Inactivation of γ-Aminobutyric Acid Aminotransferase by Various Amine Buffers

Hopkins, Mark H.; Bichler, Katherine A.; Su, Ting; Chamberlain, Cheryl L; Silverman, Richard B.

doi:10.3109/14756369209020169

Cited by 10 publications

(12 citation statements)

References 12 publications

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“…GABA-AT was purified as previously described . Succinic semialdehyde dehydrogenase (SSDH) was obtained from GABAase, a commercially available mixture of GABA-AT and SSDH, by the reported procedure . Protein assays were carried out using bovine serum albumin (BSA) and Pierce Coomassie protein assay reagent for standard curves.…”

Section: Methodsmentioning

confidence: 99%

“…35 Succinic semialdehyde dehydrogenase (SSDH) was obtained from GABAase, a commercially available mixture of GABA-AT and SSDH, by the reported procedure. 36 Protein assays were carried out using bovine serum albumin (BSA) and Pierce Coomassie protein assay reagent for standard curves. GABA-AT activity assays were carried out using a modification of the coupled assay developed by Scott and Jakoby.…”

Section: Methodsmentioning

confidence: 99%

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Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase by (S)-4-Amino-4,5-dihydro-2-thiophenecarboxylic Acid

Nikolić

Breemen

et al. 1999

J. Am. Chem. Soc.

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(S)-4-Amino-4,5-dihydro-2-thiophenecarboxylic acid ((S)-6) was previously synthesized (Adams, J. L.; Chen, T. M.; Metcalf, B. W. J. Org. Chem. 1985, 50, 2730−2736.) as a heterocyclic mimic of the natural product gabaculine (5-amino-1,3-cyclohexadienylcarboxylic acid), a mechanism-based inactivator of γ-aminobutyric acid aminotransferase (GABA-AT) (Rando, R. R. Biochemistry 1977, 16, 4604). Inactivation of GABA-AT by (S)-6 is time-dependent and protected by substrate. Two methods were utilized to demonstrate that, in addition to inactivation, about 0.7 equiv per inactivation event undergoes transamination. Inactivation results from the reaction of (S)-6 with the pyridoxal 5‘-phosphate (PLP) cofactor. The adduct was isolated and characterized by ultraviolet−visible spectroscopy, electrospray mass spectrometry, and tandem mass spectrometry. All of the results support a structure (11) that derives from the predicted aromatization inactivation mechanism (Scheme ) originally proposed by Metcalf and co-workers for this compound. This is only the third example, besides gabaculine and l-cycloserine, of an inactivator of a PLP-dependent enzyme that acts via an aromatization mechanism.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase by (S)-4-Amino-4,5-dihydro-2-thiophenecarboxylic Acid

Nikolić

Breemen

et al. 1999

J. Am. Chem. Soc.

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“…GABA aminotransferase was isolated from pig brains using the procedure of Churchich and Moses . Succinic semialdehyde dehydrogenase (SSDH) was obtained from GABAase (Boehringer Mannheim, West Germany), a mixture of GABA aminotransferase and SSDH, by the reported procedure . GABA aminotransferase activity assays were carried out using a modification of the coupled assay developed by Scott and Jakoby .…”

Section: Methodsmentioning

confidence: 99%

An Aromatization Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase for the Antibiotic l-Cycloserine

Olson

Lau

et al. 1998

J. Am. Chem. Soc.

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The mechanism of inactivation of pig brain γ-aminobutyric acid (GABA) aminotransferase by the antibiotic l-cycloserine was investigated. l-Cycloserine is a time-dependent inactivator of GABA aminotransferase; no enzyme activity returns upon gel filtration or dialysis. Treatment of GABA aminotransferase with [14C]-l-cycloserine, followed by rapid gel filtration, gives enzyme containing l.l equiv of radioactivity bound. Dialysis or denaturation by acid, base, or urea releases the radioactivity. Inactivation of [3H]pyridoxal 5‘-phosphate (PLP)-reconstituted GABA aminotransferase with l-cycloserine followed by dialysis or denaturation also leads to the release of radioactivity from the enzyme. Both the released [14C]- and [3H]-labeled adducts comigrate by HPLC, suggesting that the inactivation adduct is a condensation product of l-cycloserine with the PLP coenzyme. By HPLC comparison, it was shown that the radiolabeled adduct is not PLP, PMP, PLP oxime, or 4-[3-hydroxy-2-methyl-5-(phosphooxymethyl)-4-pyridinyl]-2-oxo-3-butenoic acid (20), the expected product of an enamine-type inactivation mechanism. On the basis of the stability of the released adduct to acid and base and its UV−visible spectrum, which has the appearance of a PMP analogue, a simple Schiff base between PLP and cycloserine also was excluded. HPLC of the cycloserine−coenzyme adduct had a retention time very similar to that of the gabaculine−coenzyme adduct. Electrospray ionization tandem mass spectrometry of the isolated cycloserine−coenzyme adduct is consistent with a structure that is one of the tautomeric forms of the Schiff base between PMP and oxidized cycloserine (21).

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“…However, GABase was not able to be used directly in our human GABA-T assay due to the presence of bacterial GABA-T. So we used the bacterial SSADH isolation method as previously described 39 , which allowed large amounts of bacterial SSADH to be isolated quickly and conveniently from GABase by irreversibly inhibiting the bacterial GABA-T activity. Five milligrams of gabaculine (Santa Cruz Biotechnology, sc-200473), a naturally occurring GABA-T irreversible inhibitor, was added to 25 ml of 1 U/mL GABase solution (reconstituted with 50 mM K 4 P 2 O 7 buffer at pH 8.6), followed by maintaining the reaction at 4 °C for 30 min.…”

Section: Methodsmentioning

confidence: 99%

High-yield synthesis and purification of recombinant human GABA transaminase for high-throughput screening assays

Park

Han

Kim

et al. 2021

Journal of Enzyme Inhibition and Medicinal Chemistry

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Many studies have focussed on modulating the activity of γ-aminobutyric acid transaminase (GABA-T), a GABA-catabolizing enzyme, for treating neurological diseases, such as epilepsy and drug addiction. Nevertheless, human GABA-T synthesis and purification have not been established. Thus, biochemical and drug design studies on GABA-T have been performed by using porcine GABA-T mostly and even bacterial GABA-T. Here we report an optimised protocol for overexpression of 6xHis-tagged human GABA-T in human cells followed by a two-step protein purification. Then, we established an optimised human GABA-T (0.5 U/mg) activity assay. Finally, we compared the difference between human and bacterial GABA-T in sensitivity to two irreversible GABA-T inhibitors, gabaculine and vigabatrin. Human GABA-T in homodimeric form showed 70-fold higher sensitivity to vigabatrin than bacterial GABA-T in multimeric form, indicating the importance of using human GABA-T. In summary, our newly developed protocol can be an important first step in developing more effective human GABA-T modulators.

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Inactivation of γ-Aminobutyric Acid Aminotransferase by Various Amine Buffers

Cited by 10 publications

References 12 publications

Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase by (S)-4-Amino-4,5-dihydro-2-thiophenecarboxylic Acid

Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase by (S)-4-Amino-4,5-dihydro-2-thiophenecarboxylic Acid

An Aromatization Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase for the Antibiotic l-Cycloserine

High-yield synthesis and purification of recombinant human GABA transaminase for high-throughput screening assays

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