Japan. J. Pharmacol.25, 693-707(1975)
SEPARATION
OF CHOLINERGIC
693
PROTEOLIPIDS
FROM
QUENCHED RAT CEREBRAL CORTEX AND RESOLUTION OF PROPERTIES AT LOW TEMPERATURE Futoshi IZUMI* and Simon FREED Departmentsof Biochemistry and Neurology, New YorkMedicalCollege , NewYork, N. Y. 10029, U.S.A. and Departmentof Chemistry, Brookhavven NationalLaboratory,Upton,N.Y. 11973,U.S.A. AcceptedSeptember3, 1975
Abstract-Proteolipidsfrom rat cerebralcortex quenchedat -196 C were extracted and fractionated at subzero temperature(-60`C). On SephadexLH-20 column chromatography,acetylcholineand cholinergicblockingagents such as dimethyl-d tubocurarineand decamethoniumwere observedto bind to differentfractionsof pro teolipidsshowingthat the receptorfractionsfor acetylcholineand that for cholinergic blockersneed not be the same. The acetylcholinesterase (EC 3.1.1.7)activity was demonstratedto be absent in both types of receptor fraction. The specificitiesof bindingbycholinergicsubstancesto proteolipidfractionspreparedat 60°C persisted at room temperaturewithsomeloss in specificityfor acetylcholine. Sincethesespeci ficities were not observedin previouslyreported experimentsat room temperature, it appears that the structuresof proteolipidsextractedat the two temperaturesdiffer. The appearanceof specificities in the proteolipidspreparedat low temperaturesuggest that their structuresare in better approximationto thosein vivothat presumablyare highlyspecific. The importanceof theproteinmoietyof proteolipidsas a discriminator for the neurotransmitteris discussed. Special hydrophobic proteolipids extractable with chloroform-methanol
(2:1 v/v) have
been reported to be found in large amounts in brain white and gray matter by Folch-Pi and Lees (1). De Robertis and his associates have demonstrated that proteolipids from mammalian brain showed high affinities for binding drugs affecting synaptic transmission, such as dimethyl-d-tubocurarine blocking agents (5, 6).
(2), atropine (3), serotonin (4) and several adrenergic
Such proteolipids were also isolated and purified from the electric
organ of Torpedo and Electrophorus (7) and were shown to bind acetylcholine and cholinergic drugs, being designated as "receptor proteolipids".
But this solvent for the extraction of
proteolipids is well known for its modification of protein structure, not infrequently to the extent of irreversible denaturation. To obtain preparations of proteolipids in better approximation extraction of the brain tissue, previously quenched at -196'C
to in vivo structure,
was carried out at -60'C.
The binding abilities of such proteolipids for acetylcholine and cholinergic blocking agents were examined at low temperature and the characteristics of the proteolipids as the possible cholinergic receptor fractions are discussed. Present address: Department of Pharmacology 11, Osaka University School of Medicine, 33, Joancho, Kitaku, Osaka, Japan, 530. Reprint requests should be sent to this address.
694
F. IZUMI & S. FREED At the same time, the authors would like to introduce the technical details of low
temperature which could be applied to studies in pharmacology. MATERIALS AND METHODS Preparation of protcolipids fi-oiia rat cerebral cortex at low temperature The cerebral cortex of Sprague-Dawley rats was quenched at -196 'C in liquid nitrogen and pulverized at this temperature. ature).
The following procedures
chromatography, at -60--C.
Fic,. 1.
The brain powder was stored at -78 °C (dry ice temper were all carried out below -60'C
except column
Extraction of tissue to acquire a total lipid extract was done
Diagram of filter for use at subzero tremperature. Container and well were precooled in dewar flask with dry ice (in methanol) to desired temperature ( 60 C). Samples were applied in the well and then filtered slowly under low vacuum. The filtrate was recovered in the container.
CHOLINERGIC
PRO TEOLIPIDS
695
FIG. 2.
Diagram of an apparatus for molecular distillation at subzero temperature. Samples for molecular distillation were put in the container precooled with dry ice as in Fig. 1. After connecting to a vacuum line which was driven by an oil pump and mercury diffusion pump, the 0-ring valve was opened slowly. The well was then filled with liquid nitrogen, thus getting a high vacuum. Using this ap paratus, 1 I of chloroform-methanol (2:1 v `v) was completely dried up in a day at 60 3C.
with chloroform-methanol
(2:1 v/v, 100 ml for 100 mg of brain powder).
was carried out for 20 hours with mixing.
