CALCIUM-DEPENDENT BINDING OF BRAIN GLUTAMATE DECARBOXYLASE TO PHOSPHOLIPID VESICLES M A S I ~ ECOVARRI:BIAS L a n d RICARDOTAPIA’ Departamento de Biologia Experimental. Instituto de Biologia. Universidad Nacional Autonoma de Mexico, Apartado Postal 70-600, Mexico 20, D. F., Mexico
The binding of glutamate decarhoxqlase (GAD). to phospholipid vesicles (liposomes) in the absence and in the presence of several Ca” and Mg” concentrations was studied. Phosphatidylcholine--phosphatidylserine(4:11 liposomes are capable of binding GAD in a Caz+-dependent manner. The per cent of GAD bound increased from 5 to 65”,, in a sigmoid shape with Ca2+ concentrations in the 0 . 2 - 4 m ~range. Mg” also induces GAD binding but is less effective than Ca”. The CaZ’depcndent binding of GAD is not the result of unspecific association of protein, since C a z t did not promote any binding of choline acetyltransferase or lactate dehydrogenase. Furthermore. the relative specific actib ity (”lserine is probahl! involved in the Ca’--dependent binding of GAD to hrain memhrancs. Phospholipid tesicles seem to he a useful experimental model for studying the mechanisms of this GAD association to mcmhranes and the possible physiological implications of the GAD-Ca2 * mcmhrane interaction regarding the release of ncwlj sjnthesized GABA from nerve endings. Abstract
i ) A M r . v A i . role of glutamate decarboxylase (GAD: ~.-glutaniate-l-carboxy~yase. EC 4.1.1.15) in CNS is ampl) recognired. GAD catalyzes the onestep synthesis of GABA. and its activity seems t o be a crucial factor regulating the function of this amino
& DE ROHLRTIS. 1965: FONNUM. 1968). These findings rise the question of the actual location of GAD iri riro. In fact. electron microscopic studies o n the localization of GAD. using specific GAD antibodies. consistently show that GAD is associated to the presynaptic membrane as well as t o the synaptic acid 21s the most widely distributed inhibitory neurotransmitter in the mammalian brain (CuRi-is. 1975; vesicles membrane and the mitochondria1 membrane RORI-KTS c’t d..1976). F r o m the results of a series (MCLALGHLIN clt a/.. 1975: WtX>D c’t a/. 1976). of experiments o n the relationship between the inhibiF r o m the foregoing d a t a it seems possible that at tion of GAD activity and the appearance of seirures. least a fraction of the GAD present in nerve endings may bind to membranes in uiro in a Ca*’-dependent we have postulated that the activity of GAD is a regulatory mechanism of cercbral excitability through the manner. Thus. the role of Ca2’ at GABAergic tercoupling of the synthesis and the release of GABA minals might be double: ( I ) to induce the binding of (TAPIA. 1974; TAPIA ct NI.. 1975). The possibility that GAD t o membranes for the synthesis-secretion GAD may bind to membranes is particularly relevant coupling of GABA and (2) to participate in the release for this synthesis-secretion coupling. Although subcel- of previously synthesized GABA through the well established depolarization-secretion coupling (RUBIN. lular fractionation sttidies have located GAD mainly 1974: BI.AUSTEIN, 1975: T A P I A & MEZA-Ru~z,1977). in the soluble component of the nerve endings (synapIn order to obtain more information o n the mechantoplasm). i t has been shown that in the presence of Ca” GAD binds strong11 t o both nerve endings ism of the Ca”-dependent binding of GAD to memmembrane and synaptic vesicles membrane (SALGAUI- branes. and with the aim of developing an experimental model for the study of the postulated synthesisAhhrwiutiotis ir.wrl: GAD. glutanlate decarhoxylase: secretion coupling of GABA. in the present paper we ChAT. choline acetqltransfcrase: LDH. lactatt dehydro- have studied the effect of Ca” o n the binding of brain GAD to phospholipid vesicles (liposomes). For genase. comparative purposes we also studied the binding of ’ To whom correspondence should he sent. cOFF.
