A convenient assay for γ-aminobutyric acid transaminase

GABA Neurotmnsmissiotl Bruin Resrcrrch Bulletin,

Vol. 5, Suppl. 2, pp. 51-55. Printed in the U.S.A

A Convenient Assay for y-Aminobutyric Acid Transaminase SETTI Department

S. RENGACHARY

AND BOB IN-YU

YANG’

of Chemistry, College of Arts and Sciences and School of Medicine University of Missouri-Kansas City, Kansas City, MO 641 IO

RENGACHARY, S. S. AND B. I.-Y. YANG. A conwnim trsstry fbr yrrminohufyric~ wid trrrn.swnintrsc. BRAIN RES. BULL. 5: Suppl. 2, 51-55, 1980.-A facile technique is reported for separating succinic semialdehyde dehydrogenase (SSADH) from y-aminobutyric acid transaminase (GABA-T) in a commercially available mixture of the two enzymes. The dehydrogenase isolated is a suitable coupling enzyme for the spectrophotometric assay of GABA-T from various sources. To obtain SSADH, the enzyme mixture was first passed through a gel filtration column in 50 mM phosphate buffer, pH 7.2, to remove interfering ions. The filtrate was then adsorbed onto an affinity column of commercially obtainable pNADP-agarose. GABA-T was removed with 50 mM phosphate buffer, pH 7.2. SSADH was then specifically desorbed with 10 mM NADP in the same buffer. A 76% yield was obtained. Contamination by GABA-T and NADPH oxidase was determined to be 0.2 and 0.04%, respectively. The enzyme so prepared was stable, e.g. retaining up to 89% of its activity after 24 days, when stored in concentrated form in the presence of glycerol plus bovine serum albumin or egg yolk lysolecithin. GABA-T activity determined by the coupled spectrophotometric assay was compared to that obtained in a direct radioisotopic method. y-Aminobutyric acid Succinic semialdehyde

y-Aminobutyric dehydrogenase

acid transaminase

y-AMINOBUTYRIC acid transaminase (GABA-T, E.C. 2.6.1.19) catalyzes the conversion of GABA to succinic semialdehyde (SSA), GABA + a-ketoglutarate * SSA +

glutamate (Equation 1). The transamination reaction, followed by SSA dehydrogenase (SSADH)-catalyzed oxidation of SSA to succinic acid, SSA+NAD(P)+* succinic acid + NAD(P)H+H+ (Equation 2), constitutes the major pathway for the degradation of the putative inhibitory neurotransmitter GABA in mammalian brain [6]. In several disorders of neuronal excitability, it has been demonstrated that the level of GABA is considerably lower than normal in the extrapyramidal regions of the brain. In the case of Huntington’s chorea, the decrease in the concentration of GABA apparently results from a dramatic decline, as much as 85%, in the activity of glutamic acid decarboxylase (GAD) [17], the enzyme responsible for the biosynthesis of GABA. Since the enzymes GAD and GABA-T control the level of GABA, it is theoretically possible to restore GABA concentration by either activating the former or inhibiting the latter enzyme. In actuality, GAD activity can not be readily raised. Thus GABA-T constitutes a likely point for therapeutic intervention. Indeed, keen interest in the transaminase has led to the isolation and purification of this enzyme from numerous sources. As early as 1959, Scott and Jakoby [ 161 purified the enzyme from Pseudomonas j7uorescens. Subsequently, GABA-T was obtained from mouse [3, 15, 18, 201, lobster WI, rat 112,191, pig [1,41, guinea pig 191, rabbit 1111, and man 15,131. In the course of isolating the enzyme, a number of

