Life Sciences Vol . 19, pp . 903-910, 1976 . Printed in the U .S .A .
Pergamon Presa
DIFFERENCES IN THE CHOLINERGIC PROTEOLIPID ISOLATED BY AFFINITY CHROMATOGRAPHY FROM NORMAL AND DENERVATED RAT LEG MUSCLE Maria C . Llorente de Carlin and E . De Robertis Instituto de Biologis Celular, Facultad de Medicine, Universidad de Bueras Aires, 1121 Buenos Aires, Argentina, (Received in final form August 3, 1976)
Summer The cholinergic proteolipid from rat leg muscle, isolated by Sephadex LH-20 column chromatography, was purified about 5 fold further by affinity chromatography in organic solvents . The affinity column consisted of p-phenyltrimethylammonium as the active group and a pulse of acetylcholine or acid wns used to desorb the specific fraction . From normal controls 36 Ng of specific protein per g fresh tissue were obtained . In the denerwated muscle there was no increase in this protein after three days, but it ~~creased by 120â after six days . Bindjng studies carried out with C-acetylcholine, H-+(-bungarotoxin and1 4C-d-tubocurarine showed that only the specific fraction was able to bind the ligands . Three days after denervation the specific acti~ity (rmoles/mg protein) for 14C-acetylcholine increased 400 and for H-~(-bungarotoxin 100X over the control ; on the contrary, there was no change in the binding of 14C-d-tubocurarlne . These results are discussed in relation to the different pharmacological properties of the functional and extrajunctional receptors in skeletal muscle . In previous investigations special proteolipids (i .e . hydrophobic proteins) were isolated from the electroplax of Electrophorus and Toroedo (1) and from skeletal muscle (2) by chromatography with Sephadex LH-20 . In~oth cases the nicotinic binding properties of these proteins were demonstrated (see,3) .Thus, it was possib to show that the ~holinergic prate ipid fraction 2-R fr~am muscle binds :~~C-d-tubocurarine,l C-hexamethoniun,~~C-decamethonium and 3H1(bungarotoxin (4) . More recently this chollnergic roteollpid was also separated by affinity chromatography in organic solvents (5~. The fraction isolated by conventional chromatography in Sephadex LH-20, from rat diaphragm, was separated into a non-specific and a specific fraction ; in the latter there was a 15 .4 fold purification bringing the total to 15,400 fold (5) . An important problem related to the cholinergic receptor of skeletal muscle is that of the hypersensitivity that occurs after denervation ; while in normal muscle receptors are confined to the end-plate region, after section of the nerve the entire cell surface becomes sensitive to acetylcholine (6,7) . In rat diaphragm, Lunt et al .(B) found that the proteolipids extracted from denervated muscle membranes had an increased turnover ; denervation also increased the incorporation of isotopically labeled leucine into the cholinergic proteolipid . After 21 days of denervation this increase was 10 fold above the control, in the non-inervated region of the muscle fiber, and 5 fold in the region containing the end-plate (2) . Such changes were accompanied by an increase in the protein content and also in the binding of acetylcholine to fraction 2-R (4) . In the present work the effect of denervation on the cholinergic proteolipid 903
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was investigated by the use of affinity chromatography to separate the choliner gic roteolipid from rat eg muscles . It will be shown that while the binding of 1 ~C-acetylcholine and ~Hy(-b~agarotoxin increases after denervation, there is no change in the binding of C-d-tubocurarine to the cholinergic proteolipid . Methods Adult Wistar rats (150-180g ; 3-4 months) were used . Denervation was carried out on the right sciatic nerve at its entrance into the thigh, while the left leg was used as control . In both legs the soleus and gastrocnemius muscles were dissected and used for extraction of the proteolipids . The tissue was homogenized in distilled water using an Ultraturrax to give a final homogenate of 10~ (w/v) and then frozen and lyophilized . For the lipid extraction 500 mg of the dried material, corresponding to 2,5g of fresh tissue, where homogenized in 20 ml of chloroform-methanol (2 :1, v/v) and, after 30 ~ninUtes, the extract was filtered through Whatman N°2 filter paper, To the filtrate, half volume of chloroform was added and the solution was evaporated under reduced pressure at 30°C to a volume of 3-4 ml . The extract was loaded onto the Sephadex LH-20 column (21 x 2 .1 cm) which was equilibrated with chloroform and the e]ution was carried out as previously described 4) . From the eluate the protein fraction that binds the cholinerglc ligands peak 2-R), was separated for further purification . The affinity column consisted of Sephadex LH-20, a spacer arm of -3,3iminobis ropylamine- and a quaternary ammonium compound (p-pher~yltrlmethylanmoniun~, of recognized cholinergic character (g), covalently linked to the free end of the spacer arm . The column of 12 x 1 .1 an was equilibrated overnight with chloroform . The extract was loaded and the elution carried out with chloroform and mtxtures of chloroform-methanol of Increasing polarity up to 4 :1 or 1 :1 (v/v) ; fractions of 2-4 ml were separated using an LKB Uvlcord fraction collector (Fig . 