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Transamination of analogues of γ-aminobutyric acid by extracts of rat brain mitochondria
Brain Research, 49 (1973) 459-462
459
c~'~ Elsewer Scientific Pubhshlng Company, Amsterdam - Printed m The Netherlands
Transamination of analogues of ),-aminobutyric acid by extracts of rat brain mitochondria
P M BEART Axo G A. R. JOHNSTON Department of Phys'tology, Austrahan Nattonal Untver~ity, Canberra (Austraha)
(~ccepted October 3rd, 1972)
The inhibitory synaptic transmitter, ),-aminobutyric acid (GABA), is catabohsed to succinic semlaldehyde in the nervous system by G A B A • 2-oxoglutarate amino transferase (GABA-T, E.C. 2.6.1 19)21. We have examined the ability of extracts of rat brain to transaminate some conformationally restricted analogues of GABA, various substituted G A B A derivatives and related compounds, using radioactive 2oxoglutarate as the amino group acceptor and measuring the production of radioactive glutamate by a convenient and sensitive procedure. Brain mltochondria contain a number of amino acid transaminases, in addition to GABA-T, which use 2-oxoglutarate as a substrate3: thus, activity in the present study does not necessarily imply that a substance is a substrate for GABA-T. On the other hand a substance which is inactive is unlikely to be a substrate for this enzyme. A crude mitochondrial preparation was isolated from rat cerebral gray matter as described previously 7. The enzymic production of glutamate from 2-oxoglutarate by this preparation was assayed as follows: 0.1 ml of a stock mixture, containing 10 m M dithiothreitol and 0 3 m M pyridoxal phosphate in 100 m M Tris buffer at p H 8.5, was mixed with 0.05 ml of 4 m M 2-oxoglutarate (containing 0.1/zC1 of [1-14C]2-oxogluta rate) and 0.1 ml of 3 m M potential amino group donor in l M, p H 8.5 Tris buffer and preincubated at 37 °C for 5 mm. The mltochondnal preparation (0.1 ml) was then added and incubation continued for 30 min. The reaction was stopped by the addition of 0.05 ml of 25 }o aqueous trichloroacetic acid and the radioactive glutamate formed determined by a procedure similar to that of Waksman and Roberts 25. The entire sample was transferred to a column (1 cm "< 4 cm) of Dowex 50W X4 [I4~] resin ( 100-200 mesh), and the column eluted with water (20 ml) and 2 M aqueous a m m o m a (2 y, 5.0 ml). A 1.0 ml aliquot of the second ammonia eluent was taken and its radioactivity measured in 10 ml of dioxan scintillator. This second ammonia eluent, when concentrated in vacuo in a rotatory evaporator and examined by thin layer chromatography on cellulose plates, contained a single ninhydrin positive spot of identical RF to an authentic sample of L-glutamate. All analogues that contained an asymmetric carbon atom were tested as racemates. The following were gifts: 2-fluoro-GABA (2-fluoro-4-aminobutanoic acid, Dr.
460
SHOR[ ( ()MVIUNI( \ t)l ~ ,,~
TABLE l SUBSTRATES
FOR T H F
lzNZ~Ml(
PI~.()DLJ(FION
()f
(,tUTAMAT[
FROM
2-OXOGI[
IAR,'.,IF
B~
t~,zkl
Pi~"
MITOCHONDRI,X Results are expressed relative to the actlwt~ of G A B A as a substrate and are mean~ S E of ~,l~e~ f r o m at least 3 experiments The following showed no measurable activity as substrate~ 2-Amlnobutano~. a~.ld, ~t,~- and ~ cm,ammocyclohexane-l-carboxyhc acid, 4 - a m m o p e n t a n m c acid, 3 - a m m o p r o p a n e s u l p h o m c a~.~d. 4 - a m m o t e t r o h c acid, 2-fluoro-GABA, gl~cme, 2-hydroxy-GABA, 3-hydroxv-GABA, mu~cm~ol, taurlne.