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MANUEI C O ~ A R K U Band I A SRICARDOTAPIA
tuo othcr soluble brain e n ~ m e s choline . acetyltransferase (ChAT: acetql-CoA : choline 0-acetyl transferiisc. EC 2.3.1.6) and lactate dehydrogenase ( L D H ; L-lactate: NAD owydoreductase. EC 1.1.1.27). C h A T is of particular interest. since it is also related t o the synthesis of :I n ~ u r o t r a n s m i t t e rand. as GAD. is concentrated in the synaptoplasm (WHITTAKER er a!.. 196.1). MATERIALS AND METHODS
En:
i.nw prrptrrtriron. The same enzyme preparation was
used for studqing the binding of GAD. ChAT and LDH. The preparation was the supernatant of mouse brain homogenatcs (lo“,, w,v). prepared in water containing 0. I mwp!rido\al 5’-phosphats. after centrifugation at 100.0Oog for 1 h at 0-4 C. A freshlj prepared preparation hiis uzcd in each experiment. Prrpururion of lipownits und bindiny incuhurion. Multilamellar liposomes were obtained by shaking in a Vortex mixer ;I 2”,,water suspension of phosphatidylcholine or ;I plio4phatidylcholine~~phosphatidylserine mixture (4:I by height). under a nitrogen atmosphere. Liposomes were incubated in pol)carbonate centrifuge tubes at 37 C for N m i n v,ith the eniyme preparation. in a final vol of 8.5 or 4.25 nil. in the presence of 0.1 mM-pyridoxal phosphate and different concentrations of calcium or magnesium acetate. I n all experiments the tubes with no Ca2+ contained 1 mhi-EGTA. Unless otherwise indicated, the relationship phospholipid protein was 1.5 ( w w. usually 30:20mg or half these amounts). and the incubation medium was water. The pH of the incubation mixture in water was 6.7-6.8, and this pH did not change during incubation. After incubation the mixture was centrifuged at 100,000g for 45 min at &4 C (50-Ti Beckman rotor). the liposomes pellet was rehuspended in 2 ml of 0.4“,, Triton-X-100 containing 0.1 mu-p!ridoxal phosphate. and GAD. ChAT and LDH acti~itiesHere measured i n both the resuspended pellet and the supernatant. In order to calculate the recovery. the enzymes activity was also measured in an aliquot of the enzyme preparation. En:j~nie.\ usauj. GAD actkity was determined by the isotopic method previously described (TAPIA& AWAPAKA. 1969). based on the measurement of the I4CO, released from [I-’4C]glutamate. with the exception that 3.6 N-sulfuric acid was used instead of trichloroacetic acid to stop the reaction and release the I4CO, from the medium. Aliquots of 0.4 ml of both the liposomes pellet and supernatant were used for GAD measurement. ChAT was measured b! the rapid radiochemical method of FONNUM (1975~). but using the larger volume and the composition of the general incubation medium described by the same author for procedures using synthetic acetyl-CoA (FoxS L M .lY75b). The medium contained also 50.(MOc.p.m. of tjl-CoA and 0.08 ml of liposomes pellet or supernatant. At the end of incubation the medium was quicklq transferred with a Pastcur pipette to scintillation cials containing the diluting buffer. and the tubes were rinsed once with the same buffer before adding the kalignost-containing acetonitrile and the toluene scintillation mixture. With this procedure. activity curves against protein concentration were similar to those published with the original technique. LDH was measured by the spectrophotometric procedure described by BERGMEYER er a/. (1963). using 0.1 ml of the liposomes pellet or supernatant convenientl) diluted (usually H o l d and 10-fold. respectively). Protein was determined by the method of LOWRY
YI a/. (1951). In the liposomes pellet protein mcasurement was not precise because of turbidit! duc to phospholipids. Therefore, the per cent protein in this pellet has obtained by subtraction of the liposomes supernatant protein from total protein in the homogenate supernatant. Matc,riu/s. L-1-Phosphatid! lcholine from egg !elk (type V-E) and phosphatidylserine (bo\ine brain extract. type 111. containing 8tb85”, phosphatidjlserine. 5~lo“,, cerebrosides and 5;; phosphatidic acid) were obtained from Sigma Chemical Co. (St. Louis. MO). A purer preparation of pi,osphatidylserine. containing onl! a small amount of phosphatidylcholine (as judged from TLC in chloroformmethanol-acetic acid-hater. 25:15:4:2. b> \oI). obtained from Applied Science Laboratories. Inc. (State College. PA). was also used in home experimcnts. h i t h identical results. DL-[ ~-i4C]Glutamicacid (sp. act. 40 60 mCi mmol) and [a~etyl-l-’~C]CoA (sp. act. 40-60 niCi mmol) were obtaincd from New England Nuclear (Boston, MA). Kalignost (sodium tetraphenylhoron) and acetonitrile were from Merck (Darmstadt. German!). All other chemicals used for enzqme assays were from Sigma.
