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Demonstration of an endogenous, competitive inhibitor(s) of [3H] diazepam binding in bovine brain
Pergamon Press
Life Sciences Vol . 22, pp . 1893-1900 Printed in the II .S .A .
DEMONSTRATION OF AN ENDOGENOUS, COMPETITIVE INHIBITOR S) OF [sli] DIAZEPAM BINDING IN BOVINE BRAIN P .J . Marangos + , S .M . Paul, P . Greenlaw, F .K . Goodwin and P . Skolnick National Institute of Mental Health and National Institute on Alcohol Abuse and Alcoholism 9000 Rockville Pike Bethesda, Maryland 20014 (Received in final form April 13, 1978) SUMMARY An endogenous inhibitor s) of [~li] diazepam binding to syhaptosomes has been demonstrated in bovine brain . The inhibitory activity of crude extracts is heat stable, dialyzable, and not affected by ether extraction . Three distinct peaks of inhibitory activity were resolved using Sephadex G-25 chromatography . The lowest molecular weight peak (<700 daltons) had the highest specific inhibitory activity and its inhibition of [~li] diazepam binding was competitive . A similar low molecular weight fraction was not observed in either muscle or liver suggesting that it may be unique to brain . Thin layer chromatography of the Sephadex G-25 fractions revealed a discrete band of inhibitory activity in the two low molecular weight peaks . Although the benzodiazepines are among the most widely used psychotherapeutic agents, their mechanism s) of action are still obscure (1) . Recently, a high affinity, stereospecific, saturable binding site for benzodiazepines has been reported in the mammalian central nervous system (CNS) (2-6) . In addition, high correlations have been demonstrated between the binding affinities of a series of benzodiazepine derivatives and their potencies as muscle relaxants (2), anxiolytics (7), and anti-convulsants (7), suggesting that this site may be a pharmacologic receptor mediating benzodiazepine action in the CNS . Since a number of putative neurotransmitters and small peptides failed to inhibit [ ~] diazepam binding to these rece for sites (6), the presence of an endogenous ligand has been ostulated (2~ . We now report the presence of a competitive inhibitor s) of g ~i] diazepam binding in extracts of bovine brain . The characteristics of this inhibitor s) are similar to those that might be associated with an endogenous ligand to the benzodiazepine receptor . METHODS Tissue re oration : Bovine tissue was obtained immediately after sacrifice an qu cc rozen on dry ice . The frozen tissue was fragmented and homogenized in 3-5 volumes of acidified acetone (0 .2 ml concentrated HC1/liter) + o w om reques s
or repr nts s ou
e mailed .
1894
Endogenous Inhibitor of Diazepam Binding
Vol . 22, No . 21, 1978
using a Waring blender . The resulting Homogenate was stirred for four hours at room temperature and filtered under vacuum (Whatman #1 pager) . The residue retained on the filter . was re-extracted in 3 tissue volumes of an acetone-H 2 0 mixture (80 :20) for four hours and filtered . The pooled filtrates of the This above extractions were evaporated under vacuum to the aqueous phase . cloudy solution was centrifuged at 25,000 x g for 20 minutes and the supernatant was filtered and lyophilized . The resulting residue was suspended in methanol (1 ml/gram tissue) and stirred for one hour at room temperature . The resulting residue was dissolved in distilled water (1 ml/lOg tissue) and centrifuged at 10,000 x g for 20 minutes . The dense lipid-like layer at the top was discarded and the remaining clear solution extracted with 5 volumes of petroleum ether . The aqueous phase was then lyophilized . This powder was resuspended in 1 .OmM Tris-HC1 buffer pH 7 .4 (1 ml/30 grams tissue) and referred to as the crude extract . Assa for inhibition of Eli diaze am bindin : Tissue extracts were assayed or e r a i ty to in n ng o diazepam (New England Nuclear, it Boston, Mass ., Sp . Act . 