The extraction
The extract was freed from residue by filtration
(Fig. 1). The organic solvent was completely removed under high vacuum by molecular distillation (Fig. 2). The dried material was dissolved with chloroform-methanol (2:1 v/v) and was used as total lipid extract for chromatography teolipids.
and for further isolation of pro
Proteolipids were precipitated from the total lipid extract by adding twice its
volume of diethyl ether (9).
The precipitate was gathered by centrifugation and the organic
solvents was removed under high vacuum. chloroform-methanol
The proteolipids were finally dissolved with
(2:1 v/v) and allowed to react with acetylcholine and cholinergic
blocking agents. Preparation of ' Sephadex LH-20 column Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala) previously washed at room temperature
according to the instruction of the manufacture
except that methanol was
substituted for acetone, was rinsed in pure chloroform (500 ml for 50 g of Sephadex LH-20) and allowed to stand for 5 hours at room temperature for complete swelling. cooled down to -60°C
It was then
with gentle stirring under low vacuum thus minimizing the dis
solution of air into the liquid at this temperature.
The cooled Sephadex LH-20 gel was
packed into the column which was put in the refrigerator cabinet (Model ULT 657, Revco) adjusted at 60"C. To acquire the homogenous packing, the gel was first transferred to the flask attached to the top of the column and packed slowly with the aid of low vacuum.
696
E IZUMI & S. FREED
On the top of the gel, 2 cm of ignited sea sand was put to avoid the floating of gel in pure chloroform.
The column was washed with 150 ml of chloroform and allowed to stand
overnight to get a constant flow rate.
The column was freshly prepared for each experiment
and the one used in this experiment had the dimensions, 1.8 cm x 45 cm and the gel was packed to a height of 40 cm. The outlet of the column made of glass tubing (2 mm caliber) ascended to a height 10 cm below the top of the gel. For the eluants, the fraction collector (ISCO Model 272) was also put in the refrigerator cabinet.
For satisfactory running of
the fraction collector, all grease at the gears was removed and a small electric heater was placed below the operating unit thermally insulated from cabinet air space. Column chromatography at low temperature (-60°C) Column chromatography at low temperature was used to detect the bindings of acetyl choline and cholinergic blocking agents to proteolipids preparation.
Proteolipids (con
taining about 5 mg of protein) dissolved in 5 ml of chloroform-methanol incubated
(2:1 v/v) were
with 14C-acetylcholine (5X10-8 moles), dimethyl-14C-d tubocurarine
(2X10-1
moles), 14C-decamethonium (2,,
SEPARATION
OF CHOLINERGIC
693
PROTEOLIPIDS
FROM
QUENCHED RAT CEREBRAL CORTEX AND RESOLUTION OF PROPERTIES AT LOW TEMPERATURE Futoshi IZUMI* and Simon FREED Departmentsof Biochemistry and Neurology, New YorkMedicalCollege , NewYork, N. Y. 10029, U.S.A. and Departmentof Chemistry, Brookhavven NationalLaboratory,Upton,N.Y. 11973,U.S.A. AcceptedSeptember3, 1975
Abstract-Proteolipidsfrom rat cerebralcortex quenchedat -196 C were extracted and fractionated at subzero temperature(-60`C). On SephadexLH-20 column chromatography,acetylcholineand cholinergicblockingagents such as dimethyl-d tubocurarineand decamethoniumwere observedto bind to differentfractionsof pro teolipidsshowingthat the receptorfractionsfor acetylcholineand that for cholinergic blockersneed not be the same. The acetylcholinesterase (EC 3.1.1.7)activity was demonstratedto be absent in both types of receptor fraction. The specificitiesof bindingbycholinergicsubstancesto proteolipidfractionspreparedat 60°C persisted at room temperaturewithsomeloss in specificityfor acetylcholine. Sincethesespeci ficities were not observedin previouslyreported experimentsat room temperature, it appears that the structuresof proteolipidsextractedat the two temperaturesdiffer. The appearanceof specificities in the proteolipidspreparedat low temperaturesuggest that their structuresare in better approximationto thosein vivothat presumablyare highlyspecific. The importanceof theproteinmoietyof proteolipidsas a discriminator for the neurotransmitteris discussed. Special hydrophobic proteolipids extractable with chloroform-methanol
(2:1 v/v) have
been reported to be found in large amounts in brain white and gray matter by Folch-Pi and Lees (1). De Robertis and his associates have demonstrated that proteolipids from mammalian brain showed high affinities for binding drugs affecting synaptic transmission, such as dimethyl-d-tubocurarine blocking agents (5, 6).
(2), atropine (3), serotonin (4) and several adrenergic
Such proteolipids were also isolated and purified from the electric
organ of Torpedo and Electrophorus (7) and were shown to bind acetylcholine and cholinergic drugs, being designated as "receptor proteolipids".