RESULTS AND DISCL‘SSION
In the absence of divalent cations. less than S’,, of GAD was bound t o the phosphatidq 1choline:phosphatidylserine liposomes. This percentage increased in a sigmoid shape with increasing concentrations of Ca’+, reaching a plateau value of about H”,at 4 mM concentration. When Mg’ was added instead of Ca”, GAD binding was also greatly stimulated. reaching also a plateau value at 4 mM concentration. but the shape of the curve was different from that of CaZ+,due mainly t o a much lower binding induced by Mg2+ than by C a ” at a 2 m concentration ~ +
(Fig. 1). In contrast to G A D , a high pcrcentage of C h A T (41:~;) was bound t o the liposomes in the absence of Ca’+ o r Mg”. and the addition of either cation. from 0.5 to 4 mM concentration. resulted in a decrease of this value t o 2(f28”, (Fig. I ) . The binding of L D H was negligible in the absence and in the presence of Ca2+ o r Mg” u p t o a 8 m concentration ~ (Fig. 7). With no divalent cations present. lo”,, of total protein was bound to the liposomes. and this value increased with increasing concentration of Ca’ or Mg2+ u p t o about 36”“ at 4 or 8 m ~ T. h e binding curve was identical with C a 2 + and with M g Z t (Fig. 2). T h e above results indicate that the binding of G A D t o phospholipid membranes is strictly dependent on Ca’+ or Mg” concentration. although M g 2 * was less effective than Ca”. At concentrations a s low a s 0.5mM-Ca”. the binding of GAD doubles that obtained in the absence of Ca”. and at 2 m w C a ” the binding is 9-fold the control value. That this binding of GAD is not the result of an unspecific association of protein to the liposomes is shown by the lack of stirnulatory effect of Ca” and Mg” on the binding of ChAT and L D H . Furthermore. when the results are expressed as relative specific activitv (‘I, of GAD bound/”’;, of protein bound). a 4.2-fold increase was observed from 0 t o Zrn~-Ca’-. whereas Mg*+ was clearly less efficient (Fig. 3). +
lZfl
Cdlcium glutamate decarboxqlase-liposomes interaction r
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FIG.I . Binding of GAD and ChAT to phosphatidylcholine-phosphatidylserine liposomes as a function of Ca' + or MgL+concentration. Liposomes were incubated with the enzyme preparation and separated by centrifugation as described in Materials and Methods. The per cent bound was calculated considering as lOO", the value of the supernatant plus that of the pellet. As compared to the enzyme preparation. recovery values at the different concentration points (CaZ+and Mg2 data calculated together, since they were very similar) varied from 70 to 84"i for GAD. ChAT recovery was 11 1 -133", for the 0 . 2 4 mM points; however. in the absence of cations the recovery was only 4232, indicating that in these conditions considerable ChAT activity is lost during the incubation and centrifugation. Each point is the mean value of the number of experiments shown in parentheses ~ s . E . M . ;3 experiments for the points without number. In other series of experiments. GAD binding was binding. In this respect it is interesting that two forms studied as a function of the phospholipid,/protein of brain GAD activity have been identified kineticallv. ratio, in the absence and in the presence of differing in their dependence on free pyridoxal phos1971; BAYONet a[., 1977). 2 m ~ - C a ' + . The results are shown in Fig. 4. The phate (TAPIA& SANDOVAL. binding of the enzyme, expressed as per cent bound, Furthermore, two peaks of GAD activity. with similar increases almost linearly from a ratio of 0 to 1.5, but kinetic and immunological properties, have been no further increase was observed with ratios higher separated by gel filtration chromatography, suggestthan 1.5. Interestingly. when the results are expressed ing that one corresponds to a high molecular weight as relative specific activity the shape of the curve was Isimilar to the per cent curve. Since the protein was w 401maintained constant at all but the last point of the curve (the actual experimental phospholipid/protein ratios were. in mg. 2.5110. 5/10, 15/10. 