Journal of the Neurological Scwnces, 1977, 34 101-108
101
© Elsevier SczentlficPubhshmg Company, Amsterdam - Printed m The Netherlands
IS THERE ACETYLCHOLINE RECEPTOR IN HUMAN THYMUS 9
GARTH A NICHOLSONand STANLEY H APPEL Duke Umverstty Medical Center, Durham, N C 27710 ( U S A )
(Received 12 Aprd, 1977)
SUMMARY Recent studies have suggested that thymus tissue of the calf may bear nicotinic acetylchohne receptors The presence of similar receptors In thymus tissue of man could thereby serve as a source of antigen for the production of antibody to the acetylchohne receptor in patients with myasthenia gravis In the present experiments, human thymus tissue was examined for the presence of acetylchohne receptor Both [lgSI]a-bungarotoxin binding and antiserum to the human acetylchohne receptor were used in tests for acetylchohne receptor in thymus glands from normal individuals and from patients with myasthenia gravis Neither normal nor myasthenlc thymus tissue were found to possess the [l~SI]a-bungarotoxln binding or the antigenic properties of the acetylcholine receptor
INTRODUCTION Myasthenla gravls (MG) is an autolmmune neuromuscular disease characterized by muscle fatlguablhty which increases with exertion and improves with rest The pathogenesls of this disorder appears related to circulating antibodies which react with the acetylchohne (ACh) receptor in the muscle neuromuscular junction and thereby impair synaptlc transmission (Lindstrom and Patrick 1973, Fambrough, Drachman and Satyamurtl 1973, Almon, Andrew and Appe11974a) Many fundamental questions about the lmtlatlon of the disease process remain unanswered including (1) how tolerance is broken to a normal constltutent such as the acetylchohne receptor, and (2) the locus of the acetylchohne receptor antigen responsible for the production of antibody to the receptor The high Incidence of thymlc hyperplasla and thymoma in myasthenla gravls (Castleman 1966), the presence of a number of muscle antigens in thymus (Strauss, Smith, Cage, Van der Geld, McFarhn and Barlow 1966) and the ability to grow striated muscle fibers from thymus tissue (Wekerle, Paterson, Ketelsen and Feldman 1975)
102 focused attention on this organ not only as a possible site for mnJatlon ol the aheled immune state but also as a possible locus for the acetylchohne receptor antigen E~dence for the presence of acetylchohne receptor antigen m mammalian thymus ~,t~ prowded by Aharonov, Tarrab-Hazdm, Abramskl and Fuchs (1975) who reported the presence of an antigen m calf thymus extracts which fixed complement m the presence of antl-electroplax ACh receptor serum and which cross-reacted w~th electroplax ACh receptor In a lymphocyte transformation assay In the present report, we have used several d~rect methods to investigate whethel ACh receptor is present m human thymus extracts from e~ther normal or myasthemc patients Four different criteria have been employed to ~dentlfy ACh receptor, three of which depend upon the binding of [125I]~t-bungarotoxm These cnterm include (1) the presence of saturable and high affimty (Ka 108L/mole) bmdmg of [lZ~l]~bungarotoxin, (2) lnhlNtlon of toxin binding by chohnerglc agomsts and antagomsts. d-tubocurarme, decamethonmm, and carbamylchollne, (3) the sedimentation of toxin thym~c extract complexes on sucrose density gradients at a similar rate to toxinskeletal muscle ACh receptor complexes (9S-10S) (Berg, Kelly, Sargent, Wflhamson and Hall 1972), and (4) the abdlty of thymlc extracts to absorb skeletal muscle ACh receptor antibody from human myasthemc serum Neither material extracted from normal thymus nor material extracted from myasthemc thymus fulfilled any of these criteria for the ACh receptor METHODS Normal thymic tissue was obtained from patients undergoing elective cardiac surgery Thymlc tissue from patients with M G was obtained from patients undergoing therapeutic thymectomy Informed consent was obtained from all patients The dmgnosls of M G was established from history and physical examination, improvement m muscle strength after injection of edrophonmm hydrochlorlde and decremental response of muscle action potentials on nerve stimulation W~ssue was maintained at 0 °C during transportation and either used fresh or stored frozen at --70 °C