Succinic semialdehyde

methods for assaying GABA-T were devised. Jakoby [lo] first described the coupled assay utilizing excess SSADH and monitored the enzyme activity spectrophotometrically as a function of the rate of pyridine nucleotide reduction. Salvador and Albers [ 141 used a fluorometric method for the determination of the condensation product of SSA with 3,5-diaminobenzoic acid. Sytinsky and Vasilijev 1181 elaborated a calorimetric procedure based on the interaction of 3-methyl-2-benzthiazolone-2-hydrazone with SSA. Waksman and Roberts [20] measured directly the production of glutamate from radioactive a-ketoglutarate. Gonnard et cd/. 171used I-‘“C-cY-ketoglutarate as the substrate and employed GAD to generate radioactive carbon dioxide from l‘“C-glutamate. Schousboe et al. [IS] coupled the transamination reaction to a dehydrogenation reaction, using excess glutamate dehydrogenase. Except for the method of Jakoby, these protocols are rather inconvenient point (stop) assays, requiring periodic removal of aliquots of the assay mixture. The fluorometric and calorimetric methods require rigid standardization and lack the specificity inherent in enzymatic determinations. The procedures involving solid or gaseous radioactive substrates are by nature less convenient albeit capable of rather high sensitivity. The coupled assay of Schousboe et al. entails point determination of glutamate produced in the transamination reaction by the catalytic action of glutamate dehydrogenase, the latter reaction being highly unfavorable thermodynamically. To overcome the unfavorable equilibrium, acetylpyridine-NAD is used in place

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Copyright

0 1980 ANKHO

International

Inc.-0361-9230/80/080051-05$01.00/O

RENGACHARY of NAD. Additionally, carbonyl trapping agents. aminoacid and hydrazine, also have to be included. Thus the Jakoby method anpears to be an attract ive one but for the fact that the preparation of the coupling enzyme SSADH is a relatively involved process. In this report we describe a convenient affinity chromatographic technique for isolating SSADH from its mixture with GABA-T; the enzyme mixture and the specific chromatographic resin being commercially available. -“.,~--+L

“nya~cx‘c

‘

AND

YANG

The pooled SSADH from the afftnity column was concentrated approximately sixfold by ultrafiltration in a collodion bag. The concentrated enzyme was made 5 mM in mercaptoethanol and 75 mM in phosphate buffer, pH 7.2. Addition of glycerol (25-.5O%, v/v), bovine serum albumin (0.5%) or lysolecithin (0.41 mgiml) was then made. The treated enzyme was assayed periodically to determine its specific

activity.

METHOD

Gabase

(a mixture

of GABA-T and SSADH from ~-NADP-agarose in which NADP ribose hydroxyl groups were attached to agarose via a six-carbon spacer, GABA, a-ketoglutarate, SSA, NADP, egg yolk t-cY-lysolecithin, bovine serum albumin Fraction V, pyridoxal 5’-phosphate, &mercaptoethanol and tris (hydroxymethyl) aminomethane (Tris) were obtained from Sigma Chemical Company. ~-Amino-~-[U-‘~C]-buty~c acid was purchased from Amersh~ Corporation. Aquasol universal liquid scintillation counting cocktail was acquired from New England Nuclear Corporation. Collodion bags, manufactured by Schleicher and Schuell, Inc. were supplied by VWR Scientific, Inc. Bio-Rad Protein Assay Kit was obtained from Bio-Rad Laboratories. All other chemicals were of the highest purity available.

P~eud~m~~~~~s

~uor~.~~cn.~),

instrumentation

All optical measurements were made in a Varian Cary 1501 uv-visible spectrophotometer equipped with slide wires for both 1.0 and 0.1 absorbance unit. Absorbance was recorded at room temperature (22 2 I’C). Amino acid analysis was done in a Jeol JLC-6AH amino acid analyzer which permitted complete diversion of column effluent, prior to the ninhydrin reaction, to a fraction collector. Radioactivity was counted in a Packard Tricarb Model 3320 liquid scintillation spectrometer.

