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Articles |
-Aminobutyric Acid (GABA) Transaminase in Isolated Leukocytes and Evidence That GABA and ß-Alanine Transaminases Are Identical
1
Metabolic Unit, Department of Clinical Chemistry, University Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
2
Department of Molecular and Medical Genetics,
Biochemical Genetics Laboratory, Oregon Health Sciences University,
Portland, OR 97201.
a Author for correspondence. Fax 31-20-4440305; e-mail
C.Jakobs{at}azvu.nl.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-aminobutyric acid transaminase (GABA-T) activity, but these methods
are either impracticable because of the use of radioisotopes or
insufficiently sensitive to determine small enzyme activities in
leukocyte extracts. We developed a direct and sensitive enzyme method.
Methods: We developed a stable-isotope dilution method for the
measurement of [15N]glutamic acid derived from
[15N]GABA and 
-ketoglutaric acid, catalyzed by
GABA-T. The method for analysis of [15N]glutamic acid
comprised a solid-phase extraction procedure to isolate this analyte
from incubation samples. After derivatization,
[15N]glutamic acid was quantified by gas
chromatography–mass spectrometry relative to its
2H5-labeled internal standard. In addition to
[15N]GABA, [15N]ß-alanine was a
cosubstrate.
Results: GABA-T-deficient lymphoblasts showed diminished enzyme activity, with both [15N]GABA and [15N]ß-alanine as substrate. Vigabatrin inhibited the enzyme activity for both substrates.
Conclusions: The activity of GABA-T can be accurately determined by our procedure using 15N-labeled substrate, measuring the formation of [15N]glutamic acid. Our results with [15N]ß-alanine indicate that GABA and ß-alanine transaminases are identical.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Aminobutyric acid
(GABA)1
is found predominantly in brain where it is an important
inhibitory neurotransmitter. However, GABA is also detected in the
peripheral system, endocrine, and several nonneural tissues where it
plays a role in oxidative metabolism (1). The enzymes
responsible for GABA metabolism in the brain are the GABA synthetic
enzyme glutamate decarboxylase (EC 4.1.1.15) and the GABA
catabolic enzymes GABA transaminase (GABA-T; EC 2.6.1.19) and succinic
semialdehyde (SSA) dehydrogenase (EC 1.2.1.24).
GABA-T is the enzyme that mediates the conversion of GABA to SSA, using
-ketoglutarate (KG) as nitrogen acceptor, yielding glutamic acid
(Fig. 1
).
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Only two patients have been described with GABA-T deficiency. These two patients had similar clinical findings, and both had increased concentrations of free and total GABA in plasma and cerebrospinal fluid (CSF). In the first patient, reported by Jaeken et al. in 1984 (2), the enzyme defect was documented in liver tissue by a radiometric assay (3). Subsequently, the GABA-T deficiency was demonstrated in lymphocytes and lymphoblasts derived from the same patient, also by a radiometric method (4). The second patient was documented in 1999 (5). Mean GABA-T activity in extracts of leukocytes of this patient was 27% of control, measured by the same method used to measure activity in leukocytes of the first patient (0.9% of control). In addition to these two patients, there were two other patients who manifested increased CSF GABA but were diagnosed as having hyper-ß-alaninemia (6)(7). This disease is named after another accumulating compound in their plasma and CSF, namely ß-alanine (ßALA). ßALA was also increased in the plasma and CSF from the first GABA-T patient (no results known for the second patient). There remains the problem of distinguishing between GABA-T deficiency and hyper-ß-alaninemia and whether they involve different enzymes or the same affected enzyme with different disease phenotypes (8).
Several methods for measuring GABA-T activity have been published
(9)(10)(11), but all of these methods are indirect, nonspecific,
and/or not sensitive enough to be used in a crude tissue preparation.
The radiometric assay, using 14C-labeled KG
and unlabeled GABA as substrates, is nonspecific, indirect, and
determines the radioactive glutamate formed by measuring the
14CO2 released by a coupled
assay using a bacterial glutamate decarboxylase (12). An
alternative method is a modification of the procedure of White
(13). [U-14C]GABA is used as
substrate, and the [U-14C]SSA formed during
incubation is measured as the methoxime derivative, which is separated
from radiolabeled GABA by reversed-phase HPLC (4).