Sub,~traIe Tran,~-4-ammocrotomc acid GABA fi-Alamne 5-Aminopentanolc acid 2-MethyI-GABA 2-Methyl-3-ammoproplomc acid 2-Chloro-GABA Homohypotaurme
4~ tt)try telattve to that o/ GABA I 7~ 0.04 I 00 - 0.04 0 90 0 04 0 ~0 0 04 0,30 0 02 0 24 ~ 0 02 0.15 0 02 0 15 0 04
P. Goldman, Bethesda), musctmol (Dr. P. Hackett, Sittingbourne), c&- and trans-3aminocyclohexane-l-carboxylic acid (Dr. D.I.B. Kerr, Adelaide), and homohypotaurine (3-ammopropanesulphtmc acid, Dr. B. Jolles-Bergeret, Orsay). The following were prepared by pubhshed procedures, trans-4-ammocrotonic aczd 19, 4-ammopentanoic acid a3, 3-aminopropanesulphomc acid ~'z, 4-aminotetrolic acid .5, 2-chloroGABA (2-chloro-4-ammobutanolc acid) '-'4, 2-hydroxy-GABA (2-hydroxy-4-ammobutanolc acid) 14, and 2-methyl-GABA (2-methyl-4-aminobutanolc acid) 1. Other analogues were purchased from chemical suppliers [l-14C]2-Oxogtutarate specific activity 24 mO/mmole, was purchased from Calatomic, Los Angeles. The results are summarised m Table 1. Trans-4-aminocrotomc acid, a conformationally restricted analogue of GABA an an extended conformanon 6, was an excellent amino group donor, being more ef~clent than GABA under the condinons investigated. None of the other conformaUonally restricted GABA analogues (4-ammotetrohc acad, musclmol, cts- and trans-3aminocyclohexane-l-carboxylic acids) showed measurable acnvlty. 4-Ammotetrohc acid, although not a substrate for GABA-T, is an inhlbator (linearly competinve wnh GABA) of GABA-T activity measured by the coupled formation of succinate fi'om GABA 7 Other workers have noted the ability of extracts of mammahan brain to transaminate/3-alanine, 5-aminopentanolc acid, and 2-methyl-3-aminoproptomc acad, and the inabihty of these extracts to transammate 3-hydroxy-GABA2,z3 TransammaUon of 2-aminobutano~c acid and glycme, which were reactive m the present study, ha~ been reported in extracts of beef brain 2. Studies on the glycme-2-oxoglutarate aminotransferase act~wty m rat brain indicate that th~s is a glycine forming rather than a glycine degrading enzymic activity tT.
SttORT COMMUNICATIONS
461
Substttuents on c a r b o n s 2 a n d / o r 4 of G A B A either considerably reduced (2methyl- a n d 2 - c h l o r o - G A B A ) or abolished (2-hydroxy-, 2-fluoro- a n d 4-methylG A B A , cis- a n d t r a n s - 3 - a m i n o c y c l o h e x a n e - l - c a r b o x y h c acid) activity in the present study. T a u r i n e , o n the basis of occurrence, postsynaptic action a n d antagonists thereof, a n d u p t a k e a n d release studies 9,11,r),15,16, m a y be an i m p o r t a n t i n h i b i t o r y t r a n s m i t t e r in m a m m a l i a n brain. Little is k n o w n of t a u r i n e c a t a b o h s m other t h a n that it is slowly converted to isethionate ~°. It ~s not t r a n s a m i n a t e d u n d e r the c o n d i t i o n s used m the present study. If a t a u r l n e : 2 - o x o g l u t a r a t e a m l n o t r a n s f e r a s e activity is present in rat b r a i n its p H o p t i m u m a n d / o r subcellular d i s t r i b u t i o n might be different from that of G A B A - T . It is interesting that while the sulphonic acids, t a u r m e and 3 - a m l n o p r o p a n e s u l p h o n l c acid, c a n n o t be t r a n s a m l n a t e d u n d e r conditions favourable to t r a n s a m i n a t i o n of the c o r r e s p o n d i n g carboxylic acids, G A B A a n d fi-alanine, the sulphinlc acid, h o m o h y p o t a u r i n e is shows some activity. M a n y of the G A B A analogues that were inactive as a m i n e d o n o r s to 2-oxoglutarate in the present study, a n d thus unlikely to be substrates for G A B A - T , are k n o w n to be able to activate bicuculline-sensltive postsynaptic receptors 4,s,u~. 3 - h y d r o x y - G A B A , 3 - a m i n o p r o p a n e s u l p h o n i c acid, muscimol, 2-ftuoro-GABA, a n d 4 - a m m o t e t r o l i c acid. We conclude that G A B A - T has a very different substrate specificity from that of blcuculline-sensltive postsynaptlc receptors.