60110 and 60/5), the relative specific activity curve indicates that the initial increase in the binding of GAD is relatively specific. In fact. the per cent of protein bound was constant (between 29.9 and 33.2) at the corresponding points. The binding curves in the absence of Ca2+ were similar in shape to those in the presence of 2 mM-Ca". However. the effect of Ca" was evident from the 0.25 ratio. and it was very notable at 0.5 or higher ratios (Fig. 4). [GI"]. or [Mg2'_]0 ( m M ) The finding that a constant proportion of protein FIG. 2. Binding of LDH and protein to phosphatidylchoand of GAD (not more than ahniit W"~!, nf protein line-phosphatidylserine liposomes as a function of Caz+ and about 4@50:,0 of GAD), was bound to the lipo- or M g Z +concentration. LDH recovery varied from 64 to somes, in spite of the great phospholipid excess 92";. When the S.E.M. is not shown. it was equal to or present at the higher points of the curve, suggests smaller than the size of the symbol. Other details as for that only a population of proteins is susceptible t o Fig. 1. '.(
315
c
MANUEL COVAKKUBIAS and
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RICARDO TAPl.4
B 4 I \
\
n
A
I I
2
ChAT
L DH
'* 4
I 3
4
(mM)
[cP"~
FIG.3. Relative specific activity (", enzqme protein) of GAD. ChAT and LDH associated to phosphatidvlcho1ine~phosphatid)Iserineliposomes. as ;i function of Ca2 o r Mg'* concentration. The points were calculated from the data of Figs. I and 2. 'lo
aggregate o f a monomer (Wr: ('I ( I / . , 1976). It remains to be elucidated whether one of the forms of GAD is able to bind to the liposomes whereas the other is not. As mentioned in Materials and Methods, the pH of the incubation mixture in water was 6.7-6.8, and it remained constant during the incubation period. When the incubation was carricd out in 5 m w N a phosphate buffer, p H 6.5, the binding of GAD to the liposomes. both in the absence and in the prcsence of 2rnM-Ca". was thc same as in water. However, at p H 7 or 7.25 the Ca" -induced binding was decreased (Fig. 5). When a 10 mwNa-phosphate buffer was used. a similar decrease in the calcium-dcpendent binding was observed at p H 7 . However. at this sodium concentration. at pH 6.5. GAD hinding in the absence of Ca2 was slightly increased, and the effect of C a 2 + was less notable than in water or 5 mM buffer (Fig. 5). That these effects are probably due to the cation is shown by other experiments in which either +
Na-acetate or K-acetate was added at a lOOmM concentration to the usual water incubation mixtures, both in the absence and in the presence of C a 2 + (the pH of such mixtures was 7.1). As shown in Table 1, in the absence of Ca" both Na' and K + produced a 3-fold increase in the relative specific activity. However, no further increase in the binding was observed when 2mM-Ca2+ was present in addition to the monovalent cations. The usefulness of phospholipid vesicles as an experimental model for studying the binding of GAD to membranes and its possible relationship to GABA transport is evident from comparison of the results of the present paper with those previously reported with membrane preparations obtained from brain tissue. including synaptosomal membranes. The GAD and LDH binding curves in the presence of Ca2+, shown in Figs. 1 and 3, are strikingly similar to those & DE ROBERTIS(1965) and reported by SALCANICOFF
TABLE1. EFFECTOF 100mM-Na'
A S D K ' O N THE BINDING OF GAD TO PHOSPHAlIDYLCHOLlNF~PHOSPHATIDYLSERlNt LIPOSOhlFS IN THF ABSENCE A N D IN THF PKESFYCE OF Ca*+
CaZ in medium +
0
2 mM
",>of GAD bound Control Na' Kt 11.9 44.0
35.1 36.2
33.5 39.7
Relative specific actit ity Control Na' K' 0.60 1.44
1.87
1.33
1.78 1.62
The experimental conditions were as described in the legend to Fig. 1. The Na' or K' acetate salts was used for these experiments. Relative specific activity is defined in the legend to Fig. 3. Mean values of 2 or 3 independent experiments.
1213
Calcium glutamate dccarboxylase-liposomes interaction I-
1
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W
I
-
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No Co
65
t
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70
PH FIG.5. Binding of GAD to phosphatidylcholine- phosphatidylserine liposomes as a function of pH. Liposomcs were incubated with the enzyme preparation in 5 mM (0.0)or 1 0 m ~(0,B) Na-phosphate buffer at the indicated pH. The triangles refer to the results obtained with unbuffered water as incubation medium (pH 6.7-6.8). The empt) symbols indicate the results obtained in the absence of Ca2 '. and the filled symbols in the presence of 2 mM-Ca' + . Mean values of 2 experiments for the buffers and 7 experiments for water.