This method is impracticable in many laboratories because it uses
considerable amounts of radioactivity. We developed a new method,
exploiting the role of KG as nitrogen acceptor in the GABA
degradative pathway. We used [15N]GABA as
substrate and analyzed [15N]glutamate (after
derivatization) by gas chromatography–mass spectrometry (GC-MS) using
[2,3,3,4,4-2H5]glutamic
acid as internal standard.
Other studies report that the mammalian enzyme GABA-T reacts with ßALA to the same extent as with GABA, whereas ßALA is poorly transaminated by the enzyme derived from bacteria or yeast (14)(15). To investigate whether this compound is metabolized by the enzyme GABA-T in human leukocytes (especially in cells of a GABA-T-deficient patient), the applicability of our new method was expanded by the use of [15N]ßALA as substrate. We applied this method to measure enzyme activity in lysates of lymphoblast lines of six control individuals and one patient, and in lysates of lymphocytes of six control individuals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chemicals
Unless otherwise stated, all chemicals and reagents were purchased
from Sigma, Merck, or Baker. [15N]GABA
was obtained from ISOTEC, [15N]ßALA from
Witega, and
[2,3,3,4,4-2H5]glutamic
acid from MSD Isotopes.
preparation of the complete buffer
The complete buffer was a 50 mmol/L potassium phosphate buffer (pH
8.0), supplemented with 0.25 mmol/L dithiothreitol, 0.05 mmol/L
disodium EDTA, and 0.1 mmol/L pyridoxal-5-phosphate.
isolation and preparation of lymphocytes and lymphoblasts
Lymphocytes were isolated from whole blood using
AccuspinTM tubes with Histopaque®
(Sigma) and washed twice with Hanks’ Balanced Salt Solution (HBSS;
Life Technologies) supplemented with 1 g/L bovine serum albumin and one
time with HBSS. Lymphoblasts were harvested by centrifugation, and the
cells were washed twice with HBSS. Pellets were resuspended in complete
buffer on ice. Cells were lysed by sonic disruption (three times
10 s at standard capacity) using a Soniprep 150 Ultrasonic
Desintegrator (MSE). Lysates were centrifuged for 5 min at 4 °C and
8800g. After the protein concentration was measured
(bicinchoninic acid protein assay; Sigma), the supernatant was used for
the enzyme assay.
enzyme assay
GABA-T activity was assayed using a modification of the procedure
of Gibson et al. (4). After optimization, the enzymatic
assay was performed as follows: in a total volume of 100 µL of
complete buffer, each incubation contained 135 nmol of -ketoglutaric
acid monopotassium salt, 500 nmol of [15N]GABA
or [15N]ßALA, and an aliquot of lymphocyte or
lymphoblast lysate corresponding to 50 µg of protein. The incubation
was carried out in a capped 1-mL vial for 4–20 h at 37 °C. The
incubation was terminated by the addition of 10 µL of 4.2 mol/L
perchloric acid. The solution was neutralized by the addition of 7
µL of 6 mol/L potassium hydroxide. After 2 nmol of
[2,3,3,4,4-2H5]glutamic
acid was added as internal standard, the denatured protein and
insoluble potassium perchlorate were removed by centrifugation. The
resulting supernatant was prepared as described below for
analysis of glutamic acid by GC-MS.
sample preparation for gc-ms measurement of glutamic acid
Glutamic acid was converted to its N-carbamyl
derivative by the addition of 900 µL of H2O,
800 µL of 1 mol/L sodium phosphate buffer (pH 11.5), and 50 µL of
methyl chloroformate to the supernatant. The mixture was incubated for
10 min at room temperature. The solution was acidified with 210 µL of
6 mol/L HCl and applied to an OASIS solid-phase extraction column (60
mg, 3 mL; Waters) that was conditioned with 1 mL of methanol followed
by 1 mL of 0.5 mol/L HCl. After the sample was passed through by
gravity, the column was rinsed with 500 µL of 0.5 mol/L HCl. The
glutamic acid-N-carbamyl derivative was eluted from the
column with two 500-µL aliquots of methanol and methylated directly
by the addition of 20 µL of 6 mol/L HCl and heating for 1 h at
120 °C. After evaporation, the residue was dissolved in 50 µL of
ethyl acetate. From this fraction, 1 µL was used for GC-MS analysis
of glutamic acid.