1 ADAMS, R., AND FLES, D., The absolute configuration of the Ca atom In retronecanone (1-methvl-
7-oxopyrrohzldlne), J. Amer chem. Soc , 81 (1959) 49464951. 2 BAXTER, C. F , AND ROBERTS, E , The 7-ammobutyrlc acld
462
,~H()Ri
~ i~MMII\I*
'~ ~ : , ,
,'
[5 JA(OBSIbN. J G , ~,~D SMtlH. 1, H , Btochemtstry and physiology of tattrmc an0 Lau~¢m ~ ~vatlves, P t n s w l Re~ , 48 ([968) 424-51t 16 JASPER, H H., AND KO',AMA, | , Rate ot release of anamo acids f r o m the cerebral corlc~ ~, ~hc cat as affected by b r a m s t e m and thalam~c %tlmu]atlon. ('attad J Pkvs'tol P]tatnlclto]. 47 ¢~'~-~tl 889 005 17 JOt-INSION, G A R . ~,ND VH,kLI. M V.. (Jlycme:2-oxogluterate transammd~e m Tat ~c,cb~al cortex, Brain Reseur~ It. [ 4 (1969) 201 -208 18 JOLLES-BERGERET, B , A new sulphmtc amino acid: h o m o h y p o t a u r m c ( ? - a m m o - p r o p a n e . u l phmlc acid), E.rop I. B w ( h e m , I0 (t969) 569-573 19 M USASHI, A , Synthe.se der ;,-Ammo-Crotonsaure und der~,'-Ammo-Butter~aure. Hoppe-~Se~/~ Z phywol Chem , 297 (1954) 71 73 20 PECK, [~. J . AND AWAPARa...1 VormaUon of t a u r m e and ~sethlomc acid m ~at brain. B~o~/..z bwphy,~ Acta (,4rest), 141 (1967) 499 -506 2l ROB~RlS, E , A~D KURIYAMA. K , Bzochemlcal-ph;yslologlcal correlatJonb m q u d l e s ol the 7ammobutyr[c acid s~stem, Brain Resear~ h. 8 (1968) 1-35 22 RuMPr, P , PrdparatJon d"ac~des ammoalco21sulfomques en rue d'une etude physlcoclmnJque comparative. Bull Chtm 5,,~ /:)ame, 5 (1938) 871-888. 23 S'~J[NSK'~, 1 A , x'~D VaSH IJl-~, g Y , Sollle catalytic properties of purified ?,-ammobut~rate-;~oxoglutarate tran~anuna~e f r o m rat brain, l;nzymologia, 39 (1970) l - I 1. 24 TAKAHASHI. H , A'qD MIZt~H,XRA. K . . - ( h l o r o - ? , - a m m o b u t y r t c acid, Jap Patent. 1965. 13847. Chem .4h,st~., 63 (1065) 13[)84b. 25 WAKSMAN, ~., AND RORI-R~[S, ~ , Tran~ammase act~wty of dmphorase, phosphorylasc A and several dehydrogenases. Biochem hwph~ ~ Re~ C o m m . n , 12 (1963) 263-267
459
c~'~ Elsewer Scientific Pubhshlng Company, Amsterdam - Printed m The Netherlands
Transamination of analogues of ),-aminobutyric acid by extracts of rat brain mitochondria
P M BEART Axo G A. R. JOHNSTON Department of Phys'tology, Austrahan Nattonal Untver~ity, Canberra (Austraha)
(~ccepted October 3rd, 1972)