39 Ci/mM) to a crude synaptosomal preparation of rat cerebral cortex by a modification of the method of Mohler and Okada (4) . The synaptosomal f~~action was prepared as previously described (2) . The assay mixture contained approximately 1 mg of synaptosomal protein, 30 or 50mM Tris-HC1 buffer pH 7 .4 and 3nM [~-I diazepam in a final volume of 1 .5 of 1 ml . Quantities of tissue fractions of up to 0 .5 ml were incorporated in the above system to assay for the inhibition of specific [sli] diazepam binding . Non-specific binding was determined by inclusion of 3uM unlabeled diazepam (Hoffmann-LaRoche) and was routinely less than 10% of total binding . Incubations were carried out on ice for 15 minutes followed by filtration and washing (10 ml ice cold 50mM Tris-HC1 pH 7 .4) using Whatman GF/B filters . The filters were counted after being thoroughly mixed in 10 ml of Aquasol (New England Nuclear, Boston, Mass .) . Under these conditions the apparent affinity (Kd) of diazepam binding is 3 .5nM which is in agreement with values obtained in other laboratories (2-6) . The ability of various fractions to inhibit [~li] diaze am binding was expressed as units of diazepam-like activity (DLA (Table I~ . Standard curves were generated for each synaptosomal preparation by incorporating increasing amounts of unlabeled diazepam into the above assay system . Linear semi-log plots were obtained in the rànge of 1-50 pmoles when percent inhibition of [ sH] diazepam binding was plotted versus the concentration of unlabeled diazepam . A unit of DLA activity is defined as that amount of inhibitory activity equivalent to 1 pmole/assay of diazepam . De termination of [ ~i] D-ala-met-enkephalin and ç ~I~ dihydroalorenolol bindin s~pec~f c~ind~ng o e b~Ti-ty~ gums 6-r~n repara ions to n ib~ â D-ala-met-enkephalin and [~i~ dihydroalprenolol was determined as previously described (8,9) . Protein determinations : The protein or peptide content of various fractions was ethem ne y t e method of Lowry et al (10) using bovine serum albumin as standard . Both small proteins and pépi~des are measured with this technique . Chromato ra hic rocedures : Sephadex G-25 fine (Pharmacie) was, used in all ge tra on exper ments . Columns (2 .5 x 100 cm) were equilibrated in 1 .OrnM Tris -HC1 buffer pH 7 .4 and run at 4° at a flow rate of 10 ml/hour .
Vol . 22, No . 21, 1978
Endogenous Inhibitor of Diazepam Binding
1895
Thin-layer chromatography (TLC) wâs performed on 20 x 20 cm silica gel plates (Kieselgel F-254 E . Merck, Darmstadt) developed in chloroform : methanol : acetic acid (80 :40:11) . The plates were viewed with a mineralite (UV), scraped in 1 cm bands and the scrapings eluted with 1 ml of 1mM HC1 . The HCL extract was lyophilized and reconstituted in 400 ul of 50rtiM TrisHC1 buffer pH 7 .4 and assayed for inhibition of [~li] diazepam binding. RESULTS Inhibition of [ ~-I] diazepam binding by tissue fractions : The relative potenc es o cru e extracts of ovine ra n an T~ve -fin inhibiting [~li] diazepam binding to synaptosomal membranes are shown in Table I . Crude brain extract is approximately 10 times more potent than the equivalent liver extract. The inhibitory activity of crude brain extracts was not affected by boilinç~ for 10 minutes or by extraction with diethylether . However, complete loss of activity was observed following dialysis, indicating a molecular weight of less than 5000 daltons . Incubation of bovine brain at 37°C for 16 hours prior to preparation of the crude extract markedly reduced the specific activity (Table I) .
Table I Diazepam Binding Inhibitory Activity of Various Tissue Fractions FRACTION Brain crude) Liver crude) Brain (incubated) Sephadex Pooled Peaks I II III
UNITS DL~mg LRM 13 .1 1 .35 2 .70 6 .60 33 .5 155 .0
ac o t e in ca e tissue extracts was a ust to risp prior to assay. Inhibition of diazepar~ binding and Lowry reactive material (LRM) were determined as described in Methods . Brain (incubated) refers to the resulting crude extract from bovine brain incubated at 37° for 16 hours prior to preparation . Chromatographic resolution of bovine brain extract on Sephadex r,-25 yields three distinct peaks of inhibitory activity (Figure 1) . The specific activities of each pooled Sephadex peak are shown in Table I . The greatest increase in specific inhibitor activity was consistently observed in the third (lowest molecular weight peak . Similar fractionation of a bovine liver extract yielded no discernable pattern of inhibitory activity, suggesting that inhibition of diazepam binding observed for the crude liver preparation was non-specific .