1) . The elution separated first all the non-specific proteollpids and when no furtherr UV absorbing material was detected at 280rm a pulse of 10 m1, either of 10 - M acetylcholine in chloroform-methanol (4 :1 or 1 :1) or of the same solvent mixture acidified .with 0 .1 N HC1, was applied . The elution was continued with the non-acidified solvent mixture until the specific cholinergic protein peak was desorbed (Fig . 2) . Both with the acetyl choline or the acid pulse exactly the same results, regarding the amount and position of the specific proteolipid peak, were obùined . The latter procedure was mainly used since it was easier to carry out the binding . If the specific fraction is desorbed with acetylcholine the excess ligand should be eliminated by rechromatography on a small column (16 x 0 .8 an) of Sephadex LH-20 . Binding studies were carried out on the ran-specific peaks as well as on the specific one . Aliquots containing 50-~00 Ng of protein were incubated with 14 C-acetylcholine, 1 C-d-tubocurarine or H-~(-bungarotoxin for 30 min and rechromatographed on small Sephadex LH-20 columns that separate the bound from the free ligand (Fig . 2) . The eluant, containing the rechramatographed proteolipld, was separated and collected for determination of protein and radioactivity . The fractions were evaporated to dryness and counted in toluene containing 0 .4~ (w/v) PPO in a Packard Tricarb or a Nuclear Chicago liquid scintilla tlon spectrometer . The protein was determined by the method of Lowry as modified by Hess and Lewin (10) and the lipid P by the method of Chen et al (11) . For every ligand, control experiments with the free drug were run in parallel . The radioactive drugs sed were (acetyl-1- 14C)-choline Iodide (3 .84 mCi/mial), tubo~urarine-d-(methyl 1 ~C) ether iodide (112 mCi/mmol), both from Amersham . The H1(-bungarotoxin (2 C1/nnale) was a generous gift of Prof . E .A . Barnard from the University of New York at Buffalo .
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Results The chloroform-methanol extract of the control muscle contained 500 ug of total proteolipid protein and 60 ug of lipid P per g fresh tissue . The elution patterns of the proteolipids, of both the control and denervated leg muscles, were essentially similar to those obtained from the rat diaphragm (see Fig .l of 4) . In the chromatogram of the Sephadex LH-20 column three main peaks of protein may be recognized by OD at 280 nm or by the Lowry's method . Peak 1 appears with the elution's volume (10-16m1) in the chloroform, peak 2-R is also eluted in the chloroform, between 25-30m1, and peak 3 in chloroformmethanol 4 :1 (80-90m1) .In the control, peak 2-R contained 190 ug of protein per g fresh tissue . This content remained without change 3 days after denewation but increased up to 320 pg/g ( 66X) at 6 days after section of the sciatic nerve . When peak 2-R is concentrated and applied to the affinity
Chloroform
ISi pa
0-11,1
6:1
4;1
Ec N W
FIG . 1 Chromatogram obtained by affinity chromatography . Fraction 2-R, was previously Isolated by a Sephadex LH-20 column from an extract of 500mg dry tissue and it contained 480 pg of protein . This fraction was loaded onto an affinity column having p-phenyltrimethylanmonium as the active group . The chromatogram shows three .non-specific frac tions as well as the specific one appearing after the pulse . In this case the specific fraction contained 90 pg of protein . C-M,chloroform methanol mixtures . column the pattern of elution observed is shown in Fig .l . There is a main peak of protein eluted between 15 and 25 ml of chloroform, acid two smaller ones in chloroform~nethanol 15 :1 and 6 :1 . It may be observed that, when no more protein is eluted with chloroform-methanol (4 :1), the pulse of acetylcholine, or of acidified solvent, is able to desorb a small peak of protein, the socalled specific peak . Binding studies with 10- 6M 1 4 C-acetylcholine carried out on the various peeks showed that only the specific peak was able to bind the ligand . The recovery for protein of the affinity column is of the order of 90X . In the control muscle the specific peak contained 36 pg of protein per g fresh tissue which corresponds to about 1/5 of the total protein contained in peak 2-R . Considering that the total protein content of the
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muscle is 200 mg/g fresh tissue the amount of protein in peak 2-R represents about 1/1000 of that value and in the specific peak, separated by affinity, about 1/5400 of the total protein .