Two to three units of Gabase in 1.O-1.5 ml of 75 mM phosphate buffer, pH 7.2 containing 25% glycerol (v/v) were passed through a 1.0 x 25 cm Sephadex G-25-40 column which had previously been equilibrated with 50 mM phosphate buffer, pH 7.2. This gel filtration step removed small ions which would otherwise interfere with the subsequent step of affinity chromatography. The eluate, 3.5-4.0 ml in volume and containing GABA-T and SSADH, was then adsorbed onto l-2 ml of P-NADP-agarose packed in a short column having a diameter of 1.0 cm. The length of time allowed for this adsorption was approximately 3 hr. The column was washed with at least 100 ml of 50 mM phosphate buffer, pH 7.2 to remove completely GABA-T. The washing is conveniently collected in a fraction collector. Five ml of 10 mM NADP in 50 mM phosphate, pH 7.2 were then used as the eluent to desorb SSADH over a period of approximately 1.5 hr. Elution with 50 mM phosphate buffer, pH 7.2 was then resumed to recover the last trace of SSADH. The SSADH-cont~ning fractions were pooled to yield a volume of 4.5-6.5 ml.

Enzyme activity was determined spectrophotometrically by following the increase in absorption at 340 nm due to the formation of NADPH (Equation 2). The procedure is essentially that of Jakoby [lo]. The preincubation time has been lengthened to 1 hr and temperature elevated to 37°C. Absorbance change was followed continuously rather than periodically. Sprc,trophotomc~tric.

Assrr~ .fbr GABA-T

GABA-T activity was determined by coupling the transaminase to an excess of SSADH (Equations 1 and 2) so that the rate of formation of NADPH, monitored spectrophotometrically, was equal to that of transamination. The assay mixture had the same composition as that of Jakoby’s protocol ]lO], except only one-tenth as much SSADH, i.e., 0.01 unit, was used as a result of using fullscale deflection of 0.1 rather than 1.O absorbance unit. ~eincubation for I hr at 37°C was necessary for obtaining linear rates. The reaction was initiated by the introduction of a-ketoglutarate and monitored continuously in the spectrophotometer. Purjfication

of y-Amino-n-[U-

Y’)

Butyric

Acid

Twenty-five &i of radioactive GABA in 0.5 ml of aqueous solution containing 2% ethanol was applied without further treatment on the short column of the amino acid analyzer. The column was eluted with citrate buffer, pH 4.70 which was obtained by adjusting the pH of a standard 0.2 N sodium citrate buffer, pH 4.25 with 5 N sodium hydroxide. Eluate from the column was not permitted to react with n~nhyd~n but was collected in a fraction collector at 1 min (0.77 ml) intervals. GABA-cont~ning fractions, now separated from the radioactive impurity which had the same retention time as SSA (Fig. 2), were pooled to give a volume of 2.3 ml.

Except for the omission of SSADH and the inclusion of purified y-amino-n-[U-i4C] butyric acid, the assay mixture is identical in composition to that used for the coupled assay. The GABA-T used was that obtained from Gabase following afftnity chromatography. The reaction was started by the addition of GABA containing approximately 8 x lofi cpm of radioactivity. From 5 ml of reaction mixture, 1 ml aliquots were removed 5, IO, 15, 20 and 25 min following initiation and mixed with 0.5 ml of 2N HCl. After 1 min, 0.4 ml of 2 N NaOH was introduced to bring the solution pH to approximately 3. This was followed by the addition of 1.0 ml of standard amino acid analysis dilution buffer, pH 1.3. Out of each aliquot thus treated, 0.8 ml was applied on the short column of the amino acid analyzer and eluted with 0.2 N sodium citrate buffer, pH 4.70. The eluate was cotlected at

ASSAY FOR y-AMINOBUTYRATE

53

TRANSAMINASE TABLE

ISOLATION

Step

OF SUCCINIC

1

SEMIALDEHYDE

DEHYDROGENASE

Activity (~mol/min/ml)

Prot. cont. (mgiml)

Specif. act. I~moliminimg)

1.0 3.8

1.3 0.3

5.32 0.91

0.25 0.31

1.3 I.1

100 80

4.4

0.2

0.48

0.47

1.0

76

Gabase Gel filtration

Total act. (pmol/min)

Yield (%)

Volume (ml)

Affinity column

1-min intervals in a fraction collector. From each fraction, 250 ~1 were withdrawn and mixed with 10 ml of Aquasol and counted in the liquid scintillation spectrometer. Concurrent with the radioisotopic measurement, GABA-T was assayed spectrophotometrically, using the same assay mixture, but with added SSADH (0.1 unit/ml) and without radioactive GABA.