1896
Endogenous Inhibitor of Diazepam Biadiag
Vol . 2 2, No . 21, 1978
% INMIOITIOM OF ~INDINO
/N/ml LRhI (-_-_)
F~ure 1 Sephadex G-25 chromatography of bovine brain extract : Fifteen ml of bovine brain extract resulting from the preparation of 800 grams of tissue was applied on a 2 .5 x 100 cm Sephadex G-25 column equilibrated in 1mM Tris-HC1 buffer pH 7 .4 . Fractions were collected (2 .5 ml) and assayed for both inhibition of [ ~] diazepam binding (400 ul) and Lowry reactive material . The void volume of the column was at tube 42 .
Kinetic anal sis of ak III inhibitor activit : Double reciprocal analysis o ono azepam n ng n t e presence of two concentrae n tions of Peak III is shown in Figure 2 . The apparent binding affinity of diazepam decreased with increasing amounts of Peak III while maximum binding was unchanged, suggesting the inhibition was competitive .
Vol . 22, No . 21, 1978
Endogenous Inhibitor of Diazepam Binding
I
[FREE DIAZEPAM]
1897
(nMx10')
Fi~c u re 2 Double reciprocal analysis of Peak III inhibitory activity : The synaptosomal membrane preparation was assayed for specific binding with increasing amounts of [ 3H] diazepam in the absence and presence of 200 ul and 400 ul of the Se hadex Peak III fraction . Ordinate : B is the amount of specifically bound [ ~] diazepam (fmol/mg protein) as defined in Methods .
Thin-la er chromato ra h of Se hadex G-25 fractions : Thin-layer chromatograp is ana ys s o eac o t e ep a ex pea raco ons is illustrated in Figure 3 . Several discrete bands of U .~1 . absorbing material were observed in Peaks II and III while Peak I yielded a diffuse region of U .V . absorption near the origin . No inhibitory activity was observed in the TLC scrapings of Peak I, whereas Peak II and III had discrete bands of inhibitory activity co-migrating with U .V . absorbing material . The Rf values of the Peak II and Peak III inhibitory activity were 0 .47 and 0 .53, respectively .
1898
Eadogenoua Inhibitor of Diazepam Binding
Figure
Vol . 22, No . 21, 1978
3
Thin-layer chromatography of the Sephadex fractions : 300 ul of pooled fractions from each of the three Sephadex peaks were lyophilized, resuspended in 100 ul of methanol-water and spotted on TLC plates . The chromatograms were developed and assayed for inhibition of [ ~] diazepam binding inhibition as described in Methods . The hatched areas indicate U .V . absorbing material and the solid areas, diazepam binding inhibitory activity .
Effects of Se hadex fractions on o late and ~-adrener is rece tors : The y roa preno o to their n ng o -a a-me -en ep a n an respective receptors were significantly inhibited by Peak I (Table II) . Peak II did not significantly affect g~adrenergic binding while specific opiate binding was inhibited by 30% . Peak III appeared to be the most specific inhibitor of [~li] diazepam binding by these criteria since it displayed little or no inhibitory activity of either ~adrenergic or opiate receptor binding .
Vol . 22, No . 21, 1978
Endogenous Inhibitor of Diazepam Binding
1899
Table II Effects of Diazepam Binding Inhibiting Fractions On the ~Adrenergic and Opiate Receptor
Peak Fraction I II III
% Inhibition of Specific Binding [~i] Diazepam 9 .8 54 .2 37
[ ~I] Dihydroalprenolol 27 0 4
[ 3H] D-ala-met-enkephalin 100 30 0
2~0ü o t e pooTeâ péâc~frâct ons were assaye or n t on o ga binding using 3 .1nM [~i] diazepam and 1 .3nM [ ~] dihydroalprenolol . For the opiate assay 100 ul of each pooled Sephadex fraction was assayed in the presence of 0 .5nM [ 3 H] D-ala-met-enkephalin .