4 2
8
I0
12
PHOSPHOLIPID/PROTElN RATIO ( w / w )
FIG.4. Binding of GAD to phosphatidylcholine-phosphatidqlserine liposomes as a function of the phospholipid; protein ratio. in the absence of Ca2' and in the presence of 2 mM-Ca*+.The upper panel shoms the per cent bound. and the lower panel the relative specific activity (", enzyme bound, "
The binding of glutamate decarhoxqlase (GAD). to phospholipid vesicles (liposomes) in the absence and in the presence of several Ca” and Mg” concentrations was studied. Phosphatidylcholine--phosphatidylserine(4:11 liposomes are capable of binding GAD in a Caz+-dependent manner. The per cent of GAD bound increased from 5 to 65”,, in a sigmoid shape with Ca2+ concentrations in the 0 . 2 - 4 m ~range. Mg” also induces GAD binding but is less effective than Ca”. The CaZ’depcndent binding of GAD is not the result of unspecific association of protein, since C a z t did not promote any binding of choline acetyltransferase or lactate dehydrogenase. Furthermore. the relative specific actib ity (”lserine is probahl! involved in the Ca’--dependent binding of GAD to hrain memhrancs. Phospholipid tesicles seem to he a useful experimental model for studying the mechanisms of this GAD association to mcmhranes and the possible physiological implications of the GAD-Ca2 * mcmhrane interaction regarding the release of ncwlj sjnthesized GABA from nerve endings. Abstract
i ) A M r . v A i . role of glutamate decarboxylase (GAD: ~.-glutaniate-l-carboxy~yase. EC 4.1.1.15) in CNS is ampl) recognired. GAD catalyzes the onestep synthesis of GABA. and its activity seems t o be a crucial factor regulating the function of this amino
& DE ROHLRTIS. 1965: FONNUM. 1968). These findings rise the question of the actual location of GAD iri riro. In fact. electron microscopic studies o n the localization of GAD. using specific GAD antibodies. consistently show that GAD is associated to the presynaptic membrane as well as t o the synaptic acid 21s the most widely distributed inhibitory neurotransmitter in the mammalian brain (CuRi-is. 1975; vesicles membrane and the mitochondria1 membrane RORI-KTS c’t d..1976). F r o m the results of a series (MCLALGHLIN clt a/.. 1975: WtX>D c’t a/. 1976). of experiments o n the relationship between the inhibiF r o m the foregoing d a t a it seems possible that at tion of GAD activity and the appearance of seirures. least a fraction of the GAD present in nerve endings may bind to membranes in uiro in a Ca*’-dependent we have postulated that the activity of GAD is a regulatory mechanism of cercbral excitability through the manner. Thus. the role of Ca2’ at GABAergic tercoupling of the synthesis and the release of GABA minals might be double: ( I ) to induce the binding of (TAPIA. 1974; TAPIA ct NI.. 1975). The possibility that GAD t o membranes for the synthesis-secretion GAD may bind to membranes is particularly relevant coupling of GABA and (2) to participate in the release for this synthesis-secretion coupling. Although subcel- of previously synthesized GABA through the well established depolarization-secretion coupling (RUBIN. lular fractionation sttidies have located GAD mainly 1974: BI.AUSTEIN, 1975: T A P I A & MEZA-Ru~z,1977). in the soluble component of the nerve endings (synapIn order to obtain more information o n the mechantoplasm). i t has been shown that in the presence of Ca” GAD binds strong11 t o both nerve endings ism of the Ca”-dependent binding of GAD to memmembrane and synaptic vesicles membrane (SALGAUI- branes. and with the aim of developing an experimental model for the study of the postulated synthesisAhhrwiutiotis ir.wrl: GAD. glutanlate decarhoxylase: secretion coupling of GABA. in the present paper we ChAT. choline acetqltransfcrase: LDH. lactatt dehydro- have studied the effect of Ca” o n the binding of brain GAD to phospholipid vesicles (liposomes). For genase. comparative purposes we also studied the binding of ’ To whom correspondence should he sent. cOFF.