After denervation the pattern obtained fran the affinity column is essentially similar to that of the control . Up to three days after section of the sciatic nerve there was no net increase in the amount of protein in the specific peak . However after six days of denervatlon there was a 120X increase in the protein content of this fraction (80pg/g fresh tissue) .
~zoo ...... . . . ~V+x-0ugarolo~n
s
E
N
FRACTION N' 2,-4~`6~~~~ ~ CONTROL
7
4 '6
DENERVATED 3 days )
FIG . 2
Rechronatography on a small Sephadex LH-20 column of the specific peak obtained as indicated in Fig .l . The protein, from both the control and the denervated muscle, was incubated with 10-6M H-d-bungarotoxin for 60 min and then rechromatographed . There is a sharp separation of the bound and the free ligand . Observe also the increased radioactivity on the denervated side .
In Fig . 2 it is shown the pattern obtained after rechromatography of the specific peak on small Sephadex LH-20 columns . The protein is eluted in a single peak between tubes 3 to 6, corresponding to 6 to 12 ml of elutee . In thi particular experiment the proteolipid was submitted to binding with 10 - ~ H-~-bungarotoxin and it is found that the bound radioactivity is eluted in coincidence with the protein peak . The free drug appears later on in the chromatogram about tube 20 . It nay also be observed that there is a considerable increase in binding in the denervated muscle . Expressed in specific radioactivity the 3H-ol-bungarotoxin bound to the proteollpid of the control muscle 1s 0 .5 nrol/mg while that of the denervated muscle is 1 .13 nmoles/mg protein, which implies an increase of about 100X .
Fig 3 shows re~ul~~ obtained, 1n control a~d enervated muscles, with C-acetylcholine the binding of 10 and 10- M 4 C-d-tubocurarine, to the specific proteolipid fraction isolated by affinity chromatography . For l4C" acetylcholine the binding in the controls is 3 .7 and 2.9 rrnoles/ng while 1n muscles denervated for 3 days the specific radioactivity is considerably increased (i .e . 15 .1 and 20 rmoles/mg protein) . In other words, after this period of denervation there is approximately a 400X increase in binding in the specific protein fraction . In a case of 6 days of denervation the specific radioactivity was also much higher than in the control .
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Of considerable interest are the results obtained with 14 C-d-tubocurarine ; in this case after three days of denepvation there was no increase in the binding and the specific radioactivity, both in the control and in the denervated side, varied between 0 .3 and 1 nmol/mg protein (Fig . 3) .
ô
5
c [IO_s M ACh]
10 - ~ M DMTC]
FIG . 3 Histograms representing the binding of 14 C-acetylcholine and 14C-dtubocurarlne to the specific proteolipid fractions obtained by affinity chromatography . The binding is expressed as specific radio activity (males/mg protein) . C, control muscle ; D 3 denervated aftër 3 days (see the description in the text) . DISCUSSION The main purpose of this study has been to compare the binding properties of the chollnergic proteolipid in the normal and denervated rat leg muscle . In this way we hoped to obtain information about the similarities or differences between the junctional receptors and the extrajunctional receptors that appear after denervation . In a previous study (4) we found that twenty days after section of the phrenic nerve there was an increase in protein in peak 2-R of about 30X, 1n the region containing the end-plate, and of about 60X in the non plate region ; at the same time the binding of acetylcholine increased respec= tively 180% and 310X in this proteolipid fraction extracted from both regions of the rat diaphragm . Here these findings are confirmed and expanded for the rat soleus and gastrocnemius muscles and for a cholinergic proteolipid that was purified by affinity chromatography five fold further . Although more experiments are needed to follow the extract time-course of the changes after denervation the observations presented here show that after 3 days of the section of the sciatic nerve there is no net increase in protein content in the cholinergic peak, inspite of the observation that the binding of 14Cacetylcholine has increased about 400% . After 6 days, the amount of choliner gic protein has doubled and the binding capacity for acetylcholine is still very high . These proposed choline . exposure
findings are difficult to interprete within the various theories to explain the hypersensitivity of the denervated muscle to acetylA simple explanation could be that in the early stages there is an of latent receptors and later on a true synthesis bf new receptors
