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Activation of thyroid adenyl cyclase by long-acting thyroid stimulator
Vol. 9, Part I, pp. 67-73, 1970. Life Sciences Printed in reat Britain
Pergamon Press
ACTIVATION OF THYROID ADENYL CYCLASE BY LONG-ACTING THYROID STIMULATOR G . S. Levey and I . Pastan National Institutes of Health, Bethesda, Maryland
20014
(Received 18 August 1969; in final form 13 October 1969) Long-acting thyroid stimulator (LATS) is found in the blood of most patients with hyperthyroidism and may be the cause of thyroid overactivity(1) . Since LATS and thyroid stimulating hormone (TSH) produce many similar effects, they may act by the same mechanism .
We have recently presented evidence that
cyclic 3',5'-AMP is an intracellular mediator of TSH action, and that TSH activates adenyl cyclase in thyroid homogenates (2) .
This report demonstrates
that LATS also activates adenyl cyclase in bovine and canine thyroid homogenates . Methods Control sera 1, 2, and 3 were obtained from normal volunteers ; 4 was from a hypophysectomized patient .
LATS preparation 1 was a gift from
Dr . Samuel Asper, 2 from Dr . Eugene Mayberry, and 3 from Dr . Donald Munro . In the McKenzie assay 0 .025 ml of LATS serum 1 or 3 produced a response greater than 900% and 0 .25 ml of serum 2 a response greater than 400% of the control at 9 hours .
Gamma globulin containing fractions of serum were
prepared by precipitation with 40% (W/V) ammonium sulfate .
The precipitate
was dissolved in 1/5 the original volume, dialyzed against 0.01 M tris-Cl pH 7 .4 and lyophilized in 0 .5 ml aliquots . reassayed for LATS activity .
The lyophilized material was not
At the beginning of each experiment 1 .5 ml of
thyroid homogenate containing 2-4 mg/ml protein was added to a tube containing a 0 .5 ml portion of the lyophilized powder .
Then 0 .025 ml of this mixture
was assayed for adenyl cyclase activity as previously described (2,3) .
67
The
68
ACTIVIATION OF ADENYL CYCLASE
Vol . 9, No. 2
time interval between dissolving the gamma globulin fraction and the onset of incubation was approximately 5 minutes .
Samples in a total volume of 0 .06 ml
were incubated at 370 C for 5 minutes unless otherwise noted with ATP, 1 .6 mM ; [ 32 P] ATP (o-labelled at 550 mCi/mMole), 2 .5 to 3 .5 x 106 8 mM ; MgC12, mg/ml .
cvm ;
theophylline,
2 .0 M; tris-HC1, 21 mM (pH 7 .7) ; and human serum albumin 0 .8
The incubations were started by adding homogenate, which had been
kept at 10 C, to the other components which were at 23 0 C . were stopped by adding 0 .1 ml of a solution containing 1 .25 limoles of cyclic 3',5'-AMP, boiled for 3 minutes .
4
The incubations
Wmoles of ATP,
and 0 .15 WCi of cyclic'3',5'-[3H] AMP and
The cyclic 3',5'-[3H] AMP served to determine the
recovery of cyclic 3',5'-AMP during the procedure ; recovery ranged from 30 to 35 per cent .
After boiling,
0 .4
ml of water was added, the precipitate
removed by centrifugation and the supernate applied to a 0 .5 x 2 .0 cm Dowex-50 column .
The column was washed with - cater, and the eluate between 3 and
6
ml
was collected and precipitated with 0 .17 M ZnSO 4 and 0 .15MBa(OH) 2 and the cyclic 3',5'-[32P] AMP and cyclic 3',5'-[3H] AMP counted in a liquid scintillation spectrometer .
The presence of cyclic 3',5'-AMP was confirmed by
thin-layer chromatography in a solvent system containing a mixture of n-butanol,
acetone, acetic acid, 5% ammonia, and water (7 :5 :3 :3 :2) .