DISCUSSION The present report describes the partial purification of a substance s) from bovine brain which specifically inhibits the binding of [ ~i] diazepam to a crude synaptosomal fraction of rat cerebral cortex . A tissue extraction procedure was devised that minimizes proteolytic degradation and specifically selects for small molecules, since it was hypothesized that an endogenous ligand for the diazepam receptor would be a small molecule such as a peptide . The homogenization of frozen tissue in cold acidified acetone was selected since these conditions would rapidly inactivate degradatioe enzymes as well as cause the precipitation of most macromolecules . A methanol extraction was employed to reduce the amount of inorganic salts . The crude extract of brain contained the equivalent of 13 pmole of diazepam-like activity per milligram of Lowry reactive material while liver contained an order to magnitude less . Partial characterization of this inhibitors) of diazepam binding indicates that it has a low molecular weight (dialyzable), is non-lipid in nature (not extracted in ether) and heat stable (100° for 10 minutes) . Incubating brain at 37° before preparation of the crude extract led to a marked reduction in its inhibitory activity . However, incubation of the crude extract with trypsin, peptidase or protease failed to decrease inhibitory activity . These preliminary results suggest that the factors) are either non-peptide in nature or that more specific enzymes are required for inactivation . Chromatographic analysis of crude bovine brain extract resolves three discrete fractions . The estimated molecular weight of the three Sephadex fractions are : Peak I = 2000, Peak II = 1000, and Peak III <700 . The largest increase in specific activity was routinely observed for Peak III . Analysis of each Sephadex fraction by thin-layer chromatography indicated that both Peak II and Peak III contain discrete substance s) capable of inhibiting [ ~] diazepam binding . The loss of inhibitory activity observed following TLC of Peak I suggests that the inhibitory activity of this fraction prior to TLC was either labile under these conditions or of a non-specific nature .
1900
Endogenous Inhibitor of Diazepam Binding
Vol . 22, No . 21, .1978
The competitive inhibition of [~i] diazepam binding exhibited by the Sephadex Peak III material (Figure 2) suggests that the active factor is reacting with the same binding site as diazepam . In addition to having the highest specific activity, Peak III appears to be the most specific for diazepam receptors since it displayed little or no inhibition of ~adrenergic or opiate binding (Table II) . However, endorphin-like material has been reported in brain extracts purified by Sephadex G-25 chromatography with molecular weights corresponding to Peaks I and II (11) . These data demonstrate the existence of a low molecular weight, competitive inhibitory factor s) of [~i] diazepam binding in bovine brain . Both the amount of inhibitor present and the Sephadex G-25 chromatographic pattern-indicate that compared to a peripheral tissue (liver), this factor may be specific to the CNS . The observation that crude extracts of liver (Tab1e .I and unpublished observations) and muscle (unpublished observations) do have inhibitory activity, but that this activity is both qualitatively and quantitatively different from brain may well indicate that the inhibitory activity from these tissues is non-specific . Actual quantitation of tissue levels of this factors) and characterization as either an agonist or antagonist at the receptor must await purification and bioassay . The evidence suggests that at least two compounds exist in bovine brain that inhibit [ ~] diazepam binding . Further purification of these factors may yield a substance s) which function as an endogenous ligand for the benzodiazepine receptor . ACKNOWLEDGMENTS The authors wish to thank Dr . C . Pert for performing opiate receptor binding assays . P . Skolnick wishes to thank Dr . J . Daly, NIAMDD, for providing facilities during these studies . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 .