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(see 12,13) ; however at the present time it is difficult to advance any definite interpretation of these changes . The binding of 3H-~-bungarotoxin increased about 100X 3 days after denervation . This finding may be correlated with the observation of Lee et al .(14), who first demonstrated that theol-toxin accumulates in the end-plate region, 1n the control, while after denervation the binding spreads along the muscle fiber . Thesleff (15) found that the number of d -toxin binding sites per muscle fiber increases by 200X 3 days after denervation and by 500X at 5 days . The most intriguing results are those obtained with the binding of 14 C-d-tubocurarine, since with this ligand there is na increase in specific radioactivity 3 days after denervation . This finding suggests that the receptors, appearing after denervation, may have different binding properties than those present at the normal end-plate . This is in line with the early observations of Waser and Hadorn (16) ; ~n the mouse diaphragm, after denervation, they did not find a spread of the 1 C-d-tubocurarine binding sites, as demonstrated by autoradiography. Pharmacological differences, particularly with respect to the action of curare, have been found by Ber6nek and Vyskocil (17) and Feltz and Maliart (18) in the receptor appearing after denervation and Lapa et al .(19) observed that ten times more d-tubocurarine is required to block these extrajunctional receptors . All these findings suggest that the extrajunctional receptors are less sensitive to d-tubocurarine . Also in this context are the observations of Miledi and Potter (20) that d-tubocurarine could only displace 50~ of the binding sites of d-bungarotoxin ; while acetylcholine reduced the binding of the d -toxin still further . Furthernare Porter et al . (21),from a study of the binding of theol-toxin in vivo and in vitro, concluded that in muscle there seems to be two classes of receptor sites : the junctional sites which bind d-bungarotoxin and d-tubocurarine and the extrajunctional sites which bind the d -toxin, but which are not protected by d-tubocurarine . In agreement with all the above mentioned 9bservations we found that after denervation the isolated cholinergic proteolipid shows an increased binding for acetylcholine and d-bungarotoxin but not for d-tubocurarine . This may imply that the extrajunctional cholinergic receptors are different 1n their binding properties than those present at the muscle end-plate. Our findings on the cholinergic proteolipid purified by affinity differ from those of Alper et al . (22) who, using a total detergent extract, reported no difference in ligand affinities between the normal and denervated diaphragm. At present we have not an explanation for this discrepancy. Acknowledgments
This paper was supported by Grants from CONICET, Buenos Aires, Argentina and National Institutes of Health (5 ROI NS 06953-09 NEUA) U .S .A . References 1 . J .L .LA TORRE, G .S . LUNT and E. DE ROBERTIS Proc . Nat. Acad .Sci .~$g 65,716720 (1970) . 2 . G .G . LUNT, E.STEFANI and E .DE ROBERTIS .J . Neurochem .18,1545-1553 (1971) . 3 . E . DE ROBERTIS, Synaptic Receptors, Iso at on a Tcular Biology . M . Dekker, New York 1-387 (1975) . 4 . E . DE ROBERTIS, M .T . MOSQUERA and S . FISZER DE PLAZAS . Life Sciences 11, 1155-1165 (1972) . 5 . F .J .BARRANTES, S . ARBILLA, M .C . L1 . de CARLIN and E . DE ROBERTIS . Biochem . Bio s . Res . Carom . 63, 194-201 (1975) . . J . Phvsiol . .~¢j, 178-193 (1959) . 6. n~YHF~I.EFF a~ÙA 7 . R . MILEDI . J . Ph siol . London 151, 1=23 1960 8 . G .G . LUNT, . a ~$TEFANI Biochen . J . 1~2 , 23p-24p (1970) . 9 . J .D . GERMAN and M. YOUNG. Proc . Nat . Acad . Sci . US A 68, 395-398 (1971) . 10 .H, HESS and F . LEWIN. J . Neurochem. ~, 205-211 (1965) . 11 . P .S . CHEN, T .Y . TORIBARA and H. WARNER (1956) Analvt .Chem .,~, 1756-1758 .
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12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 .
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L . GOTH . P iol . Rev . 48, 645-687 (1968) . R.I . CLOS s o . ev-52, 129-198 (1972) . C .Y . LEE, L . and T:H . CHIU . Nature 215, 1177-1178 (1967) . S. THESLEFF . In Drug Receptors (HP . among~d:TMac Mi }lan, London 121-135 (1973) . P .G . MASER and I . HADORN . Bibliotheca Anat .2, 155-160 (1960) . R . BERANEK and F. VYSKOCIL~s~o . ondon 188, 53-64 (1967) . A . FELTZ and A . MALLART ~J .~~oT.-Lon own i$~5-92 (1971) . 375-386 (1974) . J . LAPA, E .Y . ALBUQUERQU a~ nâ J . DAl R . MILEDI and L.T . POTTER . Nature 233, 599-603 971 . C .W . PORTER, T .H . CHIU, J . WfE~k01,f~KT and E .A . BARNARD Nature (New Biol . 241, 3-7 (1973), 1~ALPER, J . LDIiY and J . SCHMIDT . FEBS Le tters_ . 48, 130-T32 (1974) .
