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Properties of L-glutamate decarboxylase from non-neuronal tissues
Properties of L-Glutamate Decarboxylase from Non-Neuronal Tissues
WU, J.-Y. P,vqwrtic~.s of L-,q/uttrmtrtr dcr,rr,h~l.~~/tr.,~,,fionr ~IO/I-~I(,~~~.O)~(IIti.ssuc.v. BRAIN RES. BULL. 5: Suppl. 2, 3 i-36. 1980.--The activity of L-glutamate decarboxylase (EC 4.1. I. 15)(GAD) in various mouse tissues was determined by five different methods, namely, the radiometric CO, method, column separation, electrophoretic separation, the filtration method, and amino acid analysis. Results from the latter four methods agreed well, showing that brain had the highest activity, 4.27 nmoliminimg protein (lCO%), followed by heart (7.4%), kidney (6.3%) and liver (I 3%). Measurement of brain GAD using the radiometric CO2 assay method agreed with the other techniques. However, in heart, kidney, and liver, the GAD activities measured by the COZ method were about 3-4 times higher than those obtained by the GABA method, suggesting that the CO, method does not give a valid measurement of GAD activity in a crude non-neural tissue preparation. GAD activity also was detected in adrenal gland but not in pituitary, stomach, testis, muscle, uterus, lung, salivary gland or spleen. GAD has afso been purified from bovine heart to about X00-fold over the homogenate. Both crude and highly purified GAD preparations from heart were used for immunochemical studies. GAD from brain and spinal cord appear to be identical as judged from the following results: the ~mmunoprecipitin bands fused together without a spur; the enzyme activity was inhibited by anti-GAD to the same extent; and the microcomplement fixation curves were similar in both the shape of the curve and the extent of fixation. No crossreactivity was observed between GAD from heart, kidney or liver and antibody against brain GAD in all the immunochemical tests described above, suggesting that GAD in nonneuronal tissues is different from that in brain and spinal cord. L-glutamate decarboxylase
GABA
are many lines of evidence to suggest that y-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in both the invertebrate peripheral and central nervous systems and the vertebrate central nervous system (CNS) 12, 11, 15, 161. Recently, it has been reported that GABA has depolarizing actions on peripheral ganglion cells and their axons ]4]. Biosynthesis and accumulation of GABA have also been reported to occur in the inner ear and lateral line organs of frog and fish [5] and in the olfactory nerve of fish [19] suggesting that GABA might be an excitatory neurotransmitter in sensory pathways. In addition to the neurotransmitter role, GABA may be utilized as an energy source via the GABA shunt pathway 111. In light of the diverse functions of GABA, it seemed appropriate to examine the distribution and specificity of the GABA-synthesizing enzyme, L-glutamate l-carboxy-lase (EC 4.1.1. IS) (GAD) in various tissues. Although GABA and GAD were originally believed to exist exclusively in the CNS in vertebrate [17], with more sensitive methods GABA has been detected in kidney, and GAD activity also has been detected in glia and non-neural tissues such as kidney, heart, liver, and blood vessels [6-9, 12, 23, 32, 341. It has also been reported that glial GAD, which is different from neuronal GAD, has the same properties as the GAD found in non-neural tissues 16-91. Since the above observations were made with crude preparations, it is important to establish the identity or nonidentity of GAD from neuronal, glial and non-neural tissues using highly purified preparations. It is also highly desirable to compare GAD from various sources using sensitive imTHERE
Copyright
(’ 1980 ANKHO
International
munochemical techniques since neuronal GAD has been purified to homogeneity [22, 24-26, 301 and its specific antibody is aiso available [3, 20, 21, 27, 28, 331. This communication describes the distribution of GAD in various tissues by five different assay methods: the radiometric CO, method [18], the rapid filtration-ion exchange method [3], the ion exchange column method [lo], the electrophoretic separation, and amino acid analysis [14]. In addition, immunochemical and biochemical comparisons of GAD from various tissues [29,32], particularly between purified neuronal GAD and bovine heart GAD [26], are also included.
GAD activities in various tissues were first examined by the CO, method as described [1X]. As was expected, the brain had the highest GAD activity, 4.63 x lo-” prnol of CO, formed per min per mg protein (loo%), followed by spinal cord (70%), kidney (22%), heart (18%), liver (S%), and adrenal gland (2.7%). Pituitary, stomach, testis, muscle, uterus, lung, salivary and spleen tissues did not contain detectable amounts of GAD activity. GAD activities in brain, kidney, heart and liver were further examined by the formation of GABA. Four different methods, namely the rapid filtration-ion exchange method [3J, the ion exchange column method [lo], the electrophoretic separation and amino acid analysis [14] were used for GABA determination and the results are summarized in Table I.
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wu TABLE DISTRIBUTION
OF L-GLUTAMATE
DECARBOXYLASE
I ACTIVITY
IN VARIOUS
TISSUES
OF MOUSE -
Activity (units Y IWmg protein)
Tissue
CO, method
Column* separation
Brain Heart Kidney Liver
4.63 0.82 1.00 0.22
4.55 0.35 0.17 -
“GABA was measured iGABA was calculated *GABA was calculated PGABA was calculated lIGAD activity in brain
GABA method Electrophoresist Fittration$ separation method 4.68 0.30 0.35 -
Bovine
%‘I
3.60 0.32 0.33 0.07
4.27 0.315 0.268 O.&f
100 7.4 6.3 1.5
as total counts in the eluate after subtracting the counts in the control sample. based on the ratio of the area of GABA peak to total area in electrophoresis. from counts in the filtrate after subtracting counts in the control sample. from counts in fractions corresponding to GABA peak in the standard. homogenate was used as reference. 1OW.
