated in the literature. These processes include mitochondrial functions, nerve activity, cell division, oxidative phosphorylation (1), and extrathyroidal metabolism of thyroid hormones (2). However, not much has been known about the role of SH or disulfide groups in the binding of hormones to their receptors and subsequent actions. In this paper, we report on the effect of a SH-oxidizing reagent, diamide (azodicarboxylic acid-bis-dimethylamide), and a thiol-protective and disulfide-reducing reagent, dithiothreitol (DTT), on the binding of TSH and the activity of adenylate cyclase in thyroid membranes. Materials and Methods I IJIodo-TSH binding to the membranes Highly purified bovine TSH (40 U/mg; obtained through the courtesy of Dr. J. G. Pierce, UCLA) was labeled by the lactoperoxidase method. Labeled TSH was purified by binding to and elution from thyroid membranes, followed by Sephadex G-
4
Received September 25,1978. * This work was supported in part by USPHS Grants AM-17251 and AM-16155. t To whom requests for reprints should be addressed.
membranes; neither reagent appeared to influence the capacity of TSH binding by membranes. The effects of diamide and DTT on adenylate cyclase activity of thyroid plasma membranes were just the opposite of their effects on TSH binding. Thus, diamide inhibited adenylate cyclase activity of thyroid plasma membranes while DTT increased basal enzyme activity and slightly but not significantly increased TSH-stimulated enzyme activity. The data suggest a possible physiological role for SH and disulfide groups of thyroid membranes in modulation of thyroidal activity. {Endocrinology 105: 1221,1979)
100 column chromatography (3, 4). The specific activity of labeled TSH, as determined from incorporation of 125I into protein during iodination, approximated 80-100 mCi/mg. The membranes were prepared from fresh surgical specimens of human thyroid or bovine thyroid glands obtained at an abatoir. For this purpose, the thyroid was homogenized by a Polytron (Brinkmann Instruments, Westbury, NY) or glass-Teflon homogenizer in 10 vol 10 mM Tris buffer, pH 7.5, and centrifuged at 1,500 X g for 6 min. The supernate was centrifuged at 15,000 X g for 15 min and the pellet was suspended in 10 mM Tris-50 mM NaCl, pH 7.5 (300 mgeq tissue/ml), and used as the membrane fraction. Thyroid membrane (30 mgeq) was incubated with [125I]iodo-TSH (5,000 cpm; final volume, 200 fi\) at 37 C for 60 min. The reaction was stopped by cooling the incubation mixture on ice, followed by the addition of 3 mg bovine globulin and polyethylene glycol (mol wt, 6000; Schwarz/Mann, Orangeburg, NY) to a final concentration of 5% and centrifugation at 1,500 x g for 15 min. The radioactivity in the pellet was determined by scintillation counting, and the percent total radioactivity bound in the pellet was calculated. Nonspecific binding of [125I]iodo-TSH in the pellet was determined using boiled thyroid membranes. The nonspecific binding determined in this manner was essentially the same as the binding of [125I]iodo-TSH to membranes observed in the presence of 1 U nonradioactive bovine TSH (Thytropar). Nonspecific binding was subtracted from the total binding and the result was taken to reflect the specific binding. The influence of the SH reagents on the binding of TSH to membranes was studied by preincu-
1221
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 02:16 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1979 Vol 105 • No 5
OZAWA ET AL.
1222
bation of the membrane at 37 C with the reagents for 1 or 2 h before adding [I25I]iodo-TSH as well as by simultaneous incubation of the reagents and [125I]iodo-TSH with the membranes. Adenylate cyclase activity assay Adenylate cyclase activity was studied by the production of cAMP during incubation of membranes with an ATP-regenerating system consisting of 40 mM Tris-HCl (pH 7.5), 03.% bovine serum albumin, 24 mM theophylline, 24 mM creatine phosphate, 11 mM MgCl2, 4 mM ATP, and 0.8 mg/ml creatine phosphokinase. Fifty microliters of membrane (30 mgeq) suspension were preincubated with 50 /U buffer or SH reagents at 37 C for 60 min, and then 50 /il buffer, TSH, or NaF and 50 /xl ATPregenerating solution were added for incubation at 37 C for 10 min. After incubation, the reaction mixtures were cooled on ice and 300 /il absolute methanol were added, followed immediately by 2 ml ethanol to stop the reaction and to extract the cAMP generated during incubation. The mixture was centrifuged at 1000 X g for 10 min and cAMP was measured in the supernatant using a slight modification of the method of Orgiazzi et al. (5); the modification consisted of using dextran-coated charcoal instead of anion exchange resin for separation of free cAMP from that bound to the cytosol receptor. Pilot studies indicated that the SH reagents under study did not influence the cAMP assay or the recovery of cAMP from test samples.