Pergamon Presa
DIFFERENCES IN THE CHOLINERGIC PROTEOLIPID ISOLATED BY AFFINITY CHROMATOGRAPHY FROM NORMAL AND DENERVATED RAT LEG MUSCLE Maria C . Llorente de Carlin and E . De Robertis Instituto de Biologis Celular, Facultad de Medicine, Universidad de Bueras Aires, 1121 Buenos Aires, Argentina, (Received in final form August 3, 1976)
Summer The cholinergic proteolipid from rat leg muscle, isolated by Sephadex LH-20 column chromatography, was purified about 5 fold further by affinity chromatography in organic solvents . The affinity column consisted of p-phenyltrimethylammonium as the active group and a pulse of acetylcholine or acid wns used to desorb the specific fraction . From normal controls 36 Ng of specific protein per g fresh tissue were obtained . In the denerwated muscle there was no increase in this protein after three days, but it ~~creased by 120â after six days . Bindjng studies carried out with C-acetylcholine, H-+(-bungarotoxin and1 4C-d-tubocurarine showed that only the specific fraction was able to bind the ligands . Three days after denervation the specific acti~ity (rmoles/mg protein) for 14C-acetylcholine increased 400 and for H-~(-bungarotoxin 100X over the control ; on the contrary, there was no change in the binding of 14C-d-tubocurarlne . These results are discussed in relation to the different pharmacological properties of the functional and extrajunctional receptors in skeletal muscle . In previous investigations special proteolipids (i .e . hydrophobic proteins) were isolated from the electroplax of Electrophorus and Toroedo (1) and from skeletal muscle (2) by chromatography with Sephadex LH-20 . In~oth cases the nicotinic binding properties of these proteins were demonstrated (see,3) .Thus, it was possib to show that the ~holinergic prate ipid fraction 2-R fr~am muscle binds :~~C-d-tubocurarine,l C-hexamethoniun,~~C-decamethonium and 3H1(bungarotoxin (4) . More recently this chollnergic roteollpid was also separated by affinity chromatography in organic solvents (5~. The fraction isolated by conventional chromatography in Sephadex LH-20, from rat diaphragm, was separated into a non-specific and a specific fraction ; in the latter there was a 15 .4 fold purification bringing the total to 15,400 fold (5) . An important problem related to the cholinergic receptor of skeletal muscle is that of the hypersensitivity that occurs after denervation ; while in normal muscle receptors are confined to the end-plate region, after section of the nerve the entire cell surface becomes sensitive to acetylcholine (6,7) . In rat diaphragm, Lunt et al .(B) found that the proteolipids extracted from denervated muscle membranes had an increased turnover ; denervation also increased the incorporation of isotopically labeled leucine into the cholinergic proteolipid . After 21 days of denervation this increase was 10 fold above the control, in the non-inervated region of the muscle fiber, and 5 fold in the region containing the end-plate (2) . Such changes were accompanied by an increase in the protein content and also in the binding of acetylcholine to fraction 2-R (4) . In the present work the effect of denervation on the cholinergic proteolipid 903
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was investigated by the use of affinity chromatography to separate the choliner gic roteolipid from rat eg muscles . It will be shown that while the binding of 1 ~C-acetylcholine and ~Hy(-b~agarotoxin increases after denervation, there is no change in the binding of C-d-tubocurarine to the cholinergic proteolipid . Methods Adult Wistar rats (150-180g ; 3-4 months) were used . Denervation was carried out on the right sciatic nerve at its entrance into the thigh, while the left leg was used as control . In both legs the soleus and gastrocnemius muscles were dissected and used for extraction of the proteolipids . The tissue was homogenized in distilled water using an Ultraturrax to give a final homogenate of 10~ (w/v) and then frozen and lyophilized . For the lipid extraction 500 mg of the dried material, corresponding to 2,5g of fresh tissue, where homogenized in 20 ml of chloroform-methanol (2 :1, v/v) and, after 30 ~ninUtes, the extract was filtered through Whatman N°2 filter paper, To the filtrate, half volume of chloroform was added and the solution was evaporated under reduced pressure at 30°C to a volume of 3-4 ml . The extract was loaded onto the Sephadex LH-20 column (21 x 2 .1 cm) which was equilibrated with chloroform and the e]ution was carried out as previously described 4) . From the eluate the protein fraction that binds the cholinerglc ligands peak 2-R), was separated for further purification . The affinity column consisted of Sephadex LH-20, a spacer arm of -3,3iminobis ropylamine- and a quaternary ammonium compound (p-pher~yltrlmethylanmoniun~, of recognized cholinergic character (g), covalently linked to the free end of the spacer arm . The column of 12 x 1 .1 an was equilibrated overnight with chloroform . The extract was loaded and the elution carried out with chloroform and mtxtures of chloroform-methanol of Increasing polarity up to 4 :1 or 1 :1 (v/v) ; fractions of 2-4 ml were separated using an LKB Uvlcord fraction collector (Fig . 