From Table 1, it is clear that the GAD activity in brain measured by the CO, method is comparable to that obtained by the GABA method. However, in the case of non-neural tissues, e.g., heart, kidney and liver, GAD activity determined by the CO, method is about two to four times higher than the values obtained by measurement of the formation of GABA. The validity of the GABA measurements was established by paper electrophoresis and amino acid analysis by comparing the peak position of the (W)GABA formed from the GAD reaction with that of authentic GABA as shown in Figs. 1 and 2. In addition to GABA, there are several unidentified radioactive components in the eluates of the GAD reaction mixtures from various tissues as judged by electrophoresis and amino acid analysis (Figs. 1 and 2). Fu~i~~.uti(~nof GAD from
4.25 0.29 0.22 (1.06
Amino acid5 analysis
Activity assigned (units x IO:‘) mg protein
Hrcrrt
GAD has been purified from bovine heart more than 200fold over the initial homogenate by extraction with Triton X-100 followed by ammonium sulfate fractionation and column chromatography on DEAE-cellulose, calcium phosphate gel, DEAE-Sephadex and Bio-Gel A-l .S m 1261. The purification results are summarized in Table 2. The purified heart GAD appears to be highly specific regarding its substrate specificity since of the 20 naturally occurring amino acids plus D-glutamate and cY-ketoglutarate, only L-glutamate can serve as substrate for the highly purified heart GAD preparations. The a-decarboxylation of L-glutamate by the purified heart GAD preparation was established by the ident~cation of the reaction products as CO, and GABA in a molar ratio of 1:l (Fig. 3). In addition to GABA, an unidentified W-labeled product which was eluted between glycine and alanine in the amino acid analyzer was also observed. Under the same conditions, when the reaction mixture of brain GAD was analyzed in the amino acid analyzer, GABA was the only product (Fig. 3). This unidentified product is present in a lo-fold excess over GABA and may represent the product of a major reaction catalyzed by either heart GAD or some other enzymes which are still present in this highly purified heart GAD preparation. Although this unknown product has an elution profile similar to that of a-aminobutyric acid (eluted between glycine and alanine in the amino acid analyzer), it seems unlikely that it is
u-aminobutyric acid, the product of the w-decarboxylation of glutamate, because no ‘“CO, was formed when L-( I-Y) glutamate or L-(U-‘*C) giutamate was replaced by DL-(5-Y) glutamate. Fu~he~ore, should both o- and LY-decarboxylation occur, we would not expect a molar ratio of 1 for GABA and CO,. It is more likely that this unknown product is a polymerized or cyclized product of glutamate, since it behaves like a neutral compound.
Since GAD has been purified from mouse brain to homogeneity and the antibody against the purified enzyme was available in our laboratory [13, 20, 21, 24, 301, several immunochemical techniques were employed to determine the crossreactivity between non-neuronal GAD in both crude and highly purified preparations and the antibody against purified mouse brain GAD. No crossreactivity was observed as measured by several criteria. First, GAD activity in non-neuronal preparation was not affected by antiGAD IgG, while the GAD activity in either crude or purified mouse brain preparations was inhibited to an extent of 75%. Normal IgG had no effect of GAD activity in all the preparations that had been tested. Second, no precipitin band was observed with non-neuronal GAD and antibody, while a clear precipitin band was observed when crude GAD preparations from mouse brain, mouse spinal cord and bovine brain were tested on an immunodiffusion gel (Fig. 4). Third, no fixation of complement occurred with non-neuronal GAD preparation and anti-GAD IgG, while 9&95% and 4%5% fixations of complement were observed with 6 pg anti-GAD IgG and GAD preparations from mouse brain-mouse spinal cord and bovine brain, respectively (Fig. 5).
Pyruvate was a potent inhibitor of heart GAD, inhibiting the GAD activity to an extent of 95% at 10 mM. The mouse brain GAD in either the purified or the crude preparations was inhibited by about 2% by pyruvate at the same concentration. DTNB, one of the most potent inhibitors of purified mouse brain GAD (Ki= 1.1 x 10mnM) [24,3 1J, inhibited heart GAD preparations by about 30-5% at 1 mM. The GAD activity in a crude mouse brain preparation was completely
GAD IN NON-NEURONAL
--= _- ..--_. m.
