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PURIFICATION BY AFFINITY CHROHATOGRAPHYOF NICOTINIC AND HUSCARINIC HYDROPHOBIC PROTEINS SEPARATED BY SEPHADEX LH20. F.J. Barrantes, Sonia A r b i l l a , Har~a C. L. de Carlin and E. De Robertis I n s t i t u t o de Biologra Celular. Facultad de Hedicina, Buenos Aires,ArgeNtina.

Received January 2,1975 SUHMARY Hydrophobic protein f r a c t i o n s , having n i c o t i n i c or muscarinic binding properties were separated respectively from skeletal muscle and smooth intest i n a l muscle using the l i p o p h i l i c gel Sephadex LH20. Further p u r i f i c a t i o n of the s p e c i f i c f r a c t i o n s was carried out by a cholinergic a f f i n i t y column, using e l u t i o n with organic solvents, followed by a pulse of IO-~H acetylchol i n e . The results obtained confirm the v a l i d i t y of the Sephadex LH20 technique for the i s o l a t i o n and binding of cholinergic proteins proposed by De Robertis et al (4) and discard the c r i t i c i s m s raised by Levinson and Keynes (7). INTRODUCTION Special hydrophobic protein f r a c t i o n s were isolated from Torpedo and Electrophorus electroplax (1) and skeletal muscle (2) and were shown to have n i c o t i n i c binding properties. Furthermore a cholinergic protein f r a c t i o n of muscarinic type was isolated from i n t e s t i n a l smooth muscle (3). The standard procedure has been to extract the tissue with organic solvents and, a f t e r incubation with the s p e c i f i c ligand, to load i t on a column made with the l i p o p h i l i c gel Sephadex LH20 (4). This is followed by e l u t i o n with chloroform and mixtures of chloroform-methanol of increasing p o l a r i t y . In every case several protein f r a c t i o n s were separated, of which, a single s p e c i f i c peak bound the cholinergic ligand. The free drug was retained by the column or was eluted in the most polar regions of the chromatogram. For binding studies, another method used in our laboratory has been a biphasic p a r t i t i o n system in which the ligand is placed in the aqueous phase and the two phases are s t i rred u n t i l binding equilibrium is reached (5). In spite of a l l the published evidences for various receptor proteins (6)

Copyright © 19 75 by Academic Press, Inc. All rights o f reproduction in any form reserved.

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and disregarding the r e s u l t s obtained by p a r t i t i o n , Levinson and Keynes (7) have c r i t i c i z e d the binding method using Sephadex LH20, suggesting that the association of the ligand with the protein was a r t i f a c t u a l .

In the present

paper, in addition of showing that hydrophobic cholinergic proteins, having n i c o t i n i c and muscarinic binding properties, can be p u r i f i e d by a f f i n i t y chromatography, the value of the Sephadex LH2D method (4) is f u l l y confirmed. EXPERIMENTAL Lyophilised rat diaphragms were extracted with chloroform-methanol (2:1) (2).

The c i r c u l a r layer of bovine i n t e s t i n e was dissected (3) and extracted

without i y o p h i l i s a t i o n ; the t o t a l l i p i d e x t r a c t (TLE) was washed with theoret i c a l upper phase (8).

The extracts were submitted to column chromatography

on a Sephadex LH20 column and the protein f r a c t i o n s , that bind the c h o l i n e r gic drugs, were separated (Figs. 1A and 2A). The a f f i n i t y chromatography system consisted of Sephadex LH20, a spacer am -3,3'-iminobispropylamine-, and a quaternary ammonium compound p-phenyltrimethylammonlum of recognized cholinergic characte~9)covalently linked to the free end of the spacer arm (10). The column of 17 x 1.7 cm was e q u i l i b r a ted with chloroform and the material ( i . e . the previously isolated f r a c t i o n s ) was loaded onto the column in 2.5 ml samples. Eiution of the non-specific proteins was carried out by the use of chloroform and mixtures of chloroformmethanol of increasing p o l a r i t y . When no f u r t h e r UV absorbing material was detected at 280 nm~ a pulse of 10-3M acetylcholine in 5 ml of chloroformmethanol (4:1) was applied. The e l u t i o n was continued with the sane solvent mixture u n t i l

the s p e c i f i c cholinergic protein f r a c t i o n was eluted (Figs.lB

and 2B). A detailed description of the technique of cholinergic a f f i n i t y chromatography

in organic solvents is being published elsewhere (11).

RESULTS AND DISCUSSION Fig. 1A shows the t y p i c a l pattern previously described f o r skeletal muscle (2). The second protein peak (shaded area) is the s p e c i f i c one that binds

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F~No.o, o r m Cf, 0.2 E r-

CM

t

15:1 ' IO:l t 6:1'

4,.I

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I

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specific

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Two-step separation of the cholinergic binding protein from rat diaphragm (A) Conventional chromatography on Sephadex LH20 of a TLE corresponding to 200 mg of lyophilised tissue. (B) A f f i n i t y chromatography in which the specific peak of (A) ( i . e . shaded area) corresponding to 1 g of lyophilised tissuewas used. This was equivalent to 32 hemidiaphragms. The specific cholinergic binding protein ( i . e . receptor peak) is eluted after the IO-3H acetylcholine pulse (see the description in the t e x t ) ,

various n i c o t i n i c

drugs,

including[3HJoL-bungarotoxinf"

average o f 200~g p r o t e i n / g fresh t i s s u e .