Because of the limited availability of material, it was not possible to obtain age- and sex-matched thymus tissue Preparation of [l~5I]a-bungarotoxm and details of binding studies employing Sephadex G-200 chromatography were as previously described (Almon, Andrew and Appel 1974b) Sucrose density gradient centrlfugatlon was performed using a Beckman LKB65 ultracentrifuge, SW 50 1 rotor at 50,000 rpm for 10 5 hr at 5 °C on a continuous sucrose gradient (5-20 %) in 50 mM Trls, 100 mM NaCI, 1% Triton X-100, pH 7 4 Triton X-100 extracts of thymus were made as previously described for muscle homogenates (Almon et al 1974b) containing ACh receptor except for a reduction in the volume of buffer used for homogenization from four volumes to 2 volumes Binding of myasthemc serum antibody to thymlc extract was assessed by reactmn with [12aI]a-bungarotoxm-labelled thymIc extract and unlabelled extract
Method 1 MG antibody bmdmg to bungarotoxm-labelled thymtc extraet~ This method measured the abihty of thymus extracts to bind radioactive toxin by
103 precipitation of the resulting complex by myasthenlc serum followed by rabbit antlhuman-),-globuhn serum Thymlc extract was incubated with [t2~I]a-bungarotoxm (30 nM) for 16 hr at 4 °C and was partially purified and concentrated by chromatography on DEAE cellulose columns Labelled extract was added to the DEAE column in 10 mM Trls, 0 1 ~ Triton X-100 buffer pH 7 4, and washed extensively with the same buffer Bungarotoxln-labelled thymlc extract complexes were eluted from the column with 0 3 M salt solution in the above buffer as used for skeletal muscle ACh receptor a-bungarotoxln complexes (Almon and Appel 1975) The DEAE absorbed [125I] a-bungarotoxln-labelled thymic extract was substituted for [lzSI]a-bungarotoxlnlabelled ACh receptor in an immunoassay described previously by Almon and Appel (1975) Myasthenlc serum (20 ttl) was added to 20 #1 (0 5-1 0 pm) of toxin-thymus extract and incubated for 30 mln at 37 °C Any antibody bound to the [leSI]a-bungarotoxin-labelled thymlc extract complex was precipitated by the addition of rabbit antlhuman-7-globuhn serum, incubated for 3 hr at 37 °C and centrifuged at 1000 × g for 10 mln Pellet and supernatant were separated by aspiration and both were counted in a Packard gamma spectrometer Precipitated radloactxwty would indicate the presence of a substance with bungarotoxln binding, DEAE absorption and antigenic properties of the ACh receptor
Method 2 Absorption of unlabelled thymtc extract by myasthemc serum This method measured the ability ofthymlc extracts to absorb antibodies directed against the ACh receptor Thymlc Triton X-100 extract (20 #1) was Incubated for 16 hr at 4 °C with 10 #1 of a dilution of myasthenlc serum (1/16) capable of binding 5 0 ~ of the concentration of [125I]a-bungarotoxtn-labelled skeletal muscle ACh receptor used in the assay The thymus absorbed antlsera were then incubated with 10 #1 (0 5-1 0 pm) [125I]a-bungarotoxin-labelled ACh receptor which had previously been prepared by incubating Triton extracted denervated rat skeletal muscle with [125I]abungarotoxln (30 nM) for 16 hr at 4 °C The labelled receptor was purified either by Sephadex G-200 chromatography as previously described (Almon and Appel 1975) or by DEAE cellulose chromatography employing 0 3 M sodium chloride elutlons Antibody-bound [125I]ct-bungarotoxln-labelled ACh receptor was separated from free [12aI]a-bungarotoxin-labelled ACh receptor as in Method 1 RESULTS No saturable binding could be demonstrated using 10-10-10 -6 M [125I]a-bungarotoxin In the presence of 10-3 M d-tubocurarlne the binding of toxin to such extracts was inhibited only 20-30 ~ compared to greater than 90 ~ Inhibition of the same bungarotoxln preparations to extracts of rat or human skeletal muscle Higher concentrations of tubocurarlne (10 -2 M) did not produce any further inhibition Binding isotherms with normal thymus extracts did not conform to equations for the binding of hgand to a set of specific sites The material derived from myasthemc thymus slmdarly did not demonstrate either saturation or the presence of a high affinity site (Fig 1) and there were no statistically significant differences between the isotherms
104 NORMAL THYMUS
3(3 i
2.0
J
O~
N
(2_ X ,,10
!