_
TISSUES
,
FIG. I. Electrophoresis of Dowex 1column eluates of GAD reaction mixtures. For either brain or heart, enzyme activity was first determined by ‘%ZO, measurement as described previously. Each assay vessel contained 2.86 mg protein for brain and 4.24 mg protein for heart. Three such reaction mixtures for either tissue, after CO, diffusion. were combined to give a total volume of 3.9 ml. This was centrifuged at 3000 revlmin for 10 min to obtain a 3.5 ml supernatant. Dowex I chromatography and electrophoresis were then carried out as described previously. The brain electrophoretogram shown above has been attenuated by a factor of 100. I=brain, Il=heart and III-standard.
by DTNB at lo’-” M. NaCI, which is a weak inhibitor of the brain GAD (Ki=17 mM) [24,31], activated heart GAD activity by about I%--190% at 0.2 M. inhibited
CONCLUDING REMARKS Neuronal GAD and non-neuronal GAD are different in several aspects. First, they are different in their immunochemicai properties. No crossreactivity is found between non-neuronal GAD and the antibody against purified mouse brain GAD as shown by immunodiffusion, enzyme inhibition by antibody, or mi~rocompiement fixation. Second, they are different in their enzymatic properties. The heart enzyme is greatly inhibited by pyruvate (95% at JO mM) whiie the brain enzyme is only slightly inhibited (20% at 10 mM). On the other hand, the heart enzyme is only slightly
FIG. 2. Amino acid analysis of GAD reaction mixtures A=brain; B=heart; C=kidney; D=liver; E==control, i.e., reaction mixture without protein: F=purified L-(U-Y) glutamate; G=nonradioactive amino acid standards. Each tissue homogenate was assayed by the “CO, method as described previously. The protein content for each assay volume of 1.1ml was as follows: brain=0.95 mg; heart: I .41 mg; kidney= I. 12 mg and liver= 1.22mg. After lJCO, diffusion, the reaction mixture was analyzed with a Beckman Model 119 automatic amino acid analyzer using Durmm Type DC-IA resin.
inhibited by DTNB (30-W% at 1 mM), while the brain enzyme is completely inhibited even at lo-” M (Ki = 1.1x 10Mx)[24,31]. NaCl is a weak inhibitor for brain GAD (Ki= 17 mM) [24,31] but is an activator for heart GAD. Third, they differ in their substrate specificity. Among twenty naturally occurring amino acids and a-ketoglutarate, the brain
wu
34
TABLE2 PURIFlCATION
OF L-GL~rAMAT~
D~CARBOXYLASE
FROM BOVINE
Total activity
Total protein
Sample
(units)
(mg)
Specific activity (units/mg x lo’)
Homogenate extract (NH*)~SO~(2~35~) DEAE (pool) Calcium phosphate gel (pool) DEAE-Sephadex (pool) Bio-Gel A-l.5 rn$ (pool) Bio-Gel A-1.5 m# (pool)
31.5 44.6 27.4 9.5 3.8 1.6 0.61 0.46
350,000 48,000 9.450 860 120 7.P 8.Ot 10.5” t&ii 1.21 :i.os 1.9
0.009 0.093 0.29 1.1 3.1 10.2* 22SF 24.0
Triton
HEART
Recovery of activity IQ) 100 165 87 30 12 5 1.9 1.4
*The fust GAD peak from the DEAE-Seph~ex column. iThe second GAD peak from the DEAE-Sephadex column. $The sample applied to the B&gel A-l .5 m column was the first GAD peak from the DEAESephadex column. EjThe first GAD peak from Bio-Gel A-1.5 m column. Whe second GAD peak from Bio-Gel A- I .f m column.
#The sample applied to the Bio-Gel A-1.5 m column was the second GAD peak from the DEAE-Sephadex
column.
’ ‘\ !30 240 250 260 270 280 290 300 Elutlon
vol,
ml
FIG. 3. Amino acid analyzer elution profile of the GAD reaction mixture with L-glutamate as substrate. 0.5 ml of the reaction mixture plus the standards L-glutamic acid, glycine, L-alanine, @-alanine, GABA and L-histi~ne were applied to the analyzer cohnnn. Amino acids were 3; heart identified as their ninhydrin products at 570 nm (---), and radioactivity was expressed as c.p.m. (brain reaction mixture, O--, reaction mixture (O-----O).
enzyme can only catalyze a-decarboxylation of L-aspartic acid and L-glutamic acid (substrate activity of L-aspartic acid is about 3-S% of that of L-glutamic acid) [24,30], while the heart enzyme can only use L-glutamic acid as substrate. Moreover, when the reaction mixtures are examined by amino acid analysis, the brain reaction mixture contains only GABA, while the heart reaction mixture contains an unidentified radioactively-labeled product in addition to GABA. It is interesting that the GABA-degradative enzyme, GABAtransaminase (GABA-T), has been shown to be lacking in tissue-specificity [32] (also see Schousboe et al., in this volume). In light of its high substrate and tissue specificites,
GAD is undoubtedly mainly, if not exclusively, involved in the biosynthesis of GABA for use either as a transmitter or for energy metabolism. GABA-T on the other hand, may be involved in the metabolism of other amino acids, e.g., p-alanine, in addition to GABA.
ACKNOWLEDGEMENTS
This work was supported in part by a grant from the National Institutes of Health (NS-13224) and the Huntington’s Chorea Foundation.