(12).

It contains an

Fig. 1B shows the r e s u l t of a f f i n i t y

chromatography. The large n o n - s p e c l f i c peak contains most of the p r o t e i n loaded onto the column. The s p e c i f i c p r o t e i n ( i . e .

receptor peak), eluted

a f t e r the a c e t y l c h o l i n e pulse, contains 13 pg/g fresh t i s s u e . Since the recovery is 95~, t h i s amount corresponds to a 15.4 f o l d enrichement over the preceding step.

I f we consider that the t o t a l

p r o t e i n content in rat d i a -

phragm is about 200 mg/g fresh tissue ¢he t o t a l enrichement fraction

of t h i s p r o t e i n

is in the range of 15.000 f o l d , Upon rechromatography on Sephadex

LH20, in the presence of 10"6H [14C 1 a c e t y l c h o l i n e

iodide (Amersham 2.4B Ci/

mole), the n o n - s p e c i f i c f r a c t i o n did not show binding,

196

indicating

that the

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Chloroform

CM

115:1 ~10:1 f 6 : 1 '

4:1

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iChlo, form

60

80

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CM

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120

140

160

180 mJ

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0.4 o 0.3 eJ UJ

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20

Fig. 2

40

60

80

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120 mJ

Two-step separation of the muscarinic cholinergic binding protein Fraction from the c i r c u l a r layer of the bovine i n t e s t i n e . (A) Conventional chromatography on Sephadex LH20 carried out with a TLE of 16g fresh tissue. B) A f f i n i t y chromatography done with Four s p e c i f i c peaks of (A). The chol~nergic Fraction ( i . e . receptor peak) is eluted a f t e r the IO-'M acetylcholine pulse (see the description in the t e x t ) .

s p e c i f i c cholinergic binding protein had been completely removed by the affinity

column. On the other hand, a f t e r elimination of the excess of a c e t y /

choline from the receptor

peak by Sephadex LH20 chromatography, the binding

of [14C] acetylcholine to this f r a c t i o n was demonstrated by a new rechromaLography on Sephadex LH20; Fig. 3 shows the single sharp peak of protein that appears in coincidence with the radioactive I igand in the chloroform.

In

this experiment as well as in the c o n t r o l , without protein, the f r e e [ 1 4 C J acetylcholine is eluted in the more polar solvents. Similar results were obtained using as n i c o t i n i c

l igand 8/ug of /3Hl0~-bungarotoxin (2Ci/mmole)

kindly provided by Prof. E.A. Barnard. The binding of [3Hlc~-bungarotoxin was also demonstrated by the p a r t i t i o n method of Weber et a1.(5).

197

I t should

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: : E 280nm 0 - - - 4 4 C ] ACh IO-6M

7OO 60O 5OO

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Rechromatography on Sephadex LH20 of the receptor peak from rat diapbraqln (see Fig. 1B), after incubation for 30 min with lO-°R ~14C] acetylcholine. The specific r a d i o a c t i v i t y reached in this experiment was 5 nmoles/mg protein.

be emphasized that [][3HJ~-bungarotoxln

has its p a r t i t i o n coeficient 100~

favorable to water. In the case of the TLE of smooth intestinal muscle two peaks Of protein were obtained by eiution with chloroform using conventional chromatography (Fig.2A), instead of the three peaks previously found in the l~ophilised tissue (3).

Here the second peak (shaded area) is the specific one that binds

[14C] acetylcholine and [3H] atropine (Amersham, 434 Ci/mole). This protein fraction contains 95 Pg protein/g fresh tissue. After a f f i n i t y chromatography the non-specific protein is e|uted in two peaks (Fig.2A) and the specific fraction, eluted by the acetylcholine pulse, represents only 23~ of the protein applied to the column.

In this receptor peak 22 #g protein/g fresh

tissue were estimated, which corresponds to a 4.3 fold p u r i f i c a t i o n over the preceding step and a total enrichment of the specific protein of about 4,500 fold ( i . e . considering the total protein content to be about tOO mg/g protein) Upon rechromatography on Sephadex LH20 the non-specific fraction showed no binding for cholinergic drugs, while in the receptor peak the binding of ~4C]acetylcholine and of I, 3 ~ a t r o p i n e was demonstrated ) L

#

198

(Fig.4). Ochoa and

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E 2 8 0 nm

,,~ / I

5000

, 2000

,L

!

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CL L)

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/

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Rechromatography on Sephadex LH20 of the muscarinic binding protein from i n t e s t i n a l smooth muscle ~see Fig 2B), a f t e r 30 min. incubation with 1.07 x IO-QM [ 3 H l a t r o p i n e . The s p e c i f i c r a d i o a c t i v i t y reached at this low drug concentration was 0.25 nmoles/mg protein.