A boiled
enzyme preparation did not produce any measurable cyclic 3',5'-AMP. Results Three LATS-positive sera increased the accumulation of cyclic 3',5'-[32P] AMP in bovine and canine thyroid homogenates approximately 2-fold compared to four LATS-negative sera (Table 1) .
The time course of acti-
vation of adenyl cyclase by LATS in a typical experiment is shown in Table 2 . An effect of LATS was detected after three minutes of incubation but not after 1-1/2 minutes in four experiments .
Adenyl cyclase activity increased in rela-
tion to the protein content of the LATS preparation over the range of 5 to 20 mg/ml (Table 3) .
LATS did not increase adenyl cyclase activity in a
Vol . 9, No. 2
69
ACTIVIATION OF ADENYL CYCLASE
TARTE 1 Activation of Thyroid Adenyl Cyclase by LATS EXPT . NO .
SAMPLE
PROTEIN mg/ml
PICOM)LES CYCLIC 3',5'-AMP ACCUMULATED/5 MIN .*
A. BOVINE 4 .6 + 0 .9 9 .0 + 0 .7
1.
CONTROL-1 LATS-1
16 .o 16 .o
2.
CONTROL-1 LATS-1 No Added TSH (loo X/ml)
16 .o 16 .o
6 .7 17 .7 3-5 5 .4
3.
CONTRDL-1 LATS-1
14 .o 8 .o
15 .8 + 1 .4 25 .2 + 1 .2
4.
CONTROL-1 CONTROL-2 CONTROL-3 CONTROL-4 LATS-1 LATS-2
14 .o 12 .0 10 .4 14 .4 12 .0 13 .0
7 .4 + 0.2 8.3 _ 8.2 7 .6 16 .4 _ + 1 .4 14 .6
5.
CONTROL-2 LATS-2
14 .4 12 .0
4.6 + 1 .0 9.6 + 1 .0
CONTROL-4 CONTROL-4 + TSH* LATS-1 LATS-3
34 .0 34 .0 14 .o 21 .0
1.6 4.9 _+ 1 .1 4.6 4.7 + o .8
+ _+ + ±
1 .2 0.7 0-5 0 .2
B . CANINE 1.
*Values are expressed as the mean _+ SE of three samples or the mean of closely agreeing duplicate samples . *TSB present at 25 mü/mlhomogenate prepared from a mouse adrenal tumor in which ACTH Increased adenyl cyclase activity ten-fold (4) . In the assay employed increased accumulation of cyclic AMP could have resulted from inhibition of cyclic nucleotide,phosphodiesterase activity by LATS .
Therefore LATS or control protein was added to complete reaction mix
tures containing thyroid homogenate and 200 pmoles of cyclic AMP.
In five
minutes 36 .± 1 (n = 4) pmoles of cyclic AMP disappeared from the control and 38 ± 1.3 (n = 4) from the reaction mixture containing LATS .
Thus LATS does
70
ACTIVIATION OF ADENYL CYCLASE
Vol. 9, No . 2
not affect phosphodiesterase activity . TABLE 2 Activation of Canine Thyroid Adenyl Cyclase as a Function of Time
TIME (minutes)
PICOM)LES CYCLIC 3',5'-AMP ACCUMTLATHD* CONTROL-4
LATS-1
1 .5
< 1 .0
< 1 .0
3 .0
< 1 .0
2 .5
5 .0
1 .6
4 .6
10 .0
4.6
9 .0
*Values are the mean of duplicate samples .
TAS 3 Activation of Bovine Thyroid Adenyl Cyclase as a Function of LATS Concentration PFOTEIN ADDED SOURCE mg/ml
PICOM)LES CYCLIC 3',5'-AMP ACCUMJLATED/5 MIN-*
CONIHOL-1
5 .0
2 .8 + 0.8
LATS-1
5 .0
5-1 + o.6
CON ROL-1
10 .0
5 .6 + 1 .7
LATS-1
10 .0
9 .3 + 2 .7
CONTROL-1
20 .0
7 .4 + 1 .1
LATS-1
20 .0
16 .2 + 5 .6
A PICOM)LES
P$
2 .3
< 0 .01
3 .7
< 0 .05
8 .8
< 0 .05
*Values represent the mean _+ SK of 3 samples . *Statistical tests performed utilizing Student's test for paired data . In all experiments the effect of LATS Was compared to a gamma globulin preparation made from a normal serum, since the presence of increasing amounts of serum protein appeared to increase adenyl cyclase activity by itself (Tables 1 and 3) .