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MANUEI C O ~ A R K U Band I A SRICARDOTAPIA
tuo othcr soluble brain e n ~ m e s choline . acetyltransferase (ChAT: acetql-CoA : choline 0-acetyl transferiisc. EC 2.3.1.6) and lactate dehydrogenase ( L D H ; L-lactate: NAD owydoreductase. EC 1.1.1.27). C h A T is of particular interest. since it is also related t o the synthesis of :I n ~ u r o t r a n s m i t t e rand. as GAD. is concentrated in the synaptoplasm (WHITTAKER er a!.. 196.1). MATERIALS AND METHODS
En:
i.nw prrptrrtriron. The same enzyme preparation was
used for studqing the binding of GAD. ChAT and LDH. The preparation was the supernatant of mouse brain homogenatcs (lo“,, w,v). prepared in water containing 0. I mwp!rido\al 5’-phosphats. after centrifugation at 100.0Oog for 1 h at 0-4 C. A freshlj prepared preparation hiis uzcd in each experiment. Prrpururion of lipownits und bindiny incuhurion. Multilamellar liposomes were obtained by shaking in a Vortex mixer ;I 2”,,water suspension of phosphatidylcholine or ;I plio4phatidylcholine~~phosphatidylserine mixture (4:I by height). under a nitrogen atmosphere. Liposomes were incubated in pol)carbonate centrifuge tubes at 37 C for N m i n v,ith the eniyme preparation. in a final vol of 8.5 or 4.25 nil. in the presence of 0.1 mM-pyridoxal phosphate and different concentrations of calcium or magnesium acetate. I n all experiments the tubes with no Ca2+ contained 1 mhi-EGTA. Unless otherwise indicated, the relationship phospholipid protein was 1.5 ( w w. usually 30:20mg or half these amounts). and the incubation medium was water. The pH of the incubation mixture in water was 6.7-6.8, and this pH did not change during incubation. After incubation the mixture was centrifuged at 100,000g for 45 min at &4 C (50-Ti Beckman rotor). the liposomes pellet was rehuspended in 2 ml of 0.4“,, Triton-X-100 containing 0.1 mu-p!ridoxal phosphate. and GAD. ChAT and LDH acti~itiesHere measured i n both the resuspended pellet and the supernatant. In order to calculate the recovery. the enzymes activity was also measured in an aliquot of the enzyme preparation. En:j~nie.\ usauj. GAD actkity was determined by the isotopic method previously described (TAPIA& AWAPAKA. 1969). based on the measurement of the I4CO, released from [I-’4C]glutamate. with the exception that 3.6 N-sulfuric acid was used instead of trichloroacetic acid to stop the reaction and release the I4CO, from the medium. Aliquots of 0.4 ml of both the liposomes pellet and supernatant were used for GAD measurement. ChAT was measured b! the rapid radiochemical method of FONNUM (1975~). but using the larger volume and the composition of the general incubation medium described by the same author for procedures using synthetic acetyl-CoA (FoxS L M .lY75b). The medium contained also 50.(MOc.p.m. of tjl-CoA and 0.08 ml of liposomes pellet or supernatant. At the end of incubation the medium was quicklq transferred with a Pastcur pipette to scintillation cials containing the diluting buffer. and the tubes were rinsed once with the same buffer before adding the kalignost-containing acetonitrile and the toluene scintillation mixture. With this procedure. activity curves against protein concentration were similar to those published with the original technique. LDH was measured by the spectrophotometric procedure described by BERGMEYER er a/. (1963). using 0.1 ml of the liposomes pellet or supernatant convenientl) diluted (usually H o l d and 10-fold. respectively). Protein was determined by the method of LOWRY
YI a/. (1951). In the liposomes pellet protein mcasurement was not precise because of turbidit! duc to phospholipids. Therefore, the per cent protein in this pellet has obtained by subtraction of the liposomes supernatant protein from total protein in the homogenate supernatant. Matc,riu/s. L-1-Phosphatid! lcholine from egg !elk (type V-E) and phosphatidylserine (bo\ine brain extract. type 111. containing 8tb85”, phosphatidjlserine. 5~lo“,, cerebrosides and 5;; phosphatidic acid) were obtained from Sigma Chemical Co. (St. Louis. MO). A purer preparation of pi,osphatidylserine. containing onl! a small amount of phosphatidylcholine (as judged from TLC in chloroformmethanol-acetic acid-hater. 25:15:4:2. b> \oI). obtained from Applied Science Laboratories. Inc. (State College. PA). was also used in home experimcnts. h i t h identical results. DL-[ ~-i4C]Glutamicacid (sp. act. 40 60 mCi mmol) and [a~etyl-l-’~C]CoA (sp. act. 40-60 niCi mmol) were obtaincd from New England Nuclear (Boston, MA). Kalignost (sodium tetraphenylhoron) and acetonitrile were from Merck (Darmstadt. German!). All other chemicals used for enzqme assays were from Sigma.