De Robertis (3) has previously shown that the muscarinic protein from smooth muscle does not bind [ 1 4 C ] i - t u b o c u r a r i n e . Present results demonstrate that the chollnerglc a f f i n i t y zed by Barrantes (10)

column,synthesi-

is capable of separating both n i c o t i n i c and muscarinic

proteins in organic solvents and is able to improve on the p u r i f i c a t i o n obtained by conventional chromatography in Sephadex LH20. Furthermore since acetylcholinesterase is not soluble in organic solvents (13), this technique only separates the chollnergic receptor protein f r a c t i o n . The use of a cholinergic a f f i n i t y p r i n c i p l e s as those of a f f i n i t y

column, which works on the sa=ne general

chromatography in aqueous solvents (11),

confirms the value of the conventional technique based on Sephadex LH20 for the separation of receptor proteins and the study of the binding with specif i c ligands (4).

Barrantes and Weber (11) have shown that the a f f i n i t y

column is able to remove e n t i r e l y the chollnergic protein from the TLE of El ectropho[us e l e c t r o p l a x .

In a work in progress, Saraceno and De Robert|s

(14) have observed that the cholinerglc protein f r a c t i o n from the TLE of the cerebral cortex is also completely removed by the a f f i n i t y

199

column and i t is

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BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

only released by the acetylcholine pulse.

These observations demonstrate

that the cholinergic protein fractions were present in the TLE of Electrophorus and cerebral cortex.

Even more conclusive is the finding shown here that

the s p e c i f i c fractions isolated by Sephadex LH20 chromatography from skeletal and smooth i n t e s t i n a l muscle could be f u r t h e r p u r i f i e d by the a f f i n i t y

column.

The active fractions obtained by Sephadex LH20 chromatography (Flgs.lA and 2A) were f u r t h e r separated into non-speCific and receptor f r a c t i o n s ; the l a t t e r being eluted a f t e r the acetylcholine pulse (Figs. 1B and 2B). The use of the Sephadex LH20 technique under the same technical conditions, permitted to show that the non-speciflc fractions did not bind the c h o l i n e r gic ligands, while the receptor fractions were able to bind the corresponding specific ligand, i . e . e i t h e r n i c o t i n i c or muscarinic drugs. Furthermore, in these fractions the binding was carried out at drug concentrations small enough (10-8M to 10"6M) to minimize any kind of non-specific binding. The methodologic approach used, in which two d i f f e r e n t methods of chromat~ graphy, produce a d d i t i v e results regarding the p u r i f i c a t i o n of the active protein, permit to discard completely the c r i t i c i s m s Keynes (7).

raised by Levinson and

We suppose that the drastic changes introduced by the authors

in the Sephadex LH20 technique of De Robertis et al (4) were probably the cause of t h e i r a r t i f a c t s .

A complete analysis of the Sephadex LH20 technique,

that confirms our findings, has been carried out by J.F. Donnellan, K.J. Cattel and coworkers, at the Shell laboratory in the United Kingdom, in their study of the hydrophobic cholinergic receptor protein of the f l y head (Personal communication). Thus the value of this

method for the separation

of cholinergic receptor proteins and for the study of the binding with specific ligands is f u l l y confirmed. ACKNOWLEDGMENTS We express our gratitude to Prof. Gregorio Weber for c r i t i c a l l y reviewing this manuscript and for his valuable suggestions. This work has been supported by Grant from the National I n s t i t u t e s of Health (5 ROI NS 06953-09 NEUA) U.S.A.

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REFERENCES

1. 2. 3. 4,

5. 6. 7. 8. 9. 10. 11. 12.

13. 14.

La Torre, J . L . , Lunt, G.S., and De Robertis,E.(1970) Proc. Nat.Acad.Sci. U.S., 655, 716. Lunt, G.G., Stefani,E., and De Robertis,E.(1971) J. Neurochem. 18,1545. Ochoa, E.L.M., and De Robertis,E.(1973) Biochem. Biophys.Acta 2 ~ , 528. De Robertis, E., Fiszer,S., Pasquini,J., and Sote, E.F.(1969) J, Neurobiol. i, 41. Weber,G., Borris,D.P., De Robertis,E., Barrantes,F.J., La Torte J.L., and de Carlin H.C.l. (1971) Molecular Pharmacol. 7_, 530 (1971). De Robertls,E.,(1971) Science 171, 963. Levinson, S.R., and Keynes, R.D.,(1972) Biochem.Biophys.Acta 288,241. Folch-Pi,J., Lees, H., and Sloane-Stanley,B.H.(1957) J. Biol. Chem.2.26, 497. Berman, J.D. and Young, H. (1971). Proc. Nat. Acad. Sci. US. 68, 395. Barrantes, F.J. (1973) Proc. 9th Int. Cong. Biochem. Stockholm--p.443. Barrantes, F.J. and Weber, G.,Biochemistry (1975) (in press). De Robertis,E., Mosquera, H.T., and Fiszer de Plazas, S. (1972) Life Sci. 11, 1155. Iz---umi,F., and Freed, S. (1974) FEBS Letters 41, 151. Saraceno, H., and De Robertis, E. (unpublished-)-.

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