This increase in activity was not due to TSH present in the
plasma of normal subjects, since the plasma of a hypophysectomized patient
Vol. 9, No. 2
was also active .
ACTIVIATION OF ADENYL CYCLASE
71
This nonspecific effect of serum protein was also observed
with the adrenal adenyl cyclase . Discussion The mechanism by which LATS acts is important in understanding the pathophysiology of Graves' disease .
Several studies have shown that LATS and TSH
have similar effects on the thyroid .
Scott et al have reported that LATS in
creased glucose oxidation and phospholipid synthesis in sheep thyroid slices (5) and Field et al noted similar effects in canine thyroid slices (6) . Shishiba et al compared the effects of LATS and TSH on two of the early thyroid responses in mice, the appearance of colloid droplets and release of preformed colloid-stored radiciodine .
They found that although the time course of the
effects were different, the responses to LATS were indistinguishable from TSH (7) .
Burke studied the effects of LATS or TSH in mice to determine if prior
administration of either of these agents inhibited the response to the other agent (8) .
The data indicated that LATS and TSH compete with each other and
therefore probably act through a common pathway.
Gilman and Rall reported that
TSH elevated cyclic AMP levels in beef thyroid tissue but LATS did not (9) . However,
the significance of the failure of LATS to act is unclear since the
authors did not measure the potency of their LATS preparation and did not purify it or concentrate it before use.
Our report demonstrates that LATS,
like TSH, activates adenyl cyclase in thyroid homogenates .
We and others have
previously presented evidence suggesting that many of the metabolic functions observed in thyroid slices are mediated by cyclic 3',5'-AMP (10-12) .
Since TSH
does not appear to enter the thyroid cell, but binds to the cellular membrane (13), it would seem unlikely that the large LATS molecule penetrate the membrane .
(W -160,000) would
Indeed Burke has presented evidence that LATS is bound
to the thyroid cell membrane
(14) .
Therefore, any direct effects of LATS on
the thyroid cell would of necessity require an external binding site .
The
adenyl cyclase system, localized in the cell membrane, provides an appropriate
72
ACTIVIATION OF ADENYL CYCLASE
Vol . 9, No. 2
site to mediate LATS effects . It should be noted that both in vivo and in vitro LATE effects generally occur later than TSH effects .
Although we have not compared LATS and TSH in
the same experiment, we previously found that in the adenyl cyclase assay TSH induced increases in cyclic 3',5'-AMP were noted within 30 seconds (2) whereas in the current experiments LATS-induced increases are not observed until three minutes of incubation .
However, the differences in the activation kinetics in
this and other systems may reflect differences in the rates of binding to the site of action and do not necessarily imply a difference-in basic site or mechanism of action .
In our experiments we have employed two sera with very
high LATS titers, although activation with one serum with only moderate LATS levels was observed .
We have not as yet compared in a systematic way the
potency of LATS in vivo with its potency in our in vitro system . have employed high concentrations of LATS .
However, we
In general,effects of hormones
on intact tissue are detected at lower concentrations than in homogenates . For example, 015 AU/ml of TSH increases cyclic AMP levels in beef thyroid slices
(9), whereas it requires about 5mU/ml to get a detectable increase in
adenyl cyclase activity in 'beef thyroid homogenates (2) . On the basis of the data presented in this report, we feel that a mechanism has been defined which could explain the thyroid stimulating effects of LATS . Summa~' Evidence is presented demonstrating that long-acting thyroid stimulator (LATS) activates adenyl cyclase in bovine and canine thyroid homogenates . Three LATS-positive sera increased the accumulation of cyclic 3',5'-[32P] AMP approximately 2-fold compared to four LATS-negative sera .