W . HAEFELY, A . KULCSAR, H . MOHLER, L . PIERI, P . POLC, and R . SCHAFFNER, in Mechanism of Action of Benzodiaze roes, Raven Press, New York, eds . os a an . reenga , p 5) . R .F . SQUIRES and C . BRAESTRUP, Nature 266 : 732 (1977) . C . BRAESTRUP and R .F . SQUIRES, Proc . Nat . Acad . Sci . 74 : 3805 (1977) . H . MOHLER and T . OKADA, Life Sci . 20 : 2101 (1977) . H .B . BOSMANN, K .R . CASE and P . DISTE~ANO, FEBS Letters 82 : 368 1977) . C . BRAESTRUP, R . ALBRECHTSEN and R . F . SQUIRES, Nature _2b~3 : 7021977) . H . MOHLER and T . OKADA, Science 198 : 849 (1977) . C .B . PERT and D .L . BOWIE, in Endo~ins in Mental Health Research, Macmillan Press, London, e s . . s n, . . unney, r ., and N .S . Kline, 1978, in press . P . SKOLNICK, L .P . STALVEY, J .W . DALY, T .W . STONE, and D . TAYLOR, Life Sci . 21 : 1655 (1977) . O .H . LOWRY, N .J~tOSEBR000H, A .L . FARR, and R .J . RANDALL, J . Biol . Chem . 193 :265 (1951) . A .L . Z`REUNG, W .B . STAVINHOA and A . GOLDSTEIN, Life Sci . 20 : 1285 (1977) .
Life Sciences Vol . 22, pp . 1893-1900 Printed in the II .S .A .
DEMONSTRATION OF AN ENDOGENOUS, COMPETITIVE INHIBITOR S) OF [sli] DIAZEPAM BINDING IN BOVINE BRAIN P .J . Marangos + , S .M . Paul, P . Greenlaw, F .K . Goodwin and P . Skolnick National Institute of Mental Health and National Institute on Alcohol Abuse and Alcoholism 9000 Rockville Pike Bethesda, Maryland 20014 (Received in final form April 13, 1978) SUMMARY An endogenous inhibitor s) of [~li] diazepam binding to syhaptosomes has been demonstrated in bovine brain . The inhibitory activity of crude extracts is heat stable, dialyzable, and not affected by ether extraction . Three distinct peaks of inhibitory activity were resolved using Sephadex G-25 chromatography . The lowest molecular weight peak (<700 daltons) had the highest specific inhibitory activity and its inhibition of [~li] diazepam binding was competitive . A similar low molecular weight fraction was not observed in either muscle or liver suggesting that it may be unique to brain . Thin layer chromatography of the Sephadex G-25 fractions revealed a discrete band of inhibitory activity in the two low molecular weight peaks . Although the benzodiazepines are among the most widely used psychotherapeutic agents, their mechanism s) of action are still obscure (1) . Recently, a high affinity, stereospecific, saturable binding site for benzodiazepines has been reported in the mammalian central nervous system (CNS) (2-6) . In addition, high correlations have been demonstrated between the binding affinities of a series of benzodiazepine derivatives and their potencies as muscle relaxants (2), anxiolytics (7), and anti-convulsants (7), suggesting that this site may be a pharmacologic receptor mediating benzodiazepine action in the CNS . Since a number of putative neurotransmitters and small peptides failed to inhibit [ ~] diazepam binding to these rece for sites (6), the presence of an endogenous ligand has been ostulated (2~ . We now report the presence of a competitive inhibitor s) of g ~i] diazepam binding in extracts of bovine brain . The characteristics of this inhibitor s) are similar to those that might be associated with an endogenous ligand to the benzodiazepine receptor . METHODS Tissue re oration : Bovine tissue was obtained immediately after sacrifice an qu cc rozen on dry ice . The frozen tissue was fragmented and homogenized in 3-5 volumes of acidified acetone (0 .2 ml concentrated HC1/liter) + o w om reques s
or repr nts s ou
e mailed .