(.9 Z Z m
z
THYMUS FROM MYASTHENIC PATIENTS
T 20m
10-
-10
-9
-8
LOG,~ FREE o{-BUNGAROTOXlN (moOr)
Fig 1 Binding of [lZSl]a-bungarotoxm to thymus extracts from 4 normal and 5 myasthemc subjects.
Bound [125]la-bungarotoxm was separated from free [125I]a-bungarotoxm by Sephadex G-200 column chromatography Bungarotoxm binding was expressed as the difference m bmdmg m the presence ("non-specificbinding") and absence of 10-a M d-tubocurare Isotherms were determined on 3 separate occasions for each gland Points m the figure are the mean and standard dewatmn of the combined results for all thymus glands in each category at each concentration of free [tZSlJa-bungarotoxm for normal or myasthemc thymus extracts Neither 10-5 M decamethomum, 10-3 M carbamylchohne, nor 10-z M atropine had any effect on the binding of [125I]a-bungarotoxin m thymus extracts Even 10-2 M concentratmn of these hgands d~d not effect [125I]a-hungarotoxm binding No labelled matenal m thymus with sechmentat~on charactenst]cs s~mflar to A C h receptor could be demonstrated ]n the present mvest]gatmn. The [t25I]a-bungarotoxin-labelled thymus fractmn which was ]nh]b]table with 10-8 M d-tubocurarme migrated m the hghter density fract]ons of the gradient w~th a rate identical to that of free [125I]a-bungarotoxm (Fig 2) Myasthemc serum ACh receptor antibody was used to determine whether the
105
CATALASE RAT Ach R
200
HEMOGLOBIN
~
16 A i
T ISO
12 ? O
~loo
~
A,
50-
4 l
,
I 5
,
~'
I 15
,
,
FRACTION NUMBER
Fig 2 Sed]mentat]on velocity centrffugatlon distribution of bound [1251]a-bungarotoxln-labelled thymus extract from the Sephadex G-200 excluston volume and marker proteins (hemoglobin 4 3S, catalase 11 3S and [~sI]a-bungarotoxln-labelleddenervated rat ACh receptor 9 0S) using a Beckman LKB65 ultracentrifuge, SW 50 l rotor at 50,000 rpm for l0 5 hr at 5 °C on a continuous sucrose gradient (5-20 ~) m 50 mM Tns, 100 mM NaCI, 1 ~ Triton X-100, pH 7 4 [125I]a-bungarotoxm-labelled thymus extract after incubation with l0 -3 M d-tubocurarme (@ 0 ) , and without d-tubocurarme (,~_3 ©), free [125I]a-bungarotoxm m the above buffer (A
A)
[125I]a-bungarotoxln-labelled material in thymlc extracts had a n y of the i m m u n o logical properties of skeletal muscle A C h receptor M G serum did n o t precipitate a larger percentage of [125I]a-bungarotoxin t h y m u s receptor complexes t h a n n o r m a l ser u m However, when skeletal muscle was used as the source of A C h receptor M G serum precipitated significant radioactivity (Table l) The criteria for identification TABLE 1 MG ANTIBODY BINDING TO [12~I]a-BUNGAROTOXIN-LABELLED THYMIC AND MUSCLE EXTRACTS Binding of [1251]a-bungarotoxmlabelled extract a MG serum
control serum (fm ± SD)
[125I]a-BTX-labelled MG thym~c extract (53fmsltes, N = 3 )
085±045
1 16i034
[1251]a-BTX-labelled normal thym~c extract (27 fm sates, N = 3)
0 34 ± 0 07
0 44 .~ 0 09
[125I]a-BTX-labelled skeletal muscle extract (16 fm sites, N = 3)
946 ± 1 58
094 ± 0 13
a Results are expressed as femtomoles (fm) of [12~l]a-bungarotoxm bound ± one standard deviation, in the pellet of the rabbit anti-human globuhn radlolmmunoassay (Method 1) described m the text Femtomoles shown m parenthesm refer to the number of femtomoles of [125Ila-bungarotoxln labelled extract used m each experiment, N refers to the number of extracts m each group
106 TABLE 2 EFFECT OF PREINCUBATION OF MYASTHENIA GRAVIS SERUM W1TH FHYMU% AND MUSCLE EXTRACTS ON THE BINDING OF [12q]-tt-BTX LABELLED ACFTYICHOLINE RECEPTOR BY THE SERUM Premcubat~on materml
Bmdmgof[1251]a-BTX-AChR (fro ± SD)
Slgmfican~e ol difference from buffer control
101
© Elsevier SczentlficPubhshmg Company, Amsterdam - Printed m The Netherlands
IS THERE ACETYLCHOLINE RECEPTOR IN HUMAN THYMUS 9
GARTH A NICHOLSONand STANLEY H APPEL Duke Umverstty Medical Center, Durham, N C 27710 ( U S A )
(Received 12 Aprd, 1977)