The inhibitory synaptic transmitter, ),-aminobutyric acid (GABA), is catabohsed to succinic semlaldehyde in the nervous system by G A B A • 2-oxoglutarate amino transferase (GABA-T, E.C. 2.6.1 19)21. We have examined the ability of extracts of rat brain to transaminate some conformationally restricted analogues of GABA, various substituted G A B A derivatives and related compounds, using radioactive 2oxoglutarate as the amino group acceptor and measuring the production of radioactive glutamate by a convenient and sensitive procedure. Brain mltochondria contain a number of amino acid transaminases, in addition to GABA-T, which use 2-oxoglutarate as a substrate3: thus, activity in the present study does not necessarily imply that a substance is a substrate for GABA-T. On the other hand a substance which is inactive is unlikely to be a substrate for this enzyme. A crude mitochondrial preparation was isolated from rat cerebral gray matter as described previously 7. The enzymic production of glutamate from 2-oxoglutarate by this preparation was assayed as follows: 0.1 ml of a stock mixture, containing 10 m M dithiothreitol and 0 3 m M pyridoxal phosphate in 100 m M Tris buffer at p H 8.5, was mixed with 0.05 ml of 4 m M 2-oxoglutarate (containing 0.1/zC1 of [1-14C]2-oxogluta rate) and 0.1 ml of 3 m M potential amino group donor in l M, p H 8.5 Tris buffer and preincubated at 37 °C for 5 mm. The mltochondnal preparation (0.1 ml) was then added and incubation continued for 30 min. The reaction was stopped by the addition of 0.05 ml of 25 }o aqueous trichloroacetic acid and the radioactive glutamate formed determined by a procedure similar to that of Waksman and Roberts 25. The entire sample was transferred to a column (1 cm "< 4 cm) of Dowex 50W X4 [I4~] resin ( 100-200 mesh), and the column eluted with water (20 ml) and 2 M aqueous a m m o m a (2 y, 5.0 ml). A 1.0 ml aliquot of the second ammonia eluent was taken and its radioactivity measured in 10 ml of dioxan scintillator. This second ammonia eluent, when concentrated in vacuo in a rotatory evaporator and examined by thin layer chromatography on cellulose plates, contained a single ninhydrin positive spot of identical RF to an authentic sample of L-glutamate. All analogues that contained an asymmetric carbon atom were tested as racemates. The following were gifts: 2-fluoro-GABA (2-fluoro-4-aminobutanoic acid, Dr.
460
SHOR[ ( ()MVIUNI( \ t)l ~ ,,~
TABLE l SUBSTRATES
FOR T H F
lzNZ~Ml(
PI~.()DLJ(FION
()f
(,tUTAMAT[
FROM
2-OXOGI[
IAR,'.,IF
B~
t~,zkl
Pi~"
MITOCHONDRI,X Results are expressed relative to the actlwt~ of G A B A as a substrate and are mean~ S E of ~,l~e~ f r o m at least 3 experiments The following showed no measurable activity as substrate~ 2-Amlnobutano~. a~.ld, ~t,~- and ~ cm,ammocyclohexane-l-carboxyhc acid, 4 - a m m o p e n t a n m c acid, 3 - a m m o p r o p a n e s u l p h o m c a~.~d. 4 - a m m o t e t r o h c acid, 2-fluoro-GABA, gl~cme, 2-hydroxy-GABA, 3-hydroxv-GABA, mu~cm~ol, taurlne.