1) . The elution separated first all the non-specific proteollpids and when no furtherr UV absorbing material was detected at 280rm a pulse of 10 m1, either of 10 - M acetylcholine in chloroform-methanol (4 :1 or 1 :1) or of the same solvent mixture acidified .with 0 .1 N HC1, was applied . The elution was continued with the non-acidified solvent mixture until the specific cholinergic protein peak was desorbed (Fig . 2) . Both with the acetyl choline or the acid pulse exactly the same results, regarding the amount and position of the specific proteolipid peak, were obùined . The latter procedure was mainly used since it was easier to carry out the binding . If the specific fraction is desorbed with acetylcholine the excess ligand should be eliminated by rechromatography on a small column (16 x 0 .8 an) of Sephadex LH-20 . Binding studies were carried out on the ran-specific peaks as well as on the specific one . Aliquots containing 50-~00 Ng of protein were incubated with 14 C-acetylcholine, 1 C-d-tubocurarine or H-~(-bungarotoxin for 30 min and rechromatographed on small Sephadex LH-20 columns that separate the bound from the free ligand (Fig . 2) . The eluant, containing the rechramatographed proteolipld, was separated and collected for determination of protein and radioactivity . The fractions were evaporated to dryness and counted in toluene containing 0 .4~ (w/v) PPO in a Packard Tricarb or a Nuclear Chicago liquid scintilla tlon spectrometer . The protein was determined by the method of Lowry as modified by Hess and Lewin (10) and the lipid P by the method of Chen et al (11) . For every ligand, control experiments with the free drug were run in parallel . The radioactive drugs sed were (acetyl-1- 14C)-choline Iodide (3 .84 mCi/mial), tubo~urarine-d-(methyl 1 ~C) ether iodide (112 mCi/mmol), both from Amersham . The H1(-bungarotoxin (2 C1/nnale) was a generous gift of Prof . E .A . Barnard from the University of New York at Buffalo .
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Results The chloroform-methanol extract of the control muscle contained 500 ug of total proteolipid protein and 60 ug of lipid P per g fresh tissue . The elution patterns of the proteolipids, of both the control and denervated leg muscles, were essentially similar to those obtained from the rat diaphragm (see Fig .l of 4) . In the chromatogram of the Sephadex LH-20 column three main peaks of protein may be recognized by OD at 280 nm or by the Lowry's method . Peak 1 appears with the elution's volume (10-16m1) in the chloroform, peak 2-R is also eluted in the chloroform, between 25-30m1, and peak 3 in chloroformmethanol 4 :1 (80-90m1) .In the control, peak 2-R contained 190 ug of protein per g fresh tissue . This content remained without change 3 days after denewation but increased up to 320 pg/g ( 66X) at 6 days after section of the sciatic nerve . When peak 2-R is concentrated and applied to the affinity
Chloroform
ISi pa
0-11,1
6:1
4;1
Ec N W
FIG . 1 Chromatogram obtained by affinity chromatography . Fraction 2-R, was previously Isolated by a Sephadex LH-20 column from an extract of 500mg dry tissue and it contained 480 pg of protein . This fraction was loaded onto an affinity column having p-phenyltrimethylanmonium as the active group . The chromatogram shows three .non-specific frac tions as well as the specific one appearing after the pulse . In this case the specific fraction contained 90 pg of protein . C-M,chloroform methanol mixtures . column the pattern of elution observed is shown in Fig .l . There is a main peak of protein eluted between 15 and 25 ml of chloroform, acid two smaller ones in chloroform~nethanol 15 :1 and 6 :1 . It may be observed that, when no more protein is eluted with chloroform-methanol (4 :1), the pulse of acetylcholine, or of acidified solvent, is able to desorb a small peak of protein, the socalled specific peak . Binding studies with 10- 6M 1 4 C-acetylcholine carried out on the various peeks showed that only the specific peak was able to bind the ligand . The recovery for protein of the affinity column is of the order of 90X . In the control muscle the specific peak contained 36 pg of protein per g fresh tissue which corresponds to about 1/5 of the total protein contained in peak 2-R . Considering that the total protein content of the
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muscle is 200 mg/g fresh tissue the amount of protein in peak 2-R represents about 1/1000 of that value and in the specific peak, separated by affinity, about 1/5400 of the total protein .