of NADPH

Detrrtninmtion

Oxiduse

Activity

The assay mixture consisted of 1 mM NADPH, 3 mM mercaptoethanol in 0.05 M Tris buffer, pH 7.9. NADPH oxidase activity was determined spectrophotometrically by following the decrease in absorbance at 340 nm.

of Protein Concrntrution

Determincltion

The Bio-Rad Protein Assay was employed to determine protein concentration. The method is based on the dyebinding assay of Bradford [2]. RESULTS

Isolation

of SSADH

Results

of the two-step isolation scheme for SSADH in Table 1. The rather high overall yield in enzyme activity makes the procedure of practical value. The affinity chromatographic technique applied permitted complete resolution of GABA-T and SSADH in Gabase (Fig. 1). When sufficient 50 mM phosphate buffer, pH 7.2 was used to wash off GABA-T, the SSADH was contaminated by the transaminase only to the extent of 0.2%. Contamination by NADPH oxidase activity which would diminish the production of NADPH in the coupled assay for GABA-T was found to be less than 0.04% of SSADH activity.

FIG. I. Affinity chromatographic separation of GABA-T and SSADH in Gabase. The absorbance at 280 nm (0) of the first collected fractions was monitored. Succinic semialdehyde dehydrogenase was identified by assaying (0). The absorbance at 280 nm of these later fractions was excessively high due to the presence of NADP which was used for desorption.

are summarized

Stcrhilizrrtion

of SSADH

Dilute SSADH eluted from the affinity column quickly lost all its activity. However several treatments were found effective in stabilizing the enzyme (Table 2). Glycerol in high concentration enhanced the stability of the enzyme. Bovine serum albumin and lysolecithin were also found effective. Further improvement was possible by concentrating SSADH approximately sixfold following affinity chromatography. It is clear that the shelf-life of SSADH can be prolonged sufficiently to make the enzyme suitable for the coupling assay.

The purpose of the concurrent determine whether the convenient

GABA-T assays was to coupled assay reflected

FIG. 2. Chromatographic separation of radioactive SSA from GABA. Assay mixture containing y-amino-n[U-“Cl butyrate was incubated with GABA-T. Five minutes following the initiation of the reaction, a l-ml aliquot was removed, treated and chromatographed on the amino acid analyzer as described in METHOD. Note difference in abscissa scale for GABA (0) and SSA (0).

54

RENGACHA~YANDYANG TABLE 2 STABILITY OF SUCCINIC SEMIALDEHYDE DEHYDROGENASE Activity (~mollminimg) after Days Conditions* 33% glycerol+

25% glycerol, 0.5% BSAt

1

3

0.14 (lOu%f

0.10

0.08

(71%)

(56%)

0.22 (100%)

0.19 (85%)

0.15 (67%)

25% glycerol, 0.41 mg/ml lysolecithint

0.23 (108%)

50% glycerol, 0.5% BSA, concentration*

0.57 (100%)

0.18 (77%)

-

6

0.13 (57%)

14 -8

-

0.56 (97%)

21

24

-

-

0 (0%) -

-

-

0.51 GE=)

*In addition to those components indicated, all solutions contained 5 mM mercaptoethanol and 75 mM phosphate buffer, pH 7.2. +These three experiments were done using the same preparation of SSADH. *This experiment used a SSADH preparation different from that of the previous set. 6Data were not obtained.

showed a retention time of 8 min. Authentic GABA, chromatographed and identified by ninhydrin, gave an elution time of 25 min. GABA-T activity calculated from the radioisotopic experiment is compared with that of the coupled assay in Fig. 3. It is clear that the two methods yielded comparable results in the initial phase of the reaction, indicating that the continuous spectrophotometric method reflected the true activity of GABA-T. However, a significant drop in GABA-T activity assayed radioisotopitally occurred subsequently. The reasons for this decrease are as yet unclear.

6

16

10 The

20

26

lmtnf

FIG. 3. Comparison of the spectrophotomet~c and radioisotopic assays. GABA-T activity was determined as a function of time by the coupled assay (0) and the radioisotopic method (0). In the latter method, aliquots of the reaction mixture were removed at 5, 10, 15, 20 and 25 min. Increment in product radioactivity for each S-min period was used to calculate the average enzyme specific activity for that period. the true activity of the enzyme.