GAD IN NON-NEURONAL
TISSUES
FIG. 4a. Ouchterlony double diffusion tests of GAD preparations from various mouse tissues. In all cases 30 ~1 of antiserum against the purified GAD preparation from mouse brain were placed in the center well and 30 ~1 of concentrated solution of the enzyme from different tissues in the outer wells. Plate on the right shows (l), (3), (5) brain (2) kidney (4) heart (6) liver. Plate on the left shows (1). (3), (5) brain (2) spinal cord (4) kidney (6) heart. The plates were incubated for 24 hr at 4°C before the pictures were taken. FIG. 4b. The same conditions as those described in 4a with the exception of the antigens. Plate shown: (I) crude GAD (specific activity 3.0~ IO-’ units/mg) from mouse brain; (2) crude GAD (specific activity 1.6x IO-’ unitsimg) from bovine brain; (3) crude GAD from mouse brain (specific activity 3.0x 10eL unitsimg); (4) purified GAD from bovine heart (specific activity 2.1~ 10-r unitsimg); (5) purified GAD from bovine heart (specific activity 2.25x 10-l unitsimg); (6) purified GAD from bovine heart (specific activity 2.4~ IO-’ unitsimg).
t
I
40
I
80
-
160
nq GAD FIG. 5a. Microcomplement fixation curve of GAD. The amounts of complement fixed (in SC) were plotted against the amounts of GAD from different tissues. The amount of GAD (ng) was calculated from the protein concentrations and the specific activities of the samples by expressing the latter as a percentage of the specific activity of the purified enzyme. GAD preparations from mouse brain (A ---A), and spinal cord (O-0). No fixation of complement was obtained with GAD preparations from heart, kidney and liver (not shown). FIG. 5b. The same procedures as those described in Fig. 5a except that the antigens used were from different preparations. 0 -0, crude GAD preparation from mouse brain (specific activity 3.0x IO-’ unitsimg). a --.fi, crude GAD preparation from the bovine brain (specific activity I .6x 10m2units/mg). l l , purified GAD from bovine heart, specific activity 2.1 x 10-r unitsimg). A ----A, purified GAD from bovine heart, specific activity 2.25x 10-l unitsimg.
36
WU REFERENCES
1. Baxter, C. F. The nature of y-aminobuty~c
acid. In: ~11~~b~~~~~ edited by A. Lajtha. New York: Plenum Press, 1970, Vol. 3, pp. 289-353. 2. Bazemore, A. W., K. A. Elhott and E. Florey. Isolation of factor I. J. Nrurochem. 1: 334-339, 1957. 3. Chude, 0. and J.-Y. Wu. A rapid method for assaying enzymes whose substrates and products differ by charge-application to brain L-glutamate decarboxylase. J. Neurochem. 27: 83-86, o$Neurochemisfry.
1976.
DeGroat, W. C. GABA-depolarization of a sensory ganglion: antagonism by picrotoxin and bicuculline. Bruin Res. 38: 429 432, 1972. 5. Flock, A. and D. M. K. Lam. Neurotransmitter synthesis in inner ear and lateral line sense organs. Nature 249: 142-144, 1974. 6. Haber, B., K. Kuriyama and E. Roberts. Decarboxylation of glutamate by tissues other than brain, non-identity with CNS GAD. ~ecln Proc. Fedn Am. Sots. uxp. BittI. 28: 577, 1949. 7. Haber, B., K. Kuriyama and E. Roberts. Mitochond~al localization of a new L-glutamic acid decarboxylase in mouse and human brain. Brain Re.s. 22: 105-112, 1970. 8. Haber, B., K., Kuriyama and E. Roberts. L-glutamic acid decarboxylase: a new type in glial cells and human brain gliomas. fcirncf 168: 598-599, 1970. 9. Haber, B., K. Kuriyama and E. Roberts. An anion stimulated L-glutamic acid decarboxylase in non-neural tissues: occurrence and subcellular localization in mouse kidney and developing chick embryo brain. Biochrm. Pharmac. 19: 1119-1136, 1970. 10. Karvitz, E. A., P. B. Molinoff and 2. W. Hall. A comparison of the enzymes and substrates of gamma-am~nobuty~~ acid metabolism in lobster excitatory and inhibitory axons. Proc. natn. Acad. Sci. U.S.A. 54: 778-782, 1%5. Il. Kmjevic, K. Chemical nature of synaptic transmission in vertebrates. Physiol. Rev. 54: 411540, 1974. 12. Kuriyama, K., B. Haber and E. Roberts. Occurrence of a new L-gtutamic acid decarboxylase in several blood vessels of the rabbit. Brain Res. 23: 121-123, 1970. 13. Matsuda, T., J.-Y. Wu and E. Roberts. Immunochemical studies on glutamic acid decarboxylase from mouse brain. J. NCU~~JC~W. 21: 159-166, 1973. 14. Moore, S. and W. H. Stein. Chromatography of amino acids on sulfonated polystyrene resins. J. hi&. Chem. 192: 663-681, 1951. 15. Otsuka, M., L. L. Iversen, Z. W. Hall and E. A. Kravitz. Release of gamma-aminobutyric acid from inhibitory nerves of lobster. Proc. natn. Acud. Sci. U.S.A. 56: 1110-1115, 1966. 16. Roberts, E., T. N. Chase and D. B. Tower (editors). GABA in Nervous Sysiem Function. New York: Raven Press, 1976. 17. Roberts. E. and S. Franlcel. v-Aminobutyric acid in brain. Fedn Proi,. 9: 219, 1950. 18. Roberts, E. and D. G. Simonsen. Some properties of L-glutamate decarboxvlase in mouse brain. Biochem. Pharmucol. 12: 113-134, 1963.4.