1894
Endogenous Inhibitor of Diazepam Binding
Vol . 22, No . 21, 1978
using a Waring blender . The resulting Homogenate was stirred for four hours at room temperature and filtered under vacuum (Whatman #1 pager) . The residue retained on the filter . was re-extracted in 3 tissue volumes of an acetone-H 2 0 mixture (80 :20) for four hours and filtered . The pooled filtrates of the This above extractions were evaporated under vacuum to the aqueous phase . cloudy solution was centrifuged at 25,000 x g for 20 minutes and the supernatant was filtered and lyophilized . The resulting residue was suspended in methanol (1 ml/gram tissue) and stirred for one hour at room temperature . The resulting residue was dissolved in distilled water (1 ml/lOg tissue) and centrifuged at 10,000 x g for 20 minutes . The dense lipid-like layer at the top was discarded and the remaining clear solution extracted with 5 volumes of petroleum ether . The aqueous phase was then lyophilized . This powder was resuspended in 1 .OmM Tris-HC1 buffer pH 7 .4 (1 ml/30 grams tissue) and referred to as the crude extract . Assa for inhibition of Eli diaze am bindin : Tissue extracts were assayed or e r a i ty to in n ng o diazepam (New England Nuclear, it Boston, Mass ., Sp . Act . 39 Ci/mM) to a crude synaptosomal preparation of rat cerebral cortex by a modification of the method of Mohler and Okada (4) . The synaptosomal f~~action was prepared as previously described (2) . The assay mixture contained approximately 1 mg of synaptosomal protein, 30 or 50mM Tris-HC1 buffer pH 7 .4 and 3nM [~-I diazepam in a final volume of 1 .5 of 1 ml . Quantities of tissue fractions of up to 0 .5 ml were incorporated in the above system to assay for the inhibition of specific [sli] diazepam binding . Non-specific binding was determined by inclusion of 3uM unlabeled diazepam (Hoffmann-LaRoche) and was routinely less than 10% of total binding . Incubations were carried out on ice for 15 minutes followed by filtration and washing (10 ml ice cold 50mM Tris-HC1 pH 7 .4) using Whatman GF/B filters . The filters were counted after being thoroughly mixed in 10 ml of Aquasol (New England Nuclear, Boston, Mass .) . Under these conditions the apparent affinity (Kd) of diazepam binding is 3 .5nM which is in agreement with values obtained in other laboratories (2-6) . The ability of various fractions to inhibit [~li] diaze am binding was expressed as units of diazepam-like activity (DLA (Table I~ . Standard curves were generated for each synaptosomal preparation by incorporating increasing amounts of unlabeled diazepam into the above assay system . Linear semi-log plots were obtained in the rànge of 1-50 pmoles when percent inhibition of [ sH] diazepam binding was plotted versus the concentration of unlabeled diazepam . A unit of DLA activity is defined as that amount of inhibitory activity equivalent to 1 pmole/assay of diazepam . De termination of [ ~i] D-ala-met-enkephalin and ç ~I~ dihydroalorenolol bindin s~pec~f c~ind~ng o e b~Ti-ty~ gums 6-r~n repara ions to n ib~ â D-ala-met-enkephalin and [~i~ dihydroalprenolol was determined as previously described (8,9) . Protein determinations : The protein or peptide content of various fractions was ethem ne y t e method of Lowry et al (10) using bovine serum albumin as standard . Both small proteins and pépi~des are measured with this technique . Chromato ra hic rocedures : Sephadex G-25 fine (Pharmacie) was, used in all ge tra on exper ments . Columns (2 .5 x 100 cm) were equilibrated in 1 .OrnM Tris -HC1 buffer pH 7 .4 and run at 4° at a flow rate of 10 ml/hour .
Vol . 22, No . 21, 1978
Endogenous Inhibitor of Diazepam Binding
1895
Thin-layer chromatography (TLC) wâs performed on 20 x 20 cm silica gel plates (Kieselgel F-254 E . Merck, Darmstadt) developed in chloroform : methanol : acetic acid (80 :40:11) . The plates were viewed with a mineralite (UV), scraped in 1 cm bands and the scrapings eluted with 1 ml of 1mM HC1 . The HCL extract was lyophilized and reconstituted in 400 ul of 50rtiM TrisHC1 buffer pH 7 .4 and assayed for inhibition of [~li] diazepam binding. RESULTS Inhibition of [ ~-I] diazepam binding by tissue fractions : The relative potenc es o cru e extracts of ovine ra n an T~ve -fin inhibiting [~li] diazepam binding to synaptosomal membranes are shown in Table I . Crude brain extract is approximately 10 times more potent than the equivalent liver extract. The inhibitory activity of crude brain extracts was not affected by boilinç~ for 10 minutes or by extraction with diethylether . However, complete loss of activity was observed following dialysis, indicating a molecular weight of less than 5000 daltons . Incubation of bovine brain at 37°C for 16 hours prior to preparation of the crude extract markedly reduced the specific activity (Table I) .