zyme inhibitor) altered the effect of diamide on the binding of TSH to thyroid membranes. It had no effect, suggesting that the TSH binding-enhancing effect of diamide was probably not due to any inhibition of proteolytic enzymes that may have been contaminating our membrane preparations. Diamide enhanced the binding of [125I]iodo-TSH to thyroid membranes in a dose-dependent manner over the concentration range of 10~5-10~2 M. DTT inhibited the binding in a dose-dependent manner from 10~5-10"3 M (Fig. 2). The effects of 10~3 M diamide and 10"4 M DTT on the binding of labeled and unlabeled TSH to human thyroid membranes are shown in Fig. 3. Scatchard analysis of these data gave nonlinear plots, as has been observed in other reports (3,4). The curve was steeper in the presence of diamide, while it was more gently sloping in the presence of DTT. This suggested that diamide enhanced 15r
Vol. 105, No. 5
Printed in U.S.A.
The Role of Sulfhydryl Groups in Thyrotropin Binding and Adenylate Cyclase Activities of Thyroid Plasma Membranes* YASUNORI OZAWA, INDER J. CHOPRA,f DAVID H. SOLOMON, AND FRANCHISE SMITH Department of Medicine, University of California at Los Angeles, Center for the Health Sciences, Los Angeles, California 90024
ABSTRACT. The effects of sulfhydryl (SH) reagents on the binding of TSH to thyroid membranes and on membrane adenylate cyclase activity were studied. Diamide, a SH-oxidizing agent, enhanced [125I]iodo-TSH binding to thyroid membranes in a dose-dependent manner. On the contrary, dithiothreitol (DTT), a disulfide-reducing reagent, markedly inhibited this activity in a dose-dependent manner. These effects could not be attributed to a direct interaction between the reagents and the radioactive TSH. Scatchard plot analysis of the data was limited by curvilinear plots but suggested that diamide increases while DTT decreases the affinity of the receptors for TSH in thyroid
I
N THE PAST two decades, data on the important role of sulfhydryl groups (SH) in many physiological and biochemical processes have accumulated in the literature. These processes include mitochondrial functions, nerve activity, cell division, oxidative phosphorylation (1), and extrathyroidal metabolism of thyroid hormones (2). However, not much has been known about the role of SH or disulfide groups in the binding of hormones to their receptors and subsequent actions. In this paper, we report on the effect of a SH-oxidizing reagent, diamide (azodicarboxylic acid-bis-dimethylamide), and a thiol-protective and disulfide-reducing reagent, dithiothreitol (DTT), on the binding of TSH and the activity of adenylate cyclase in thyroid membranes. Materials and Methods I IJIodo-TSH binding to the membranes Highly purified bovine TSH (40 U/mg; obtained through the courtesy of Dr. J. G. Pierce, UCLA) was labeled by the lactoperoxidase method. Labeled TSH was purified by binding to and elution from thyroid membranes, followed by Sephadex G-
4
Received September 25,1978. * This work was supported in part by USPHS Grants AM-17251 and AM-16155. t To whom requests for reprints should be addressed.