After denervation the pattern obtained fran the affinity column is essentially similar to that of the control . Up to three days after section of the sciatic nerve there was no net increase in the amount of protein in the specific peak . However after six days of denervatlon there was a 120X increase in the protein content of this fraction (80pg/g fresh tissue) .
~zoo ...... . . . ~V+x-0ugarolo~n
s
E
N
FRACTION N' 2,-4~`6~~~~ ~ CONTROL
7
4 '6
DENERVATED 3 days )
FIG . 2
Rechronatography on a small Sephadex LH-20 column of the specific peak obtained as indicated in Fig .l . The protein, from both the control and the denervated muscle, was incubated with 10-6M H-d-bungarotoxin for 60 min and then rechromatographed . There is a sharp separation of the bound and the free ligand . Observe also the increased radioactivity on the denervated side .
In Fig . 2 it is shown the pattern obtained after rechromatography of the specific peak on small Sephadex LH-20 columns . The protein is eluted in a single peak between tubes 3 to 6, corresponding to 6 to 12 ml of elutee . In thi particular experiment the proteolipid was submitted to binding with 10 - ~ H-~-bungarotoxin and it is found that the bound radioactivity is eluted in coincidence with the protein peak . The free drug appears later on in the chromatogram about tube 20 . It nay also be observed that there is a considerable increase in binding in the denervated muscle . Expressed in specific radioactivity the 3H-ol-bungarotoxin bound to the proteollpid of the control muscle 1s 0 .5 nrol/mg while that of the denervated muscle is 1 .13 nmoles/mg protein, which implies an increase of about 100X .
Fig 3 shows re~ul~~ obtained, 1n control a~d enervated muscles, with C-acetylcholine the binding of 10 and 10- M 4 C-d-tubocurarine, to the specific proteolipid fraction isolated by affinity chromatography . For l4C" acetylcholine the binding in the controls is 3 .7 and 2.9 rrnoles/ng while 1n muscles denervated for 3 days the specific radioactivity is considerably increased (i .e . 15 .1 and 20 rmoles/mg protein) . In other words, after this period of denervation there is approximately a 400X increase in binding in the specific protein fraction . In a case of 6 days of denervation the specific radioactivity was also much higher than in the control .
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Of considerable interest are the results obtained with 14 C-d-tubocurarine ; in this case after three days of denepvation there was no increase in the binding and the specific radioactivity, both in the control and in the denervated side, varied between 0 .3 and 1 nmol/mg protein (Fig . 3) .
ô
5
c [IO_s M ACh]
10 - ~ M DMTC]
FIG . 3 Histograms representing the binding of 14 C-acetylcholine and 14C-dtubocurarlne to the specific proteolipid fractions obtained by affinity chromatography . The binding is expressed as specific radio activity (males/mg protein) . C, control muscle ; D 3 denervated aftër 3 days (see the description in the text) . DISCUSSION The main purpose of this study has been to compare the binding properties of the chollnergic proteolipid in the normal and denervated rat leg muscle . In this way we hoped to obtain information about the similarities or differences between the junctional receptors and the extrajunctional receptors that appear after denervation . In a previous study (4) we found that twenty days after section of the phrenic nerve there was an increase in protein in peak 2-R of about 30X, 1n the region containing the end-plate, and of about 60X in the non plate region ; at the same time the binding of acetylcholine increased respec= tively 180% and 310X in this proteolipid fraction extracted from both regions of the rat diaphragm . Here these findings are confirmed and expanded for the rat soleus and gastrocnemius muscles and for a cholinergic proteolipid that was purified by affinity chromatography five fold further . Although more experiments are needed to follow the extract time-course of the changes after denervation the observations presented here show that after 3 days of the section of the sciatic nerve there is no net increase in protein content in the cholinergic peak, inspite of the observation that the binding of 14Cacetylcholine has increased about 400% . After 6 days, the amount of choliner gic protein has doubled and the binding capacity for acetylcholine is still very high . These proposed choline . exposure
findings are difficult to interprete within the various theories to explain the hypersensitivity of the denervated muscle to acetylA simple explanation could be that in the early stages there is an of latent receptors and later on a true synthesis bf new receptors