RESULTS AND DISCL‘SSION
In the absence of divalent cations. less than S’,, of GAD was bound t o the phosphatidq 1choline:phosphatidylserine liposomes. This percentage increased in a sigmoid shape with increasing concentrations of Ca’+, reaching a plateau value of about H”,at 4 mM concentration. When Mg’ was added instead of Ca”, GAD binding was also greatly stimulated. reaching also a plateau value at 4 mM concentration. but the shape of the curve was different from that of CaZ+,due mainly t o a much lower binding induced by Mg2+ than by C a ” at a 2 m concentration ~ +
(Fig. 1). In contrast to G A D , a high pcrcentage of C h A T (41:~;) was bound t o the liposomes in the absence of Ca’+ o r Mg”. and the addition of either cation. from 0.5 to 4 mM concentration. resulted in a decrease of this value t o 2(f28”, (Fig. I ) . The binding of L D H was negligible in the absence and in the presence of Ca2+ o r Mg” u p t o a 8 m concentration ~ (Fig. 7). With no divalent cations present. lo”,, of total protein was bound to the liposomes. and this value increased with increasing concentration of Ca’ or Mg2+ u p t o about 36”“ at 4 or 8 m ~ T. h e binding curve was identical with C a 2 + and with M g Z t (Fig. 2). T h e above results indicate that the binding of G A D t o phospholipid membranes is strictly dependent on Ca’+ or Mg” concentration. although M g 2 * was less effective than Ca”. At concentrations a s low a s 0.5mM-Ca”. the binding of GAD doubles that obtained in the absence of Ca”. and at 2 m w C a ” the binding is 9-fold the control value. That this binding of GAD is not the result of an unspecific association of protein to the liposomes is shown by the lack of stirnulatory effect of Ca” and Mg” on the binding of ChAT and L D H . Furthermore. when the results are expressed as relative specific activitv (‘I, of GAD bound/”’;, of protein bound). a 4.2-fold increase was observed from 0 t o Zrn~-Ca’-. whereas Mg*+ was clearly less efficient (Fig. 3). +
lZfl
Cdlcium glutamate decarboxqlase-liposomes interaction r
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3
4
FIG.I . Binding of GAD and ChAT to phosphatidylcholine-phosphatidylserine liposomes as a function of Ca' + or MgL+concentration. Liposomes were incubated with the enzyme preparation and separated by centrifugation as described in Materials and Methods. The per cent bound was calculated considering as lOO", the value of the supernatant plus that of the pellet. As compared to the enzyme preparation. recovery values at the different concentration points (CaZ+and Mg2 data calculated together, since they were very similar) varied from 70 to 84"i for GAD. ChAT recovery was 11 1 -133", for the 0 . 2 4 mM points; however. in the absence of cations the recovery was only 4232, indicating that in these conditions considerable ChAT activity is lost during the incubation and centrifugation. Each point is the mean value of the number of experiments shown in parentheses ~ s . E . M . ;3 experiments for the points without number. In other series of experiments. GAD binding was binding. In this respect it is interesting that two forms studied as a function of the phospholipid,/protein of brain GAD activity have been identified kineticallv. ratio, in the absence and in the presence of differing in their dependence on free pyridoxal phos1971; BAYONet a[., 1977). 2 m ~ - C a ' + . The results are shown in Fig. 4. The phate (TAPIA& SANDOVAL. binding of the enzyme, expressed as per cent bound, Furthermore, two peaks of GAD activity. with similar increases almost linearly from a ratio of 0 to 1.5, but kinetic and immunological properties, have been no further increase was observed with ratios higher separated by gel filtration chromatography, suggestthan 1.5. Interestingly. when the results are expressed ing that one corresponds to a high molecular weight as relative specific activity the shape of the curve was Isimilar to the per cent curve. Since the protein was w 401maintained constant at all but the last point of the curve (the actual experimental phospholipid/protein ratios were. in mg. 2.5110. 5/10, 15/10. 