gc-ms
GC-MS analyses were performed on a Hewlett Packard system
consisting of a Hewlett Packard 5890 series II gas chromatograph and
Hewlett Packard Engine 5989B mass spectrometer. GC separation of the
di-methyl-N-carbamyl derivative was achieved on a very polar
column (BPX70; 25 m x 0.32 mm; film thickness, 0.25 µm;
Scientific Glass Engineering) using helium as carrier gas. The injector
temperature was 240 °C, and the injection volume was 1 µL. After 1
min at 100 °C, the column temperature was increased at 20 °C/min
to 280 °C. The interface temperature was set at 290 °C, the
source temperature was 200 °C. The mass spectrometer was used in the
electron-impact (EI) mode. Selected-ion monitoring of the
m/z 174, 175, and 179 ions was performed for glutamic acid,
[15N]glutamic acid, and the labeled internal
standard, respectively.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The [M - 59] fragment (m/z 174) in the EI mass
spectrum of the unlabeled compound is most likely attributable to the
loss of -COOCH3 from the carbamyl moiety. The
m/z 175 fragment in this spectrum is derived from the
naturally occurring isotope of the unlabeled glutamic acid (8.44%) and
interferes with the ion peak area of
[15N]glutamic acid in the samples. In the mass
spectrum of the labeled internal standard, the [M - 59] fragment
(m/z 179) is of the same origin as the m/z 174
fragment in the unlabeled analog. A small m/z 175 fragment
is visible, the origin of which is not clear, but we have observed it
as a constant amount of 2.98% from the ion peak area of the
m/z 179 fragment. Selected-ion monitoring mass
fragmentograms obtained from a control lymphoblast line and from the
GABA-T-deficient patient are shown in Fig. 3
.
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calculations
Enzyme activity is directly related to the formation of
[15N]glutamic acid during the incubation with
[15N]GABA or
[15N]ßALA. To quantify the amount of
[15N]glutamic acid formed during the assay, it
was necessary to correct the area of the m/z 175 peak for
interference of the internal standard (2.98% of the area from the
m/z 179 peak) and of the natural occurring isotope abundance
(8.44% of area from the m/z 174 peak). Hereafter,
[15N]glutamic acid was quantified relative to
its 2H5-labeled internal
standard.
assay conditions
Although we started from the procedure of Gibson et al.
(4) to assay GABA-T, it seemed to be worthwhile to optimize
the assay conditions; formation of a different reaction product was
measured ([15N]glutamic acid instead of
[U-14C]SSA) and other conditions required
optimization to determine maximum activity of the enzyme. Moreover,
[15N]ßALA was introduced as a new substrate,
and optimal assay conditions needed to be established.
With the assay conditions described in Materials and
Methods, the production of [15N]glutamic
acid after incubation with 500 nmol of
[15N]GABA or
[15N]ßALA was linear up to 100 µg of
protein, linear with time up to 20 h, and optimal at pH
8.0 (not shown). For the dependence of the enzyme activity on the
amount of [15N]GABA or
[15N]ßALA, it was found that full activity
was achieved when at least 5 mmol/L
[15N]GABA was used, whereas incubating with 12
mmol/L [15N]ßALA was not sufficient to attain
optimal activity (Fig. 4
). The mathematical treatment of the direct linear plot
(16) yielded apparent Michaelis constants
(Kms) of 1.7 and 4.4 mmol/L for
[15N]GABA and
[15N]ßALA, respectively. Both substrates had
similar activities at 500 nmol, the amount used for standard reaction
conditions. Formation of [15N]glutamic acid was
optimal at an KG concentration of 1.35 mmol/L. Pyridoxal 5-phosphate
at a concentration of 0.1 mmol/L was sufficient. Higher concentrations
of KG inhibited the reaction (results not shown). On the basis of
the experiments described above, we selected the standard reaction
conditions described in Materials and Methods. In addition
to differences in optimal substrate concentrations, both substrates
required similar conditions to achieve optimal enzyme activity.
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gaba-t activity measurements in lysates of lymphoblasts and
lymphocytes
The results of assays with [15N]GABA and
[15N]ßALA for GABA-T are shown in Table 1
. The mean activity in extracts of six control lymphoblast
lines, three assayed on two different occasions, was 46
± 13 pmol·min-1·mg
protein-1. The activity in lysates of
lymphocytes obtained from six control individuals was approximately
threefold higher. No difference in enzyme activity was measured between
the two substrates [15N]GABA and
[15N]ßALA (see controls C and G–L in Table 1
) because 500 nmol of each was used. Lymphoblasts derived from the
index patient showed deficient activity with both substrates; <0.5%
of control enzyme activity was observed.