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(see 12,13) ; however at the present time it is difficult to advance any definite interpretation of these changes . The binding of 3H-~-bungarotoxin increased about 100X 3 days after denervation . This finding may be correlated with the observation of Lee et al .(14), who first demonstrated that theol-toxin accumulates in the end-plate region, 1n the control, while after denervation the binding spreads along the muscle fiber . Thesleff (15) found that the number of d -toxin binding sites per muscle fiber increases by 200X 3 days after denervation and by 500X at 5 days . The most intriguing results are those obtained with the binding of 14 C-d-tubocurarine, since with this ligand there is na increase in specific radioactivity 3 days after denervation . This finding suggests that the receptors, appearing after denervation, may have different binding properties than those present at the normal end-plate . This is in line with the early observations of Waser and Hadorn (16) ; ~n the mouse diaphragm, after denervation, they did not find a spread of the 1 C-d-tubocurarine binding sites, as demonstrated by autoradiography. Pharmacological differences, particularly with respect to the action of curare, have been found by Ber6nek and Vyskocil (17) and Feltz and Maliart (18) in the receptor appearing after denervation and Lapa et al .(19) observed that ten times more d-tubocurarine is required to block these extrajunctional receptors . All these findings suggest that the extrajunctional receptors are less sensitive to d-tubocurarine . Also in this context are the observations of Miledi and Potter (20) that d-tubocurarine could only displace 50~ of the binding sites of d-bungarotoxin ; while acetylcholine reduced the binding of the d -toxin still further . Furthernare Porter et al . (21),from a study of the binding of theol-toxin in vivo and in vitro, concluded that in muscle there seems to be two classes of receptor sites : the junctional sites which bind d-bungarotoxin and d-tubocurarine and the extrajunctional sites which bind the d -toxin, but which are not protected by d-tubocurarine . In agreement with all the above mentioned 9bservations we found that after denervation the isolated cholinergic proteolipid shows an increased binding for acetylcholine and d-bungarotoxin but not for d-tubocurarine . This may imply that the extrajunctional cholinergic receptors are different 1n their binding properties than those present at the muscle end-plate. Our findings on the cholinergic proteolipid purified by affinity differ from those of Alper et al . (22) who, using a total detergent extract, reported no difference in ligand affinities between the normal and denervated diaphragm. At present we have not an explanation for this discrepancy. Acknowledgments
This paper was supported by Grants from CONICET, Buenos Aires, Argentina and National Institutes of Health (5 ROI NS 06953-09 NEUA) U .S .A . References 1 . J .L .LA TORRE, G .S . LUNT and E. DE ROBERTIS Proc . Nat. Acad .Sci .~$g 65,716720 (1970) . 2 . G .G . LUNT, E.STEFANI and E .DE ROBERTIS .J . Neurochem .18,1545-1553 (1971) . 3 . E . DE ROBERTIS, Synaptic Receptors, Iso at on a Tcular Biology . M . Dekker, New York 1-387 (1975) . 4 . E . DE ROBERTIS, M .T . MOSQUERA and S . FISZER DE PLAZAS . Life Sciences 11, 1155-1165 (1972) . 5 . F .J .BARRANTES, S . ARBILLA, M .C . L1 . de CARLIN and E . DE ROBERTIS . Biochem . Bio s . Res . Carom . 63, 194-201 (1975) . . J . Phvsiol . .~¢j, 178-193 (1959) . 6. n~YHF~I.EFF a~ÙA 7 . R . MILEDI . J . Ph siol . London 151, 1=23 1960 8 . G .G . LUNT, . a ~$TEFANI Biochen . J . 1~2 , 23p-24p (1970) . 9 . J .D . GERMAN and M. YOUNG. Proc . Nat . Acad . Sci . US A 68, 395-398 (1971) . 10 .H, HESS and F . LEWIN. J . Neurochem. ~, 205-211 (1965) . 11 . P .S . CHEN, T .Y . TORIBARA and H. WARNER (1956) Analvt .Chem .,~, 1756-1758 .
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L . GOTH . P iol . Rev . 48, 645-687 (1968) . R.I . CLOS s o . ev-52, 129-198 (1972) . C .Y . LEE, L . and T:H . CHIU . Nature 215, 1177-1178 (1967) . S. THESLEFF . In Drug Receptors (HP . among~d:TMac Mi }lan, London 121-135 (1973) . P .G . MASER and I . HADORN . Bibliotheca Anat .2, 155-160 (1960) . R . BERANEK and F. VYSKOCIL~s~o . ondon 188, 53-64 (1967) . A . FELTZ and A . MALLART ~J .~~oT.-Lon own i$~5-92 (1971) . 375-386 (1974) . J . LAPA, E .Y . ALBUQUERQU a~ nâ J . DAl R . MILEDI and L.T . POTTER . Nature 233, 599-603 971 . C .W . PORTER, T .H . CHIU, J . WfE~k01,f~KT and E .A . BARNARD Nature (New Biol . 241, 3-7 (1973), 1~ALPER, J . LDIiY and J . SCHMIDT . FEBS Le tters_ . 48, 130-T32 (1974) .