The data may pro-
vide the mechanism by which LATS stimulates the thyroid in Graves' disease . Acknoeled~ement The authors thank Drs . Eugene Mayberry, Donald Munro and Samuel Asper for
Vol. 9, No . 2
ACTIVIATION OF ADENYL CYCLASE
73
their gifts of LATE positive sera, and Dr . Roger Wynen for assay of same of these sera . References
424 (1965)-
1.
J . M. McKenzie, J. Clin . Endocrinol . ?~,
2.
I . Pastan, and R . Katzen, Biochem . Biophys . Res . Commun . ?2,
3-
G . Krishna, B . Weiss, and B . B . Brodie, J. Pharmacol. Exptl . Therap .
792 (1967)-
1~a, 379 (1968)244, 247 (1969) .
4.
0 . D . Taunton, J . Roth, and I. Pastan, J . Biol . Chem .
5.
T.W . Scott, B .F . Good, and K. A . Ferguson, Endocrinology
6.
J. B. Field, A . Remer, G . Bloom, and J . P. Kriss, J. Clin . Invest .
,
949 (1966) .
1553 (1968) 7.
Y.
Shishiba, D .-H.
Solomon, and G . N. Beall, Endocrinology
80 ,
957 (1967)8.
G . Burke, J . Clin . Endocrinol .
28, 286 (1968) .
9.
G . A . Gilman, and T . W . Rell, J . Biol . Chem .
243, 5867 (1968)-
242, 5757 (1967)-
10 .
I . Pastan and V. Macchia, J. Biol . Chem .
11 .
G . S . Levey, J . Roth, and I . Pastan, Endocrinology
12 .
B . Wilson, E . Raghupathy, T . Tonoue, and W. Tong, Endocrinology
84, 1009 (1969) .
877 (1968)13-
I . Pastan, J . Roth, and V. Macchia, Proc . Nat . Acad . Sci .
(1966) . 14 .
G . Burke, Endocrinology
§J, 1210 (1968) .
56, 1802
,
Pergamon Press
ACTIVATION OF THYROID ADENYL CYCLASE BY LONG-ACTING THYROID STIMULATOR G . S. Levey and I . Pastan National Institutes of Health, Bethesda, Maryland
20014
(Received 18 August 1969; in final form 13 October 1969) Long-acting thyroid stimulator (LATS) is found in the blood of most patients with hyperthyroidism and may be the cause of thyroid overactivity(1) . Since LATS and thyroid stimulating hormone (TSH) produce many similar effects, they may act by the same mechanism .
We have recently presented evidence that
cyclic 3',5'-AMP is an intracellular mediator of TSH action, and that TSH activates adenyl cyclase in thyroid homogenates (2) .
This report demonstrates
that LATS also activates adenyl cyclase in bovine and canine thyroid homogenates . Methods Control sera 1, 2, and 3 were obtained from normal volunteers ; 4 was from a hypophysectomized patient .
LATS preparation 1 was a gift from
Dr . Samuel Asper, 2 from Dr . Eugene Mayberry, and 3 from Dr . Donald Munro . In the McKenzie assay 0 .025 ml of LATS serum 1 or 3 produced a response greater than 900% and 0 .25 ml of serum 2 a response greater than 400% of the control at 9 hours .
Gamma globulin containing fractions of serum were
prepared by precipitation with 40% (W/V) ammonium sulfate .
The precipitate
was dissolved in 1/5 the original volume, dialyzed against 0.01 M tris-Cl pH 7 .4 and lyophilized in 0 .5 ml aliquots . reassayed for LATS activity .
The lyophilized material was not
At the beginning of each experiment 1 .5 ml of
thyroid homogenate containing 2-4 mg/ml protein was added to a tube containing a 0 .5 ml portion of the lyophilized powder .
Then 0 .025 ml of this mixture
was assayed for adenyl cyclase activity as previously described (2,3) .