membranes; neither reagent appeared to influence the capacity of TSH binding by membranes. The effects of diamide and DTT on adenylate cyclase activity of thyroid plasma membranes were just the opposite of their effects on TSH binding. Thus, diamide inhibited adenylate cyclase activity of thyroid plasma membranes while DTT increased basal enzyme activity and slightly but not significantly increased TSH-stimulated enzyme activity. The data suggest a possible physiological role for SH and disulfide groups of thyroid membranes in modulation of thyroidal activity. {Endocrinology 105: 1221,1979)
100 column chromatography (3, 4). The specific activity of labeled TSH, as determined from incorporation of 125I into protein during iodination, approximated 80-100 mCi/mg. The membranes were prepared from fresh surgical specimens of human thyroid or bovine thyroid glands obtained at an abatoir. For this purpose, the thyroid was homogenized by a Polytron (Brinkmann Instruments, Westbury, NY) or glass-Teflon homogenizer in 10 vol 10 mM Tris buffer, pH 7.5, and centrifuged at 1,500 X g for 6 min. The supernate was centrifuged at 15,000 X g for 15 min and the pellet was suspended in 10 mM Tris-50 mM NaCl, pH 7.5 (300 mgeq tissue/ml), and used as the membrane fraction. Thyroid membrane (30 mgeq) was incubated with [125I]iodo-TSH (5,000 cpm; final volume, 200 fi\) at 37 C for 60 min. The reaction was stopped by cooling the incubation mixture on ice, followed by the addition of 3 mg bovine globulin and polyethylene glycol (mol wt, 6000; Schwarz/Mann, Orangeburg, NY) to a final concentration of 5% and centrifugation at 1,500 x g for 15 min. The radioactivity in the pellet was determined by scintillation counting, and the percent total radioactivity bound in the pellet was calculated. Nonspecific binding of [125I]iodo-TSH in the pellet was determined using boiled thyroid membranes. The nonspecific binding determined in this manner was essentially the same as the binding of [125I]iodo-TSH to membranes observed in the presence of 1 U nonradioactive bovine TSH (Thytropar). Nonspecific binding was subtracted from the total binding and the result was taken to reflect the specific binding. The influence of the SH reagents on the binding of TSH to membranes was studied by preincu-
1221
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 02:16 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1979 Vol 105 • No 5
OZAWA ET AL.
1222
bation of the membrane at 37 C with the reagents for 1 or 2 h before adding [I25I]iodo-TSH as well as by simultaneous incubation of the reagents and [125I]iodo-TSH with the membranes. Adenylate cyclase activity assay Adenylate cyclase activity was studied by the production of cAMP during incubation of membranes with an ATP-regenerating system consisting of 40 mM Tris-HCl (pH 7.5), 03.% bovine serum albumin, 24 mM theophylline, 24 mM creatine phosphate, 11 mM MgCl2, 4 mM ATP, and 0.8 mg/ml creatine phosphokinase. Fifty microliters of membrane (30 mgeq) suspension were preincubated with 50 /U buffer or SH reagents at 37 C for 60 min, and then 50 /il buffer, TSH, or NaF and 50 /xl ATPregenerating solution were added for incubation at 37 C for 10 min. After incubation, the reaction mixtures were cooled on ice and 300 /il absolute methanol were added, followed immediately by 2 ml ethanol to stop the reaction and to extract the cAMP generated during incubation. The mixture was centrifuged at 1000 X g for 10 min and cAMP was measured in the supernatant using a slight modification of the method of Orgiazzi et al. (5); the modification consisted of using dextran-coated charcoal instead of anion exchange resin for separation of free cAMP from that bound to the cytosol receptor. Pilot studies indicated that the SH reagents under study did not influence the cAMP assay or the recovery of cAMP from test samples.
zyme inhibitor) altered the effect of diamide on the binding of TSH to thyroid membranes. It had no effect, suggesting that the TSH binding-enhancing effect of diamide was probably not due to any inhibition of proteolytic enzymes that may have been contaminating our membrane preparations. Diamide enhanced the binding of [125I]iodo-TSH to thyroid membranes in a dose-dependent manner over the concentration range of 10~5-10~2 M. DTT inhibited the binding in a dose-dependent manner from 10~5-10"3 M (Fig. 2). The effects of 10~3 M diamide and 10"4 M DTT on the binding of labeled and unlabeled TSH to human thyroid membranes are shown in Fig. 3. Scatchard analysis of these data gave nonlinear plots, as has been observed in other reports (3,4). The curve was steeper in the presence of diamide, while it was more gently sloping in the presence of DTT. This suggested that diamide enhanced 15r