Figure 2 illustrates the progress of the transamination reaction in the reaction mixture containing y-~ino-ff-[~-i4C] butyrate. Radioactive succinic semialdehyde, eluted at pH 4.70 from the column 8 min following sample application was completeiy separated from GABA which had a much longer retention time. In fact, the standard amino acid analysis eiution buffer at pH 4.25 failed to desorb GABA although it readily eluted SSA. The clear separation of substrate and product made it possible to obtain reaction rates by the direct radioisotopic method. Authentic, non-radioactive SSA, when chromatographed on the analyzer and identified by 2,4-dinitrophenylhydrazine,

DISCUSSION

Bovine serum albumin has been found effective in stabilizi~ SSADH, especially when the protein is in concentrated form and with the addition of glycerol. It is quite possible that in some experiments the introduction of a foreign protein is undesirable. In such instances, egg yolk lysolecithin may be substituted. The radioisotopic assay of GABA-T yielded progressively lower enzyme activity. Several phenomena may account for this observation. First, accumulation of SSA might inhibit the forward reaction. This inhibition would not be observed in the spectrophotometric assay because of the coupling conversion of SSA to succinic acid. Second, the presence of SSADH might stabilize GABA-T during transamination. The r~ioisotopic assay mixture contained no SSADH. These possibilities are currently under investigation. Gabase from P. ~~o~~~~~n~ may also be procured from Boehringer Manheim Biochemicals. ACKNOWLEDGEMENT We wish to thank Dr. William B. Jakoby for suggesting enzyme concentration as a means of stabilizing SSADH and for alerting us to the possibility of contamination of SSADH by NADPH oxidase.

ASSAY

FOR y-AMINOBUTYRATE

55

TRANSAMINASE

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11. John, R. A. and L. J. Fowler. Kinetic and spectral properties of rabbit brain 4-aminobutyrate aminotransferase. Biochrm. J. 155: 645-651, 1976. 12. Maitre, M.. L. Ciesielski, C. Cash and P. Mandel. Purification and studies on some properties of the 4-aminobutyrate: 2-oxoglutarate transaminase from rat brain. Elrr. J. BiochPm. 52: 157-169, 1975. 13. Maitre, M., L. Ciesielski, C. Cash and P. Mandel. Comparison of the structural characteristics of the 4-aminobutyrate 2-oxoglutarate transaminase from rat and human brain, and of their affinities for certain inhibitors. Biochim. hiophys. Ac,tcr 522: 385-399,

1978.

14. Salvador, R. A. and R. W. Albers. The distribution of glutamic-y-aminobutyric transaminase in the nervous system of the rhesus monkey. J. Bid. Chc~m. 234: 922-925, 1959. 15. Schousboe, A., J.-Y. Wu and E. Roberts. Purification and characterization of the 4-aminobutyrate-a-ketoglutarate transaminase from mouse brain. Biochemistry 12: 2868-2873, 1973. 16. Scott, E. M. and W. B. Jakoby. Soluble y-aminobutyricglutamic transaminase from Psrudomonf~s JIuo~~~.T~w~s.J. Bid. Chmr. 234: 932-936, 1959. 17. Stahl, W. L. and P. D. Swanson. Biochemical abnormalities in Huntington’s chorea brains. Neurology 24: 813-819, 1974. 18. Sytinsky, I. A. and V. Y. Vasilijev. Some catalytic properties of purified y-aminobutyric-a-ketoglutarate transaminase from the rat brain. Enc,ymologitr 39: l-1 1, 1970. 19. Vasilijev, V. Y. and V. P. Eremin. Purification and properties of y-aminobutyrate-glutamate transaminase. Biohhimiya 33: 1143-1149, 1968. 20. Waksman, A. and E. Roberts. Purification and some properties of mouse brain y-aminobutyric-a-ketoglutaric acid transaminase. Bioc~hcmistry 10: 2132-2139, 1965.