But this solvent for the extraction of
proteolipids is well known for its modification of protein structure, not infrequently to the extent of irreversible denaturation. To obtain preparations of proteolipids in better approximation extraction of the brain tissue, previously quenched at -196'C
to in vivo structure,
was carried out at -60'C.
The binding abilities of such proteolipids for acetylcholine and cholinergic blocking agents were examined at low temperature and the characteristics of the proteolipids as the possible cholinergic receptor fractions are discussed. Present address: Department of Pharmacology 11, Osaka University School of Medicine, 33, Joancho, Kitaku, Osaka, Japan, 530. Reprint requests should be sent to this address.
694
F. IZUMI & S. FREED At the same time, the authors would like to introduce the technical details of low
temperature which could be applied to studies in pharmacology. MATERIALS AND METHODS Preparation of protcolipids fi-oiia rat cerebral cortex at low temperature The cerebral cortex of Sprague-Dawley rats was quenched at -196 'C in liquid nitrogen and pulverized at this temperature. ature).
The following procedures
chromatography, at -60--C.
Fic,. 1.
The brain powder was stored at -78 °C (dry ice temper were all carried out below -60'C
except column
Extraction of tissue to acquire a total lipid extract was done
Diagram of filter for use at subzero tremperature. Container and well were precooled in dewar flask with dry ice (in methanol) to desired temperature ( 60 C). Samples were applied in the well and then filtered slowly under low vacuum. The filtrate was recovered in the container.
CHOLINERGIC
PRO TEOLIPIDS
695
FIG. 2.
Diagram of an apparatus for molecular distillation at subzero temperature. Samples for molecular distillation were put in the container precooled with dry ice as in Fig. 1. After connecting to a vacuum line which was driven by an oil pump and mercury diffusion pump, the 0-ring valve was opened slowly. The well was then filled with liquid nitrogen, thus getting a high vacuum. Using this ap paratus, 1 I of chloroform-methanol (2:1 v `v) was completely dried up in a day at 60 3C.
with chloroform-methanol
(2:1 v/v, 100 ml for 100 mg of brain powder).
was carried out for 20 hours with mixing.
The extraction
The extract was freed from residue by filtration
(Fig. 1). The organic solvent was completely removed under high vacuum by molecular distillation (Fig. 2). The dried material was dissolved with chloroform-methanol (2:1 v/v) and was used as total lipid extract for chromatography teolipids.
and for further isolation of pro
Proteolipids were precipitated from the total lipid extract by adding twice its
volume of diethyl ether (9).
The precipitate was gathered by centrifugation and the organic
solvents was removed under high vacuum. chloroform-methanol
The proteolipids were finally dissolved with
(2:1 v/v) and allowed to react with acetylcholine and cholinergic
blocking agents. Preparation of ' Sephadex LH-20 column Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala) previously washed at room temperature
according to the instruction of the manufacture
except that methanol was
substituted for acetone, was rinsed in pure chloroform (500 ml for 50 g of Sephadex LH-20) and allowed to stand for 5 hours at room temperature for complete swelling. cooled down to -60°C
It was then
with gentle stirring under low vacuum thus minimizing the dis
solution of air into the liquid at this temperature.
The cooled Sephadex LH-20 gel was
packed into the column which was put in the refrigerator cabinet (Model ULT 657, Revco) adjusted at 60"C. To acquire the homogenous packing, the gel was first transferred to the flask attached to the top of the column and packed slowly with the aid of low vacuum.
696
E IZUMI & S. FREED
On the top of the gel, 2 cm of ignited sea sand was put to avoid the floating of gel in pure chloroform.
The column was washed with 150 ml of chloroform and allowed to stand
overnight to get a constant flow rate.
The column was freshly prepared for each experiment
and the one used in this experiment had the dimensions, 1.8 cm x 45 cm and the gel was packed to a height of 40 cm. The outlet of the column made of glass tubing (2 mm caliber) ascended to a height 10 cm below the top of the gel. For the eluants, the fraction collector (ISCO Model 272) was also put in the refrigerator cabinet.
For satisfactory running of
the fraction collector, all grease at the gears was removed and a small electric heater was placed below the operating unit thermally insulated from cabinet air space. Column chromatography at low temperature (-60°C) Column chromatography at low temperature was used to detect the bindings of acetyl choline and cholinergic blocking agents to proteolipids preparation.
Proteolipids (con
taining about 5 mg of protein) dissolved in 5 ml of chloroform-methanol incubated
(2:1 v/v) were
with 14C-acetylcholine (5X10-8 moles), dimethyl-14C-d tubocurarine
(2X10-1
moles), 14C-decamethonium (2,,