19. Roskoski. R.. Jr., L. D. Ryan and F. J. P. Diecke. y-Aminobutyric acid synthesized in the olfactory nerve. Nature 251: 526-529, 1974. 20. Saito, K. Immunochemical studies of GAD and GABA-T. In: CABA in Nervous System E’unc~tion, edited by E. Roberts, T. M. Chase and D. B. Tower. New York: Raven Press, 1976, pp~ 103-I 12. 21. Saito, K., J.-Y. Wu and E. Roberts. Immunochemical comparisons of vertebrate glutamic acid decarboxylase. Brtrin Rrs. 65: 277-285, 1974. 22. Su, Y. Y. T., J.-Y. Wu and D. M. K. Lam. Purification of L-glutamic acid decarboxylase from cattish brain. 1. N~,~r[~~~lern,33: 169179, 1979. 23. Whelan, D. T., C. R. Striver and F. Mohyuddin. Glutamic acid decarboxylase and gamma-aminobutyric acid in mammalian kidney. Nuturc, Land. 224: 916-917, 1969. 24. Wu, J.-Y. Purification and properties of L-glutamate decarboxylase (GAD) and GABA-aminotransferase (GABA-T). ln: GABA in Nrvvous System F~n~ti~~~, edited by E. Roberts, T. N. Chase and D. Tower. New York: Raven Press, 1976, pp. 7-155. 25. Wu, J.-Y. Purification of L-glutamate decarboxylase and cysteic acid decarboxylase from bovine brain. Proc. lnf. S~,C~. Ncurochem. 6: 632, 1977. 26. Wu, J.-Y. A comparative study of L-glutamate decarboxylase from brain and heart with purified preparations. .I. N~~,~~~~~j~~,,~. 28: 1359-1367. 1977. 27. Wu, J.-Y. Microanalytical methods for neuronal analysis. Physiol. Ret’. 58: 863-904, 1978. 28. Wu, J.-Y., M. S. Chen and W. M. Huang. Purification and immunoche~cal studies of L-glutamate decarboxylase and cysteic acid decarboxylase from bovine brain. Neitrorci. Ahstr. 4: 454, 1978. 29. Wu, J.-Y., 0. Chude, J. Wein, E. Roberts, K. Saito and E. Wong. Distribution and tissue specificity of glutamate decarboxylase (EC 4.1.1.15). J. Nrurochem. 30: 849-857, 1978. 30. Wu, J.-Y., T. Matsuda and E. Roberts. Purification and characterization of glutamate decarboxylase from mouse brain. .i. hiol. Chem. 248: 3029-3034, 1973. 31. Wu. J.-Y. and E. Roberts. Properties of brain L-glutamate decarboxylase: inhibition studies. .I. Ncurochcm. 23: 759-767, 1974.
Wu. J.-Y., K. Saito, E. Wong and E. Roberts. Studies of L-glutamate decarboxylase and y-aminobuty~c tr~sarni~~se from various tissues and species. Trcins. Am. SOC. Nrtrrcxhrm. 5: 112, 1974. 33. Wu, J.-Y., Y. Y. T. Su, C. Brandon, D. M. K. Lam, M. S. Chen and W. M. Huang. Purification and immunochemical studies of GABA-, acetylcholine-, and taurine-synthesizing enzymes from bovine and tish brains. Abstract of the ~~~~~,~~nth~nt~,rr~~i32.
tirtrrnl Meeting 34. Zac~hmon. M., y-uminohut,yric
of the
ISN, p. 662, 1979. P. Tocc,i and W. L. Nyhon. acid in human tissuc~.s other
C’hcm. 241: 3355-1358, 1966.
The occ’urren(‘e of’ thutl bruin. J. hiol.
WU, J.-Y. P,vqwrtic~.s of L-,q/uttrmtrtr dcr,rr,h~l.~~/tr.,~,,fionr ~IO/I-~I(,~~~.O)~(IIti.ssuc.v. BRAIN RES. BULL. 5: Suppl. 2, 3 i-36. 1980.--The activity of L-glutamate decarboxylase (EC 4.1. I. 15)(GAD) in various mouse tissues was determined by five different methods, namely, the radiometric CO, method, column separation, electrophoretic separation, the filtration method, and amino acid analysis. Results from the latter four methods agreed well, showing that brain had the highest activity, 4.27 nmoliminimg protein (lCO%), followed by heart (7.4%), kidney (6.3%) and liver (I 3%). Measurement of brain GAD using the radiometric CO2 assay method agreed with the other techniques. However, in heart, kidney, and liver, the GAD activities measured by the COZ method were about 3-4 times higher than those obtained by the GABA method, suggesting that the CO, method does not give a valid measurement of GAD activity in a crude non-neural tissue preparation. GAD activity also was detected in adrenal gland but not in pituitary, stomach, testis, muscle, uterus, lung, salivary gland or spleen. GAD has afso been purified from bovine heart to about X00-fold over the homogenate. Both crude and highly purified GAD preparations from heart were used for immunochemical studies. GAD from brain and spinal cord appear to be identical as judged from the following results: the ~mmunoprecipitin bands fused together without a spur; the enzyme activity was inhibited by anti-GAD to the same extent; and the microcomplement fixation curves were similar in both the shape of the curve and the extent of fixation. No crossreactivity was observed between GAD from heart, kidney or liver and antibody against brain GAD in all the immunochemical tests described above, suggesting that GAD in nonneuronal tissues is different from that in brain and spinal cord. L-glutamate decarboxylase