67
The
68
ACTIVIATION OF ADENYL CYCLASE
Vol . 9, No. 2
time interval between dissolving the gamma globulin fraction and the onset of incubation was approximately 5 minutes .
Samples in a total volume of 0 .06 ml
were incubated at 370 C for 5 minutes unless otherwise noted with ATP, 1 .6 mM ; [ 32 P] ATP (o-labelled at 550 mCi/mMole), 2 .5 to 3 .5 x 106 8 mM ; MgC12, mg/ml .
cvm ;
theophylline,
2 .0 M; tris-HC1, 21 mM (pH 7 .7) ; and human serum albumin 0 .8
The incubations were started by adding homogenate, which had been
kept at 10 C, to the other components which were at 23 0 C . were stopped by adding 0 .1 ml of a solution containing 1 .25 limoles of cyclic 3',5'-AMP, boiled for 3 minutes .
4
The incubations
Wmoles of ATP,
and 0 .15 WCi of cyclic'3',5'-[3H] AMP and
The cyclic 3',5'-[3H] AMP served to determine the
recovery of cyclic 3',5'-AMP during the procedure ; recovery ranged from 30 to 35 per cent .
After boiling,
0 .4
ml of water was added, the precipitate
removed by centrifugation and the supernate applied to a 0 .5 x 2 .0 cm Dowex-50 column .
The column was washed with - cater, and the eluate between 3 and
6
ml
was collected and precipitated with 0 .17 M ZnSO 4 and 0 .15MBa(OH) 2 and the cyclic 3',5'-[32P] AMP and cyclic 3',5'-[3H] AMP counted in a liquid scintillation spectrometer .
The presence of cyclic 3',5'-AMP was confirmed by
thin-layer chromatography in a solvent system containing a mixture of n-butanol,
acetone, acetic acid, 5% ammonia, and water (7 :5 :3 :3 :2) .
A boiled
enzyme preparation did not produce any measurable cyclic 3',5'-AMP. Results Three LATS-positive sera increased the accumulation of cyclic 3',5'-[32P] AMP in bovine and canine thyroid homogenates approximately 2-fold compared to four LATS-negative sera (Table 1) .
The time course of acti-
vation of adenyl cyclase by LATS in a typical experiment is shown in Table 2 . An effect of LATS was detected after three minutes of incubation but not after 1-1/2 minutes in four experiments .
Adenyl cyclase activity increased in rela-
tion to the protein content of the LATS preparation over the range of 5 to 20 mg/ml (Table 3) .
LATS did not increase adenyl cyclase activity in a
Vol . 9, No. 2
69
ACTIVIATION OF ADENYL CYCLASE
TARTE 1 Activation of Thyroid Adenyl Cyclase by LATS EXPT . NO .
SAMPLE
PROTEIN mg/ml
PICOM)LES CYCLIC 3',5'-AMP ACCUMULATED/5 MIN .*
A. BOVINE 4 .6 + 0 .9 9 .0 + 0 .7
1.
CONTROL-1 LATS-1
16 .o 16 .o
2.
CONTROL-1 LATS-1 No Added TSH (loo X/ml)
16 .o 16 .o
6 .7 17 .7 3-5 5 .4
3.
CONTRDL-1 LATS-1
14 .o 8 .o
15 .8 + 1 .4 25 .2 + 1 .2
4.
CONTROL-1 CONTROL-2 CONTROL-3 CONTROL-4 LATS-1 LATS-2
14 .o 12 .0 10 .4 14 .4 12 .0 13 .0
7 .4 + 0.2 8.3 _ 8.2 7 .6 16 .4 _ + 1 .4 14 .6
5.
CONTROL-2 LATS-2
14 .4 12 .0
4.6 + 1 .0 9.6 + 1 .0
CONTROL-4 CONTROL-4 + TSH* LATS-1 LATS-3
34 .0 34 .0 14 .o 21 .0
1.6 4.9 _+ 1 .1 4.6 4.7 + o .8
+ _+ + ±
1 .2 0.7 0-5 0 .2
B . CANINE 1.