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effect of vigabatrin on gaba-t activity
Vigabatrin (-amino-5-hexenoic acid) is known to irreversibly
inhibit GABA-T activity of bacterial origin (17). To
investigate the effect of vigabatrin on our assay, we performed the
assay in the presence of 22, 44, and 220 µmol/L vigabatrin. As shown
in Fig. 5
, degradation of [15N]GABA and
[15N]ßALA in lymphoblasts is almost fully
inhibited at a concentration of 220 µmol/L vigabatrin. Vigabatrin was
more inhibitory in the reaction using
[15N]ßALA than
[15N]GABA.
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effect of unlabeled gaba and ßala on gaba-t activity
To investigate the difference in affinity of GABA-T for both
substrates, we incubated lymphoblast lysates with 400 nmol of
[15N]GABA and 400 nmol of unlabeled ßALA, and
with 400 nmol of [15N]ßALA and 400 nmol of
unlabeled GABA. The effect of unlabeled ßALA on
[15N]GABA degradation was minor (remaining
activity, 88% of control), whereas the effect of unlabeled
GABA on [15N]ßALA metabolism was significant
(remaining activity, 34% of control; Table 2
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The similarity in substrate utilization, including [15N]GABA and [15N]ßALA, by the enzyme GABA-T is an important finding in our work. For both patient and controls, there was no difference in enzyme activity for [15N]ßALA compared with [15N]GABA. Optimal conditions were approximately the same for both substrates. Vigabatrin inhibited enzyme activity, decreasing enzyme activity with both substrates. Unlabeled GABA and ßALA decreased the degradation of [15N]ßALA and [15N]GABA, respectively. These results, especially the finding that the patient was deficient for both substrates, add additional evidence to the concept that GABA-T and ßALA transaminase are identical enzymes. Hopefully, more patients will be diagnosed in the future, which will help us to determine whether GABA-T deficiency and hyper-ß-alaninemia are the same disorder.
In contrast to the similarities in substrate utilization by the enzyme GABA-T, there are some remarkable differences. The dependency of enzyme activity on the amounts of [15N]GABA and [15N]ßALA was different. At <500 nmol, the activity with [15N]GABA was higher, whereas at higher substrate amounts, the activity with [15N]ßALA was higher. In addition, although vigabatrin inhibited the degradation of both substrates, it appeared that there was still residual enzyme activity with [15N]GABA. Finally, from the experiment with unlabeled substrates, we conclude that GABA-T has higher affinity for [15N]GABA than for [15N]ßALA. This was confirmed by the finding that GABA-T had a lower Km for [15N]GABA than for [15N]ßALA.
In conclusion, we present a sensitive and accurate enzymatic assay
for GABA-T. This new method can also be used to measure the activity of
any transaminase that uses KG as nitrogen acceptor, provided that
optimal conditions are determined.
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Acknowledgments |
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Footnotes |
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-aminobutyric acid; GABA-T, GABA transaminase; SSA, succinic semialdehyde; KG, -ketoglutarate; CSF, cerebrospinal fluid; ßALA, ß-alanine; GC-MS, gas chromatography–mass spectrometry; HBSS, Hanks’ Balanced Salt Solution; and EI, electron impact. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Aminobutyric acid (GABA) metabolism in mammalian neural and nonneural tissues [Review]. Comp Biochem Physiol 1995;112:247-263.
-Aminobutyric acid-transaminase deficiency: a newly recognized inborn error of neurotransmitter metabolism. Neuropediatrics 1984;15:165-169.[ISI][Medline]
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-aminobutyricaciduria, somnolence and seizures. N Engl J Med 1966;274:636-643.
-amino
acids in free and peptide-linked forms. In: Scriver CR, Beaudet AL, Sly
WS, Valle D, eds. The metabolic and molecular bases of inherited
disease, 9th ed. New York: McGraw-Hill, in press..
-aminobutyric--oxoglutaric transaminase. J Neurochem 1965;12:93-101.[ISI][Medline]
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-acetylenic GABA, -ethynyl GABA) a catalytic inhibitor of GABA transaminase, on brain GABA metabolism in vivo. J Neurochem 1977;28:717-723.[ISI][Medline]
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-aminobutyric acid--ketoglutarate transaminase of bacterial origin by 4-aminohex-5-ynoic acid, a substrate analog. Biochem Biophys Res Commun 1975;67:301-306.[ISI][Medline]
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