GABA
are many lines of evidence to suggest that y-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in both the invertebrate peripheral and central nervous systems and the vertebrate central nervous system (CNS) 12, 11, 15, 161. Recently, it has been reported that GABA has depolarizing actions on peripheral ganglion cells and their axons ]4]. Biosynthesis and accumulation of GABA have also been reported to occur in the inner ear and lateral line organs of frog and fish [5] and in the olfactory nerve of fish [19] suggesting that GABA might be an excitatory neurotransmitter in sensory pathways. In addition to the neurotransmitter role, GABA may be utilized as an energy source via the GABA shunt pathway 111. In light of the diverse functions of GABA, it seemed appropriate to examine the distribution and specificity of the GABA-synthesizing enzyme, L-glutamate l-carboxy-lase (EC 4.1.1. IS) (GAD) in various tissues. Although GABA and GAD were originally believed to exist exclusively in the CNS in vertebrate [17], with more sensitive methods GABA has been detected in kidney, and GAD activity also has been detected in glia and non-neural tissues such as kidney, heart, liver, and blood vessels [6-9, 12, 23, 32, 341. It has also been reported that glial GAD, which is different from neuronal GAD, has the same properties as the GAD found in non-neural tissues 16-91. Since the above observations were made with crude preparations, it is important to establish the identity or nonidentity of GAD from neuronal, glial and non-neural tissues using highly purified preparations. It is also highly desirable to compare GAD from various sources using sensitive imTHERE
Copyright
(’ 1980 ANKHO
International
munochemical techniques since neuronal GAD has been purified to homogeneity [22, 24-26, 301 and its specific antibody is aiso available [3, 20, 21, 27, 28, 331. This communication describes the distribution of GAD in various tissues by five different assay methods: the radiometric CO, method [18], the rapid filtration-ion exchange method [3], the ion exchange column method [lo], the electrophoretic separation, and amino acid analysis [14]. In addition, immunochemical and biochemical comparisons of GAD from various tissues [29,32], particularly between purified neuronal GAD and bovine heart GAD [26], are also included.
GAD activities in various tissues were first examined by the CO, method as described [1X]. As was expected, the brain had the highest GAD activity, 4.63 x lo-” prnol of CO, formed per min per mg protein (loo%), followed by spinal cord (70%), kidney (22%), heart (18%), liver (S%), and adrenal gland (2.7%). Pituitary, stomach, testis, muscle, uterus, lung, salivary and spleen tissues did not contain detectable amounts of GAD activity. GAD activities in brain, kidney, heart and liver were further examined by the formation of GABA. Four different methods, namely the rapid filtration-ion exchange method [3J, the ion exchange column method [lo], the electrophoretic separation and amino acid analysis [14] were used for GABA determination and the results are summarized in Table I.
Inc .-036
l-9230/80/08003
l-06$0 1.1 O/O
wu TABLE DISTRIBUTION
OF L-GLUTAMATE
DECARBOXYLASE
I ACTIVITY
IN VARIOUS
TISSUES
OF MOUSE -
Activity (units Y IWmg protein)
Tissue
CO, method
Column* separation
Brain Heart Kidney Liver
4.63 0.82 1.00 0.22
4.55 0.35 0.17 -
“GABA was measured iGABA was calculated *GABA was calculated PGABA was calculated lIGAD activity in brain
GABA method Electrophoresist Fittration$ separation method 4.68 0.30 0.35 -
Bovine
%‘I
3.60 0.32 0.33 0.07
4.27 0.315 0.268 O.&f
100 7.4 6.3 1.5
as total counts in the eluate after subtracting the counts in the control sample. based on the ratio of the area of GABA peak to total area in electrophoresis. from counts in the filtrate after subtracting counts in the control sample. from counts in fractions corresponding to GABA peak in the standard. homogenate was used as reference. 1OW.