*Values are expressed as the mean _+ SE of three samples or the mean of closely agreeing duplicate samples . *TSB present at 25 mü/mlhomogenate prepared from a mouse adrenal tumor in which ACTH Increased adenyl cyclase activity ten-fold (4) . In the assay employed increased accumulation of cyclic AMP could have resulted from inhibition of cyclic nucleotide,phosphodiesterase activity by LATS .
Therefore LATS or control protein was added to complete reaction mix
tures containing thyroid homogenate and 200 pmoles of cyclic AMP.
In five
minutes 36 .± 1 (n = 4) pmoles of cyclic AMP disappeared from the control and 38 ± 1.3 (n = 4) from the reaction mixture containing LATS .
Thus LATS does
70
ACTIVIATION OF ADENYL CYCLASE
Vol. 9, No . 2
not affect phosphodiesterase activity . TABLE 2 Activation of Canine Thyroid Adenyl Cyclase as a Function of Time
TIME (minutes)
PICOM)LES CYCLIC 3',5'-AMP ACCUMTLATHD* CONTROL-4
LATS-1
1 .5
< 1 .0
< 1 .0
3 .0
< 1 .0
2 .5
5 .0
1 .6
4 .6
10 .0
4.6
9 .0
*Values are the mean of duplicate samples .
TAS 3 Activation of Bovine Thyroid Adenyl Cyclase as a Function of LATS Concentration PFOTEIN ADDED SOURCE mg/ml
PICOM)LES CYCLIC 3',5'-AMP ACCUMJLATED/5 MIN-*
CONIHOL-1
5 .0
2 .8 + 0.8
LATS-1
5 .0
5-1 + o.6
CON ROL-1
10 .0
5 .6 + 1 .7
LATS-1
10 .0
9 .3 + 2 .7
CONTROL-1
20 .0
7 .4 + 1 .1
LATS-1
20 .0
16 .2 + 5 .6
A PICOM)LES
P$
2 .3
< 0 .01
3 .7
< 0 .05
8 .8
< 0 .05
*Values represent the mean _+ SK of 3 samples . *Statistical tests performed utilizing Student's test for paired data . In all experiments the effect of LATS Was compared to a gamma globulin preparation made from a normal serum, since the presence of increasing amounts of serum protein appeared to increase adenyl cyclase activity by itself (Tables 1 and 3) .
This increase in activity was not due to TSH present in the
plasma of normal subjects, since the plasma of a hypophysectomized patient
Vol. 9, No. 2
was also active .
ACTIVIATION OF ADENYL CYCLASE
71
This nonspecific effect of serum protein was also observed
with the adrenal adenyl cyclase . Discussion The mechanism by which LATS acts is important in understanding the pathophysiology of Graves' disease .
Several studies have shown that LATS and TSH
have similar effects on the thyroid .
Scott et al have reported that LATS in
creased glucose oxidation and phospholipid synthesis in sheep thyroid slices (5) and Field et al noted similar effects in canine thyroid slices (6) . Shishiba et al compared the effects of LATS and TSH on two of the early thyroid responses in mice, the appearance of colloid droplets and release of preformed colloid-stored radiciodine .
They found that although the time course of the
effects were different, the responses to LATS were indistinguishable from TSH (7) .
Burke studied the effects of LATS or TSH in mice to determine if prior
administration of either of these agents inhibited the response to the other agent (8) .
The data indicated that LATS and TSH compete with each other and
therefore probably act through a common pathway.
Gilman and Rall reported that
TSH elevated cyclic AMP levels in beef thyroid tissue but LATS did not (9) . However,
the significance of the failure of LATS to act is unclear since the
authors did not measure the potency of their LATS preparation and did not purify it or concentrate it before use.
Our report demonstrates that LATS,
like TSH, activates adenyl cyclase in thyroid homogenates .
We and others have
previously presented evidence suggesting that many of the metabolic functions observed in thyroid slices are mediated by cyclic 3',5'-AMP (10-12) .