Sub,~traIe Tran,~-4-ammocrotomc acid GABA fi-Alamne 5-Aminopentanolc acid 2-MethyI-GABA 2-Methyl-3-ammoproplomc acid 2-Chloro-GABA Homohypotaurme
4~ tt)try telattve to that o/ GABA I 7~ 0.04 I 00 - 0.04 0 90 0 04 0 ~0 0 04 0,30 0 02 0 24 ~ 0 02 0.15 0 02 0 15 0 04
P. Goldman, Bethesda), musctmol (Dr. P. Hackett, Sittingbourne), c&- and trans-3aminocyclohexane-l-carboxylic acid (Dr. D.I.B. Kerr, Adelaide), and homohypotaurine (3-ammopropanesulphtmc acid, Dr. B. Jolles-Bergeret, Orsay). The following were prepared by pubhshed procedures, trans-4-ammocrotonic aczd 19, 4-ammopentanoic acid a3, 3-aminopropanesulphomc acid ~'z, 4-aminotetrolic acid .5, 2-chloroGABA (2-chloro-4-ammobutanolc acid) '-'4, 2-hydroxy-GABA (2-hydroxy-4-ammobutanolc acid) 14, and 2-methyl-GABA (2-methyl-4-aminobutanolc acid) 1. Other analogues were purchased from chemical suppliers [l-14C]2-Oxogtutarate specific activity 24 mO/mmole, was purchased from Calatomic, Los Angeles. The results are summarised m Table 1. Trans-4-aminocrotomc acid, a conformationally restricted analogue of GABA an an extended conformanon 6, was an excellent amino group donor, being more ef~clent than GABA under the condinons investigated. None of the other conformaUonally restricted GABA analogues (4-ammotetrohc acad, musclmol, cts- and trans-3aminocyclohexane-l-carboxylic acids) showed measurable acnvlty. 4-Ammotetrohc acid, although not a substrate for GABA-T, is an inhlbator (linearly competinve wnh GABA) of GABA-T activity measured by the coupled formation of succinate fi'om GABA 7 Other workers have noted the ability of extracts of mammahan brain to transaminate/3-alanine, 5-aminopentanolc acid, and 2-methyl-3-aminoproptomc acad, and the inabihty of these extracts to transammate 3-hydroxy-GABA2,z3 TransammaUon of 2-aminobutano~c acid and glycme, which were reactive m the present study, ha~ been reported in extracts of beef brain 2. Studies on the glycme-2-oxoglutarate aminotransferase act~wty m rat brain indicate that th~s is a glycine forming rather than a glycine degrading enzymic activity tT.
SttORT COMMUNICATIONS
461
Substttuents on c a r b o n s 2 a n d / o r 4 of G A B A either considerably reduced (2methyl- a n d 2 - c h l o r o - G A B A ) or abolished (2-hydroxy-, 2-fluoro- a n d 4-methylG A B A , cis- a n d t r a n s - 3 - a m i n o c y c l o h e x a n e - l - c a r b o x y h c acid) activity in the present study. T a u r i n e , o n the basis of occurrence, postsynaptic action a n d antagonists thereof, a n d u p t a k e a n d release studies 9,11,r),15,16, m a y be an i m p o r t a n t i n h i b i t o r y t r a n s m i t t e r in m a m m a l i a n brain. Little is k n o w n of t a u r i n e c a t a b o h s m other t h a n that it is slowly converted to isethionate ~°. It ~s not t r a n s a m i n a t e d u n d e r the c o n d i t i o n s used m the present study. If a t a u r l n e : 2 - o x o g l u t a r a t e a m l n o t r a n s f e r a s e activity is present in rat b r a i n its p H o p t i m u m a n d / o r subcellular d i s t r i b u t i o n might be different from that of G A B A - T . It is interesting that while the sulphonic acids, t a u r m e and 3 - a m l n o p r o p a n e s u l p h o n l c acid, c a n n o t be t r a n s a m l n a t e d u n d e r conditions favourable to t r a n s a m i n a t i o n of the c o r r e s p o n d i n g carboxylic acids, G A B A a n d fi-alanine, the sulphinlc acid, h o m o h y p o t a u r i n e is shows some activity. M a n y of the G A B A analogues that were inactive as a m i n e d o n o r s to 2-oxoglutarate in the present study, a n d thus unlikely to be substrates for G A B A - T , are k n o w n to be able to activate bicuculline-sensltive postsynaptic receptors 4,s,u~. 3 - h y d r o x y - G A B A , 3 - a m i n o p r o p a n e s u l p h o n i c acid, muscimol, 2-ftuoro-GABA, a n d 4 - a m m o t e t r o l i c acid. We conclude that G A B A - T has a very different substrate specificity from that of blcuculline-sensltive postsynaptlc receptors.