From Table 1, it is clear that the GAD activity in brain measured by the CO, method is comparable to that obtained by the GABA method. However, in the case of non-neural tissues, e.g., heart, kidney and liver, GAD activity determined by the CO, method is about two to four times higher than the values obtained by measurement of the formation of GABA. The validity of the GABA measurements was established by paper electrophoresis and amino acid analysis by comparing the peak position of the (W)GABA formed from the GAD reaction with that of authentic GABA as shown in Figs. 1 and 2. In addition to GABA, there are several unidentified radioactive components in the eluates of the GAD reaction mixtures from various tissues as judged by electrophoresis and amino acid analysis (Figs. 1 and 2). Fu~i~~.uti(~nof GAD from
4.25 0.29 0.22 (1.06
Amino acid5 analysis
Activity assigned (units x IO:‘) mg protein
Hrcrrt
GAD has been purified from bovine heart more than 200fold over the initial homogenate by extraction with Triton X-100 followed by ammonium sulfate fractionation and column chromatography on DEAE-cellulose, calcium phosphate gel, DEAE-Sephadex and Bio-Gel A-l .S m 1261. The purification results are summarized in Table 2. The purified heart GAD appears to be highly specific regarding its substrate specificity since of the 20 naturally occurring amino acids plus D-glutamate and cY-ketoglutarate, only L-glutamate can serve as substrate for the highly purified heart GAD preparations. The a-decarboxylation of L-glutamate by the purified heart GAD preparation was established by the ident~cation of the reaction products as CO, and GABA in a molar ratio of 1:l (Fig. 3). In addition to GABA, an unidentified W-labeled product which was eluted between glycine and alanine in the amino acid analyzer was also observed. Under the same conditions, when the reaction mixture of brain GAD was analyzed in the amino acid analyzer, GABA was the only product (Fig. 3). This unidentified product is present in a lo-fold excess over GABA and may represent the product of a major reaction catalyzed by either heart GAD or some other enzymes which are still present in this highly purified heart GAD preparation. Although this unknown product has an elution profile similar to that of a-aminobutyric acid (eluted between glycine and alanine in the amino acid analyzer), it seems unlikely that it is
u-aminobutyric acid, the product of the w-decarboxylation of glutamate, because no ‘“CO, was formed when L-( I-Y) glutamate or L-(U-‘*C) giutamate was replaced by DL-(5-Y) glutamate. Fu~he~ore, should both o- and LY-decarboxylation occur, we would not expect a molar ratio of 1 for GABA and CO,. It is more likely that this unknown product is a polymerized or cyclized product of glutamate, since it behaves like a neutral compound.
Since GAD has been purified from mouse brain to homogeneity and the antibody against the purified enzyme was available in our laboratory [13, 20, 21, 24, 301, several immunochemical techniques were employed to determine the crossreactivity between non-neuronal GAD in both crude and highly purified preparations and the antibody against purified mouse brain GAD. No crossreactivity was observed as measured by several criteria. First, GAD activity in non-neuronal preparation was not affected by antiGAD IgG, while the GAD activity in either crude or purified mouse brain preparations was inhibited to an extent of 75%. Normal IgG had no effect of GAD activity in all the preparations that had been tested. Second, no precipitin band was observed with non-neuronal GAD and antibody, while a clear precipitin band was observed when crude GAD preparations from mouse brain, mouse spinal cord and bovine brain were tested on an immunodiffusion gel (Fig. 4). Third, no fixation of complement occurred with non-neuronal GAD preparation and anti-GAD IgG, while 9&95% and 4%5% fixations of complement were observed with 6 pg anti-GAD IgG and GAD preparations from mouse brain-mouse spinal cord and bovine brain, respectively (Fig. 5).
Pyruvate was a potent inhibitor of heart GAD, inhibiting the GAD activity to an extent of 95% at 10 mM. The mouse brain GAD in either the purified or the crude preparations was inhibited by about 2% by pyruvate at the same concentration. DTNB, one of the most potent inhibitors of purified mouse brain GAD (Ki= 1.1 x 10mnM) [24,3 1J, inhibited heart GAD preparations by about 30-5% at 1 mM. The GAD activity in a crude mouse brain preparation was completely
GAD IN NON-NEURONAL
--= _- ..--_. m.
_
TISSUES
,
FIG. I. Electrophoresis of Dowex 1column eluates of GAD reaction mixtures. For either brain or heart, enzyme activity was first determined by ‘%ZO, measurement as described previously. Each assay vessel contained 2.86 mg protein for brain and 4.24 mg protein for heart. Three such reaction mixtures for either tissue, after CO, diffusion. were combined to give a total volume of 3.9 ml. This was centrifuged at 3000 revlmin for 10 min to obtain a 3.5 ml supernatant. Dowex I chromatography and electrophoresis were then carried out as described previously. The brain electrophoretogram shown above has been attenuated by a factor of 100. I=brain, Il=heart and III-standard.
by DTNB at lo’-” M. NaCI, which is a weak inhibitor of the brain GAD (Ki=17 mM) [24,31], activated heart GAD activity by about I%--190% at 0.2 M. inhibited
CONCLUDING REMARKS Neuronal GAD and non-neuronal GAD are different in several aspects. First, they are different in their immunochemicai properties. No crossreactivity is found between non-neuronal GAD and the antibody against purified mouse brain GAD as shown by immunodiffusion, enzyme inhibition by antibody, or mi~rocompiement fixation. Second, they are different in their enzymatic properties. The heart enzyme is greatly inhibited by pyruvate (95% at JO mM) whiie the brain enzyme is only slightly inhibited (20% at 10 mM). On the other hand, the heart enzyme is only slightly
FIG. 2. Amino acid analysis of GAD reaction mixtures A=brain; B=heart; C=kidney; D=liver; E==control, i.e., reaction mixture without protein: F=purified L-(U-Y) glutamate; G=nonradioactive amino acid standards. Each tissue homogenate was assayed by the “CO, method as described previously. The protein content for each assay volume of 1.1ml was as follows: brain=0.95 mg; heart: I .41 mg; kidney= I. 12 mg and liver= 1.22mg. After lJCO, diffusion, the reaction mixture was analyzed with a Beckman Model 119 automatic amino acid analyzer using Durmm Type DC-IA resin.