Since TSH
does not appear to enter the thyroid cell, but binds to the cellular membrane (13), it would seem unlikely that the large LATS molecule penetrate the membrane .
(W -160,000) would
Indeed Burke has presented evidence that LATS is bound
to the thyroid cell membrane
(14) .
Therefore, any direct effects of LATS on
the thyroid cell would of necessity require an external binding site .
The
adenyl cyclase system, localized in the cell membrane, provides an appropriate
72
ACTIVIATION OF ADENYL CYCLASE
Vol . 9, No. 2
site to mediate LATS effects . It should be noted that both in vivo and in vitro LATE effects generally occur later than TSH effects .
Although we have not compared LATS and TSH in
the same experiment, we previously found that in the adenyl cyclase assay TSH induced increases in cyclic 3',5'-AMP were noted within 30 seconds (2) whereas in the current experiments LATS-induced increases are not observed until three minutes of incubation .
However, the differences in the activation kinetics in
this and other systems may reflect differences in the rates of binding to the site of action and do not necessarily imply a difference-in basic site or mechanism of action .
In our experiments we have employed two sera with very
high LATS titers, although activation with one serum with only moderate LATS levels was observed .
We have not as yet compared in a systematic way the
potency of LATS in vivo with its potency in our in vitro system . have employed high concentrations of LATS .
However, we
In general,effects of hormones
on intact tissue are detected at lower concentrations than in homogenates . For example, 015 AU/ml of TSH increases cyclic AMP levels in beef thyroid slices
(9), whereas it requires about 5mU/ml to get a detectable increase in
adenyl cyclase activity in 'beef thyroid homogenates (2) . On the basis of the data presented in this report, we feel that a mechanism has been defined which could explain the thyroid stimulating effects of LATS . Summa~' Evidence is presented demonstrating that long-acting thyroid stimulator (LATS) activates adenyl cyclase in bovine and canine thyroid homogenates . Three LATS-positive sera increased the accumulation of cyclic 3',5'-[32P] AMP approximately 2-fold compared to four LATS-negative sera .
The data may pro-
vide the mechanism by which LATS stimulates the thyroid in Graves' disease . Acknoeled~ement The authors thank Drs . Eugene Mayberry, Donald Munro and Samuel Asper for
Vol. 9, No . 2
ACTIVIATION OF ADENYL CYCLASE
73
their gifts of LATE positive sera, and Dr . Roger Wynen for assay of same of these sera . References
424 (1965)-
1.
J . M. McKenzie, J. Clin . Endocrinol . ?~,
2.
I . Pastan, and R . Katzen, Biochem . Biophys . Res . Commun . ?2,
3-
G . Krishna, B . Weiss, and B . B . Brodie, J. Pharmacol. Exptl . Therap .
792 (1967)-
1~a, 379 (1968)244, 247 (1969) .
4.
0 . D . Taunton, J . Roth, and I. Pastan, J . Biol . Chem .
5.
T.W . Scott, B .F . Good, and K. A . Ferguson, Endocrinology
6.
J. B. Field, A . Remer, G . Bloom, and J . P. Kriss, J. Clin . Invest .
,
949 (1966) .
1553 (1968) 7.
Y.
Shishiba, D .-H.
Solomon, and G . N. Beall, Endocrinology
80 ,
957 (1967)8.
G . Burke, J . Clin . Endocrinol .
28, 286 (1968) .
9.
G . A . Gilman, and T . W . Rell, J . Biol . Chem .
243, 5867 (1968)-
242, 5757 (1967)-
10 .
I . Pastan and V. Macchia, J. Biol . Chem .
11 .
G . S . Levey, J . Roth, and I . Pastan, Endocrinology
12 .
B . Wilson, E . Raghupathy, T . Tonoue, and W. Tong, Endocrinology
84, 1009 (1969) .
877 (1968)13-
I . Pastan, J . Roth, and V. Macchia, Proc . Nat . Acad . Sci .
(1966) . 14 .
G . Burke, Endocrinology
§J, 1210 (1968) .
56, 1802
,