SUMMARY Recent studies have suggested that thymus tissue of the calf may bear nicotinic acetylchohne receptors The presence of similar receptors In thymus tissue of man could thereby serve as a source of antigen for the production of antibody to the acetylchohne receptor in patients with myasthenia gravis In the present experiments, human thymus tissue was examined for the presence of acetylchohne receptor Both [lgSI]a-bungarotoxin binding and antiserum to the human acetylchohne receptor were used in tests for acetylchohne receptor in thymus glands from normal individuals and from patients with myasthenia gravis Neither normal nor myasthenlc thymus tissue were found to possess the [l~SI]a-bungarotoxln binding or the antigenic properties of the acetylcholine receptor
INTRODUCTION Myasthenla gravls (MG) is an autolmmune neuromuscular disease characterized by muscle fatlguablhty which increases with exertion and improves with rest The pathogenesls of this disorder appears related to circulating antibodies which react with the acetylchohne (ACh) receptor in the muscle neuromuscular junction and thereby impair synaptlc transmission (Lindstrom and Patrick 1973, Fambrough, Drachman and Satyamurtl 1973, Almon, Andrew and Appe11974a) Many fundamental questions about the lmtlatlon of the disease process remain unanswered including (1) how tolerance is broken to a normal constltutent such as the acetylchohne receptor, and (2) the locus of the acetylchohne receptor antigen responsible for the production of antibody to the receptor The high Incidence of thymlc hyperplasla and thymoma in myasthenla gravls (Castleman 1966), the presence of a number of muscle antigens in thymus (Strauss, Smith, Cage, Van der Geld, McFarhn and Barlow 1966) and the ability to grow striated muscle fibers from thymus tissue (Wekerle, Paterson, Ketelsen and Feldman 1975)
102 focused attention on this organ not only as a possible site for mnJatlon ol the aheled immune state but also as a possible locus for the acetylchohne receptor antigen E~dence for the presence of acetylchohne receptor antigen m mammalian thymus ~,t~ prowded by Aharonov, Tarrab-Hazdm, Abramskl and Fuchs (1975) who reported the presence of an antigen m calf thymus extracts which fixed complement m the presence of antl-electroplax ACh receptor serum and which cross-reacted w~th electroplax ACh receptor In a lymphocyte transformation assay In the present report, we have used several d~rect methods to investigate whethel ACh receptor is present m human thymus extracts from e~ther normal or myasthemc patients Four different criteria have been employed to ~dentlfy ACh receptor, three of which depend upon the binding of [125I]~t-bungarotoxm These cnterm include (1) the presence of saturable and high affimty (Ka 108L/mole) bmdmg of [lZ~l]~bungarotoxin, (2) lnhlNtlon of toxin binding by chohnerglc agomsts and antagomsts. d-tubocurarme, decamethonmm, and carbamylchollne, (3) the sedimentation of toxin thym~c extract complexes on sucrose density gradients at a similar rate to toxinskeletal muscle ACh receptor complexes (9S-10S) (Berg, Kelly, Sargent, Wflhamson and Hall 1972), and (4) the abdlty of thymlc extracts to absorb skeletal muscle ACh receptor antibody from human myasthemc serum Neither material extracted from normal thymus nor material extracted from myasthemc thymus fulfilled any of these criteria for the ACh receptor METHODS Normal thymic tissue was obtained from patients undergoing elective cardiac surgery Thymlc tissue from patients with M G was obtained from patients undergoing therapeutic thymectomy Informed consent was obtained from all patients The dmgnosls of M G was established from history and physical examination, improvement m muscle strength after injection of edrophonmm hydrochlorlde and decremental response of muscle action potentials on nerve stimulation W~ssue was maintained at 0 °C during transportation and either used fresh or stored frozen at --70 °C Because of the limited availability of material, it was not possible to obtain age- and sex-matched thymus tissue Preparation of [l~5I]a-bungarotoxm and details of binding studies employing Sephadex G-200 chromatography were as previously