Table I Diazepam Binding Inhibitory Activity of Various Tissue Fractions FRACTION Brain crude) Liver crude) Brain (incubated) Sephadex Pooled Peaks I II III
UNITS DL~mg LRM 13 .1 1 .35 2 .70 6 .60 33 .5 155 .0
ac o t e in ca e tissue extracts was a ust to risp prior to assay. Inhibition of diazepar~ binding and Lowry reactive material (LRM) were determined as described in Methods . Brain (incubated) refers to the resulting crude extract from bovine brain incubated at 37° for 16 hours prior to preparation . Chromatographic resolution of bovine brain extract on Sephadex r,-25 yields three distinct peaks of inhibitory activity (Figure 1) . The specific activities of each pooled Sephadex peak are shown in Table I . The greatest increase in specific inhibitor activity was consistently observed in the third (lowest molecular weight peak . Similar fractionation of a bovine liver extract yielded no discernable pattern of inhibitory activity, suggesting that inhibition of diazepam binding observed for the crude liver preparation was non-specific .
1896
Endogenous Inhibitor of Diazepam Biadiag
Vol . 2 2, No . 21, 1978
% INMIOITIOM OF ~INDINO
/N/ml LRhI (-_-_)
F~ure 1 Sephadex G-25 chromatography of bovine brain extract : Fifteen ml of bovine brain extract resulting from the preparation of 800 grams of tissue was applied on a 2 .5 x 100 cm Sephadex G-25 column equilibrated in 1mM Tris-HC1 buffer pH 7 .4 . Fractions were collected (2 .5 ml) and assayed for both inhibition of [ ~] diazepam binding (400 ul) and Lowry reactive material . The void volume of the column was at tube 42 .
Kinetic anal sis of ak III inhibitor activit : Double reciprocal analysis o ono azepam n ng n t e presence of two concentrae n tions of Peak III is shown in Figure 2 . The apparent binding affinity of diazepam decreased with increasing amounts of Peak III while maximum binding was unchanged, suggesting the inhibition was competitive .
Vol . 22, No . 21, 1978
Endogenous Inhibitor of Diazepam Binding
I
[FREE DIAZEPAM]
1897
(nMx10')
Fi~c u re 2 Double reciprocal analysis of Peak III inhibitory activity : The synaptosomal membrane preparation was assayed for specific binding with increasing amounts of [ 3H] diazepam in the absence and presence of 200 ul and 400 ul of the Se hadex Peak III fraction . Ordinate : B is the amount of specifically bound [ ~] diazepam (fmol/mg protein) as defined in Methods .
Thin-la er chromato ra h of Se hadex G-25 fractions : Thin-layer chromatograp is ana ys s o eac o t e ep a ex pea raco ons is illustrated in Figure 3 . Several discrete bands of U .~1 . absorbing material were observed in Peaks II and III while Peak I yielded a diffuse region of U .V . absorption near the origin . No inhibitory activity was observed in the TLC scrapings of Peak I, whereas Peak II and III had discrete bands of inhibitory activity co-migrating with U .V . absorbing material . The Rf values of the Peak II and Peak III inhibitory activity were 0 .47 and 0 .53, respectively .
1898
Eadogenoua Inhibitor of Diazepam Binding
Figure
Vol . 22, No . 21, 1978
3
Thin-layer chromatography of the Sephadex fractions : 300 ul of pooled fractions from each of the three Sephadex peaks were lyophilized, resuspended in 100 ul of methanol-water and spotted on TLC plates . The chromatograms were developed and assayed for inhibition of [ ~] diazepam binding inhibition as described in Methods . The hatched areas indicate U .V . absorbing material and the solid areas, diazepam binding inhibitory activity .
Effects of Se hadex fractions on o late and ~-adrener is rece tors : The y roa preno o to their n ng o -a a-me -en ep a n an respective receptors were significantly inhibited by Peak I (Table II) . Peak II did not significantly affect g~adrenergic binding while specific opiate binding was inhibited by 30% . Peak III appeared to be the most specific inhibitor of [~li] diazepam binding by these criteria since it displayed little or no inhibitory activity of either ~adrenergic or opiate receptor binding .