inhibited by DTNB (30-W% at 1 mM), while the brain enzyme is completely inhibited even at lo-” M (Ki = 1.1x 10Mx)[24,31]. NaCl is a weak inhibitor for brain GAD (Ki= 17 mM) [24,31] but is an activator for heart GAD. Third, they differ in their substrate specificity. Among twenty naturally occurring amino acids and a-ketoglutarate, the brain
wu
34
TABLE2 PURIFlCATION
OF L-GL~rAMAT~
D~CARBOXYLASE
FROM BOVINE
Total activity
Total protein
Sample
(units)
(mg)
Specific activity (units/mg x lo’)
Homogenate extract (NH*)~SO~(2~35~) DEAE (pool) Calcium phosphate gel (pool) DEAE-Sephadex (pool) Bio-Gel A-l.5 rn$ (pool) Bio-Gel A-1.5 m# (pool)
31.5 44.6 27.4 9.5 3.8 1.6 0.61 0.46
350,000 48,000 9.450 860 120 7.P 8.Ot 10.5” t&ii 1.21 :i.os 1.9
0.009 0.093 0.29 1.1 3.1 10.2* 22SF 24.0
Triton
HEART
Recovery of activity IQ) 100 165 87 30 12 5 1.9 1.4
*The fust GAD peak from the DEAE-Seph~ex column. iThe second GAD peak from the DEAE-Sephadex column. $The sample applied to the B&gel A-l .5 m column was the first GAD peak from the DEAESephadex column. EjThe first GAD peak from Bio-Gel A-1.5 m column. Whe second GAD peak from Bio-Gel A- I .f m column.
#The sample applied to the Bio-Gel A-1.5 m column was the second GAD peak from the DEAE-Sephadex
column.
’ ‘\ !30 240 250 260 270 280 290 300 Elutlon
vol,
ml
FIG. 3. Amino acid analyzer elution profile of the GAD reaction mixture with L-glutamate as substrate. 0.5 ml of the reaction mixture plus the standards L-glutamic acid, glycine, L-alanine, @-alanine, GABA and L-histi~ne were applied to the analyzer cohnnn. Amino acids were 3; heart identified as their ninhydrin products at 570 nm (---), and radioactivity was expressed as c.p.m. (brain reaction mixture, O--, reaction mixture (O-----O).
enzyme can only catalyze a-decarboxylation of L-aspartic acid and L-glutamic acid (substrate activity of L-aspartic acid is about 3-S% of that of L-glutamic acid) [24,30], while the heart enzyme can only use L-glutamic acid as substrate. Moreover, when the reaction mixtures are examined by amino acid analysis, the brain reaction mixture contains only GABA, while the heart reaction mixture contains an unidentified radioactively-labeled product in addition to GABA. It is interesting that the GABA-degradative enzyme, GABAtransaminase (GABA-T), has been shown to be lacking in tissue-specificity [32] (also see Schousboe et al., in this volume). In light of its high substrate and tissue specificites,
GAD is undoubtedly mainly, if not exclusively, involved in the biosynthesis of GABA for use either as a transmitter or for energy metabolism. GABA-T on the other hand, may be involved in the metabolism of other amino acids, e.g., p-alanine, in addition to GABA.
ACKNOWLEDGEMENTS
This work was supported in part by a grant from the National Institutes of Health (NS-13224) and the Huntington’s Chorea Foundation.
GAD IN NON-NEURONAL
TISSUES
FIG. 4a. Ouchterlony double diffusion tests of GAD preparations from various mouse tissues. In all cases 30 ~1 of antiserum against the purified GAD preparation from mouse brain were placed in the center well and 30 ~1 of concentrated solution of the enzyme from different tissues in the outer wells. Plate on the right shows (l), (3), (5) brain (2) kidney (4) heart (6) liver. Plate on the left shows (1). (3), (5) brain (2) spinal cord (4) kidney (6) heart. The plates were incubated for 24 hr at 4°C before the pictures were taken. FIG. 4b. The same conditions as those described in 4a with the exception of the antigens. Plate shown: (I) crude GAD (specific activity 3.0~ IO-’ units/mg) from mouse brain; (2) crude GAD (specific activity 1.6x IO-’ unitsimg) from bovine brain; (3) crude GAD from mouse brain (specific activity 3.0x 10eL unitsimg); (4) purified GAD from bovine heart (specific activity 2.1~ 10-r unitsimg); (5) purified GAD from bovine heart (specific activity 2.25x 10-l unitsimg); (6) purified GAD from bovine heart (specific activity 2.4~ IO-’ unitsimg).
t
I
40
I
80
-
160
nq GAD FIG. 5a. Microcomplement fixation curve of GAD. The amounts of complement fixed (in SC) were plotted against the amounts of GAD from different tissues. The amount of GAD (ng) was calculated from the protein concentrations and the specific activities of the samples by expressing the latter as a percentage of the specific activity of the purified enzyme. GAD preparations from mouse brain (A ---A), and spinal cord (O-0). No fixation of complement was obtained with GAD preparations from heart, kidney and liver (not shown). FIG. 5b. The same procedures as those described in Fig. 5a except that the antigens used were from different preparations. 0 -0, crude GAD preparation from mouse brain (specific activity 3.0x IO-’ unitsimg). a --.fi, crude GAD preparation from the bovine brain (specific activity I .6x 10m2units/mg). l l , purified GAD from bovine heart, specific activity 2.1 x 10-r unitsimg). A ----A, purified GAD from bovine heart, specific activity 2.25x 10-l unitsimg.
36
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