described (Almon, Andrew and Appel 1974b) Sucrose density gradient centrlfugatlon was performed using a Beckman LKB65 ultracentrifuge, SW 50 1 rotor at 50,000 rpm for 10 5 hr at 5 °C on a continuous sucrose gradient (5-20 %) in 50 mM Trls, 100 mM NaCI, 1% Triton X-100, pH 7 4 Triton X-100 extracts of thymus were made as previously described for muscle homogenates (Almon et al 1974b) containing ACh receptor except for a reduction in the volume of buffer used for homogenization from four volumes to 2 volumes Binding of myasthemc serum antibody to thymlc extract was assessed by reactmn with [12aI]a-bungarotoxm-labelled thymIc extract and unlabelled extract
Method 1 MG antibody bmdmg to bungarotoxm-labelled thymtc extraet~ This method measured the abihty of thymus extracts to bind radioactive toxin by
103 precipitation of the resulting complex by myasthenlc serum followed by rabbit antlhuman-),-globuhn serum Thymlc extract was incubated with [t2~I]a-bungarotoxm (30 nM) for 16 hr at 4 °C and was partially purified and concentrated by chromatography on DEAE cellulose columns Labelled extract was added to the DEAE column in 10 mM Trls, 0 1 ~ Triton X-100 buffer pH 7 4, and washed extensively with the same buffer Bungarotoxln-labelled thymlc extract complexes were eluted from the column with 0 3 M salt solution in the above buffer as used for skeletal muscle ACh receptor a-bungarotoxln complexes (Almon and Appel 1975) The DEAE absorbed [125I] a-bungarotoxln-labelled thymic extract was substituted for [lzSI]a-bungarotoxlnlabelled ACh receptor in an immunoassay described previously by Almon and Appel (1975) Myasthenlc serum (20 ttl) was added to 20 #1 (0 5-1 0 pm) of toxin-thymus extract and incubated for 30 mln at 37 °C Any antibody bound to the [leSI]a-bungarotoxin-labelled thymlc extract complex was precipitated by the addition of rabbit antlhuman-7-globuhn serum, incubated for 3 hr at 37 °C and centrifuged at 1000 × g for 10 mln Pellet and supernatant were separated by aspiration and both were counted in a Packard gamma spectrometer Precipitated radloactxwty would indicate the presence of a substance with bungarotoxln binding, DEAE absorption and antigenic properties of the ACh receptor
Method 2 Absorption of unlabelled thymtc extract by myasthemc serum This method measured the ability ofthymlc extracts to absorb antibodies directed against the ACh receptor Thymlc Triton X-100 extract (20 #1) was Incubated for 16 hr at 4 °C with 10 #1 of a dilution of myasthenlc serum (1/16) capable of binding 5 0 ~ of the concentration of [125I]a-bungarotoxtn-labelled skeletal muscle ACh receptor used in the assay The thymus absorbed antlsera were then incubated with 10 #1 (0 5-1 0 pm) [125I]a-bungarotoxin-labelled ACh receptor which had previously been prepared by incubating Triton extracted denervated rat skeletal muscle with [125I]abungarotoxln (30 nM) for 16 hr at 4 °C The labelled receptor was purified either by Sephadex G-200 chromatography as previously described (Almon and Appel 1975) or by DEAE cellulose chromatography employing 0 3 M sodium chloride elutlons Antibody-bound [125I]ct-bungarotoxln-labelled ACh receptor was separated from free [12aI]a-bungarotoxin-labelled ACh receptor as in Method 1 RESULTS No saturable binding could be demonstrated using 10-10-10 -6 M [125I]a-bungarotoxin In the presence of 10-3 M d-tubocurarlne the binding of toxin to such extracts was inhibited only 20-30 ~ compared to greater than 90 ~ Inhibition of the same bungarotoxln preparations to extracts of rat or human skeletal muscle Higher concentrations of tubocurarlne (10 -2 M) did not produce any further inhibition Binding isotherms with normal thymus extracts did not conform to equations for the binding of hgand to a set of specific sites The material derived from myasthemc thymus slmdarly did not demonstrate either saturation or the presence of a high affinity site (Fig 1) and there were no statistically significant differences between the isotherms
104 NORMAL THYMUS
3(3 i
2.0
J
O~
N
(2_ X ,,10
!