1 ADAMS, R., AND FLES, D., The absolute configuration of the Ca atom In retronecanone (1-methvl-
7-oxopyrrohzldlne), J. Amer chem. Soc , 81 (1959) 49464951. 2 BAXTER, C. F , AND ROBERTS, E , The 7-ammobutyrlc acld
462
,~H()Ri
~ i~MMII\I*
'~ ~ : , ,
,'
[5 JA(OBSIbN. J G , ~,~D SMtlH. 1, H , Btochemtstry and physiology of tattrmc an0 Lau~¢m ~ ~vatlves, P t n s w l Re~ , 48 ([968) 424-51t 16 JASPER, H H., AND KO',AMA, | , Rate ot release of anamo acids f r o m the cerebral corlc~ ~, ~hc cat as affected by b r a m s t e m and thalam~c %tlmu]atlon. ('attad J Pkvs'tol P]tatnlclto]. 47 ¢~'~-~tl 889 005 17 JOt-INSION, G A R . ~,ND VH,kLI. M V.. (Jlycme:2-oxogluterate transammd~e m Tat ~c,cb~al cortex, Brain Reseur~ It. [ 4 (1969) 201 -208 18 JOLLES-BERGERET, B , A new sulphmtc amino acid: h o m o h y p o t a u r m c ( ? - a m m o - p r o p a n e . u l phmlc acid), E.rop I. B w ( h e m , I0 (t969) 569-573 19 M USASHI, A , Synthe.se der ;,-Ammo-Crotonsaure und der~,'-Ammo-Butter~aure. Hoppe-~Se~/~ Z phywol Chem , 297 (1954) 71 73 20 PECK, [~. J . AND AWAPARa...1 VormaUon of t a u r m e and ~sethlomc acid m ~at brain. B~o~/..z bwphy,~ Acta (,4rest), 141 (1967) 499 -506 2l ROB~RlS, E , A~D KURIYAMA. K , Bzochemlcal-ph;yslologlcal correlatJonb m q u d l e s ol the 7ammobutyr[c acid s~stem, Brain Resear~ h. 8 (1968) 1-35 22 RuMPr, P , PrdparatJon d"ac~des ammoalco21sulfomques en rue d'une etude physlcoclmnJque comparative. Bull Chtm 5,,~ /:)ame, 5 (1938) 871-888. 23 S'~J[NSK'~, 1 A , x'~D VaSH IJl-~, g Y , Sollle catalytic properties of purified ?,-ammobut~rate-;~oxoglutarate tran~anuna~e f r o m rat brain, l;nzymologia, 39 (1970) l - I 1. 24 TAKAHASHI. H , A'qD MIZt~H,XRA. K . . - ( h l o r o - ? , - a m m o b u t y r t c acid, Jap Patent. 1965. 13847. Chem .4h,st~., 63 (1065) 13[)84b. 25 WAKSMAN, ~., AND RORI-R~[S, ~ , Tran~ammase act~wty of dmphorase, phosphorylasc A and several dehydrogenases. Biochem hwph~ ~ Re~ C o m m . n , 12 (1963) 263-267