60110 and 60/5), the relative specific activity curve indicates that the initial increase in the binding of GAD is relatively specific. In fact. the per cent of protein bound was constant (between 29.9 and 33.2) at the corresponding points. The binding curves in the absence of Ca2+ were similar in shape to those in the presence of 2 mM-Ca". However. the effect of Ca" was evident from the 0.25 ratio. and it was very notable at 0.5 or higher ratios (Fig. 4). [GI"]. or [Mg2'_]0 ( m M ) The finding that a constant proportion of protein FIG. 2. Binding of LDH and protein to phosphatidylchoand of GAD (not more than ahniit W"~!, nf protein line-phosphatidylserine liposomes as a function of Caz+ and about 4@50:,0 of GAD), was bound to the lipo- or M g Z +concentration. LDH recovery varied from 64 to somes, in spite of the great phospholipid excess 92";. When the S.E.M. is not shown. it was equal to or present at the higher points of the curve, suggests smaller than the size of the symbol. Other details as for that only a population of proteins is susceptible t o Fig. 1. '.(
315
c
MANUEL COVAKKUBIAS and
1212
RICARDO TAPl.4
B 4 I \
\
n
A
I I
2
ChAT
L DH
'* 4
I 3
4
(mM)
[cP"~
FIG.3. Relative specific activity (", enzqme protein) of GAD. ChAT and LDH associated to phosphatidvlcho1ine~phosphatid)Iserineliposomes. as ;i function of Ca2 o r Mg'* concentration. The points were calculated from the data of Figs. I and 2. 'lo
aggregate o f a monomer (Wr: ('I ( I / . , 1976). It remains to be elucidated whether one of the forms of GAD is able to bind to the liposomes whereas the other is not. As mentioned in Materials and Methods, the pH of the incubation mixture in water was 6.7-6.8, and it remained constant during the incubation period. When the incubation was carricd out in 5 m w N a phosphate buffer, p H 6.5, the binding of GAD to the liposomes. both in the absence and in the prcsence of 2rnM-Ca". was thc same as in water. However, at p H 7 or 7.25 the Ca" -induced binding was decreased (Fig. 5). When a 10 mwNa-phosphate buffer was used. a similar decrease in the calcium-dcpendent binding was observed at p H 7 . However. at this sodium concentration. at pH 6.5. GAD hinding in the absence of Ca2 was slightly increased, and the effect of C a 2 + was less notable than in water or 5 mM buffer (Fig. 5). That these effects are probably due to the cation is shown by other experiments in which either +
Na-acetate or K-acetate was added at a lOOmM concentration to the usual water incubation mixtures, both in the absence and in the presence of C a 2 + (the pH of such mixtures was 7.1). As shown in Table 1, in the absence of Ca" both Na' and K + produced a 3-fold increase in the relative specific activity. However, no further increase in the binding was observed when 2mM-Ca2+ was present in addition to the monovalent cations. The usefulness of phospholipid vesicles as an experimental model for studying the binding of GAD to membranes and its possible relationship to GABA transport is evident from comparison of the results of the present paper with those previously reported with membrane preparations obtained from brain tissue. including synaptosomal membranes. The GAD and LDH binding curves in the presence of Ca2+, shown in Figs. 1 and 3, are strikingly similar to those & DE ROBERTIS(1965) and reported by SALCANICOFF
TABLE1. EFFECTOF 100mM-Na'
A S D K ' O N THE BINDING OF GAD TO PHOSPHAlIDYLCHOLlNF~PHOSPHATIDYLSERlNt LIPOSOhlFS IN THF ABSENCE A N D IN THF PKESFYCE OF Ca*+
CaZ in medium +
0
2 mM
",>of GAD bound Control Na' Kt 11.9 44.0
35.1 36.2
33.5 39.7
Relative specific actit ity Control Na' K' 0.60 1.44
1.87
1.33
1.78 1.62
The experimental conditions were as described in the legend to Fig. 1. The Na' or K' acetate salts was used for these experiments. Relative specific activity is defined in the legend to Fig. 3. Mean values of 2 or 3 independent experiments.
1213
Calcium glutamate dccarboxylase-liposomes interaction I-
1
T
W
I
-
c -
No Co
65
t
2 1
70
PH FIG.5. Binding of GAD to phosphatidylcholine- phosphatidylserine liposomes as a function of pH. Liposomcs were incubated with the enzyme preparation in 5 mM (0.0)or 1 0 m ~(0,B) Na-phosphate buffer at the indicated pH. The triangles refer to the results obtained with unbuffered water as incubation medium (pH 6.7-6.8). The empt) symbols indicate the results obtained in the absence of Ca2 '. and the filled symbols in the presence of 2 mM-Ca' + . Mean values of 2 experiments for the buffers and 7 experiments for water.
4 2
8
I0
12
PHOSPHOLIPID/PROTElN RATIO ( w / w )
FIG.4. Binding of GAD to phosphatidylcholine-phosphatidqlserine liposomes as a function of the phospholipid; protein ratio. in the absence of Ca2' and in the presence of 2 mM-Ca*+.The upper panel shoms the per cent bound. and the lower panel the relative specific activity (", enzyme bound, "