(.9 Z Z m
z
THYMUS FROM MYASTHENIC PATIENTS
T 20m
10-
-10
-9
-8
LOG,~ FREE o{-BUNGAROTOXlN (moOr)
Fig 1 Binding of [lZSl]a-bungarotoxm to thymus extracts from 4 normal and 5 myasthemc subjects.
Bound [125]la-bungarotoxm was separated from free [125I]a-bungarotoxm by Sephadex G-200 column chromatography Bungarotoxm binding was expressed as the difference m bmdmg m the presence ("non-specificbinding") and absence of 10-a M d-tubocurare Isotherms were determined on 3 separate occasions for each gland Points m the figure are the mean and standard dewatmn of the combined results for all thymus glands in each category at each concentration of free [tZSlJa-bungarotoxm for normal or myasthemc thymus extracts Neither 10-5 M decamethomum, 10-3 M carbamylchohne, nor 10-z M atropine had any effect on the binding of [125I]a-bungarotoxin m thymus extracts Even 10-2 M concentratmn of these hgands d~d not effect [125I]a-hungarotoxm binding No labelled matenal m thymus with sechmentat~on charactenst]cs s~mflar to A C h receptor could be demonstrated ]n the present mvest]gatmn. The [t25I]a-bungarotoxin-labelled thymus fractmn which was ]nh]b]table with 10-8 M d-tubocurarme migrated m the hghter density fract]ons of the gradient w~th a rate identical to that of free [125I]a-bungarotoxm (Fig 2) Myasthemc serum ACh receptor antibody was used to determine whether the
105
CATALASE RAT Ach R
200
HEMOGLOBIN
~
16 A i
T ISO
12 ? O
~loo
~
A,
50-
4 l
,
I 5
,
~'
I 15
,
,
FRACTION NUMBER
Fig 2 Sed]mentat]on velocity centrffugatlon distribution of bound [1251]a-bungarotoxln-labelled thymus extract from the Sephadex G-200 excluston volume and marker proteins (hemoglobin 4 3S, catalase 11 3S and [~sI]a-bungarotoxln-labelleddenervated rat ACh receptor 9 0S) using a Beckman LKB65 ultracentrifuge, SW 50 l rotor at 50,000 rpm for l0 5 hr at 5 °C on a continuous sucrose gradient (5-20 ~) m 50 mM Tns, 100 mM NaCI, 1 ~ Triton X-100, pH 7 4 [125I]a-bungarotoxm-labelled thymus extract after incubation with l0 -3 M d-tubocurarme (@ 0 ) , and without d-tubocurarme (,~_3 ©), free [125I]a-bungarotoxm m the above buffer (A
A)
[125I]a-bungarotoxln-labelled material in thymlc extracts had a n y of the i m m u n o logical properties of skeletal muscle A C h receptor M G serum did n o t precipitate a larger percentage of [125I]a-bungarotoxin t h y m u s receptor complexes t h a n n o r m a l ser u m However, when skeletal muscle was used as the source of A C h receptor M G serum precipitated significant radioactivity (Table l) The criteria for identification TABLE 1 MG ANTIBODY BINDING TO [12~I]a-BUNGAROTOXIN-LABELLED THYMIC AND MUSCLE EXTRACTS Binding of [1251]a-bungarotoxmlabelled extract a MG serum
control serum (fm ± SD)
[125I]a-BTX-labelled MG thym~c extract (53fmsltes, N = 3 )
085±045
1 16i034
[1251]a-BTX-labelled normal thym~c extract (27 fm sates, N = 3)
0 34 ± 0 07
0 44 .~ 0 09
[125I]a-BTX-labelled skeletal muscle extract (16 fm sites, N = 3)
946 ± 1 58
094 ± 0 13
a Results are expressed as femtomoles (fm) of [12~l]a-bungarotoxm bound ± one standard deviation, in the pellet of the rabbit anti-human globuhn radlolmmunoassay (Method 1) described m the text Femtomoles shown m parenthesm refer to the number of femtomoles of [125Ila-bungarotoxln labelled extract used m each experiment, N refers to the number of extracts m each group
106 TABLE 2 EFFECT OF PREINCUBATION OF MYASTHENIA GRAVIS SERUM W1TH FHYMU% AND MUSCLE EXTRACTS ON THE BINDING OF [12q]-tt-BTX LABELLED ACFTYICHOLINE RECEPTOR BY THE SERUM Premcubat~on materml
Bmdmgof[1251]a-BTX-AChR (fro ± SD)
Slgmfican~e ol difference from buffer control