Vol . 22, No . 21, 1978
Endogenous Inhibitor of Diazepam Binding
1899
Table II Effects of Diazepam Binding Inhibiting Fractions On the ~Adrenergic and Opiate Receptor
Peak Fraction I II III
% Inhibition of Specific Binding [~i] Diazepam 9 .8 54 .2 37
[ ~I] Dihydroalprenolol 27 0 4
[ 3H] D-ala-met-enkephalin 100 30 0
2~0ü o t e pooTeâ péâc~frâct ons were assaye or n t on o ga binding using 3 .1nM [~i] diazepam and 1 .3nM [ ~] dihydroalprenolol . For the opiate assay 100 ul of each pooled Sephadex fraction was assayed in the presence of 0 .5nM [ 3 H] D-ala-met-enkephalin .
DISCUSSION The present report describes the partial purification of a substance s) from bovine brain which specifically inhibits the binding of [ ~i] diazepam to a crude synaptosomal fraction of rat cerebral cortex . A tissue extraction procedure was devised that minimizes proteolytic degradation and specifically selects for small molecules, since it was hypothesized that an endogenous ligand for the diazepam receptor would be a small molecule such as a peptide . The homogenization of frozen tissue in cold acidified acetone was selected since these conditions would rapidly inactivate degradatioe enzymes as well as cause the precipitation of most macromolecules . A methanol extraction was employed to reduce the amount of inorganic salts . The crude extract of brain contained the equivalent of 13 pmole of diazepam-like activity per milligram of Lowry reactive material while liver contained an order to magnitude less . Partial characterization of this inhibitors) of diazepam binding indicates that it has a low molecular weight (dialyzable), is non-lipid in nature (not extracted in ether) and heat stable (100° for 10 minutes) . Incubating brain at 37° before preparation of the crude extract led to a marked reduction in its inhibitory activity . However, incubation of the crude extract with trypsin, peptidase or protease failed to decrease inhibitory activity . These preliminary results suggest that the factors) are either non-peptide in nature or that more specific enzymes are required for inactivation . Chromatographic analysis of crude bovine brain extract resolves three discrete fractions . The estimated molecular weight of the three Sephadex fractions are : Peak I = 2000, Peak II = 1000, and Peak III <700 . The largest increase in specific activity was routinely observed for Peak III . Analysis of each Sephadex fraction by thin-layer chromatography indicated that both Peak II and Peak III contain discrete substance s) capable of inhibiting [ ~] diazepam binding . The loss of inhibitory activity observed following TLC of Peak I suggests that the inhibitory activity of this fraction prior to TLC was either labile under these conditions or of a non-specific nature .
1900
Endogenous Inhibitor of Diazepam Binding
Vol . 22, No . 21, .1978
The competitive inhibition of [~i] diazepam binding exhibited by the Sephadex Peak III material (Figure 2) suggests that the active factor is reacting with the same binding site as diazepam . In addition to having the highest specific activity, Peak III appears to be the most specific for diazepam receptors since it displayed little or no inhibition of ~adrenergic or opiate binding (Table II) . However, endorphin-like material has been reported in brain extracts purified by Sephadex G-25 chromatography with molecular weights corresponding to Peaks I and II (11) . These data demonstrate the existence of a low molecular weight, competitive inhibitory factor s) of [~i] diazepam binding in bovine brain . Both the amount of inhibitor present and the Sephadex G-25 chromatographic pattern-indicate that compared to a peripheral tissue (liver), this factor may be specific to the CNS . The observation that crude extracts of liver (Tab1e .I and unpublished observations) and muscle (unpublished observations) do have inhibitory activity, but that this activity is both qualitatively and quantitatively different from brain may well indicate that the inhibitory activity from these tissues is non-specific . Actual quantitation of tissue levels of this factors) and characterization as either an agonist or antagonist at the receptor must await purification and bioassay . The evidence suggests that at least two compounds exist in bovine brain that inhibit [ ~] diazepam binding . Further purification of these factors may yield a substance s) which function as an endogenous ligand for the benzodiazepine receptor . ACKNOWLEDGMENTS The authors wish to thank Dr . C . Pert for performing opiate receptor binding assays . P . Skolnick wishes to thank Dr . J . Daly, NIAMDD, for providing facilities during these studies . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 .
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