Characterization of Dog Glutathione Transferase P1-1, an Enzyme Relevant to Veterinary Medicine
Abstract
:1. Introduction
2. Results
2.1. Expression, Purification, and Characterization of Dog GST P1-1
2.2. Primary Structure
2.3. Tertiary Structure
2.4. Activities with Alternative Substrates
2.5. Effects of Different GST Inhibitors
2.6. Activation of the Prodrug Telcyta
3. Discussion
4. Materials and Methods
4.1. Heterologous Expression and Purification of Dog GST P1-1
4.2. SDS-PAGE
4.3. Enzyme Activity Measurements
4.4. Thin-Layer Chromatography to Assay Activity with the Prodrug Telcyta
4.5. Modeling of Protein Structures
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
GSH | Glutathione |
GST | Glutathione transferases |
Clu | Canis lupus familiaris |
CDNB | 1-Chloro-2,4-dinitrobenzene |
PEITC | Phenethyl Isothiocyanate |
BITC | Benzyl isothiocyanate |
AITC | Allyl isothiocyanate |
PITC | Propyl isothiocyanate |
cHITC | Cyclohexyl isothiocyanate |
CuOOH | Cumene hydroperoxide |
EA | Ethacrynic acid |
AD | Δ5-Androstene-3,17-dione |
Telcyta (TLK286) | γ-l-Glutamyl-α-amino-β-(2-ethyl-N,N,N′,N′-tetrakis(2-chloroethyl)phosphorodiamidate)sulfonylpropionyl-R(−)phenylglycine |
TLK117 | γ-l-Glutamyl-S-(benzyl)-l-cysteinyl-R(−)-phenylglycine |
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Specific Activity (µmol/min/mg) | ||
---|---|---|
Substrate | Dog GST P1-1 | Human GST P1-1/Ile105 1 |
1-Chloro-2,4-dinitrobenzene (CDNB) | 23.0 ± 1.3 | 106 |
Cumene hydroperoxide (CuOOH) | 0.07 ± 0.01 | 0.03 |
Phenethyl isothiocyanate (PEITC) | 6.37 ± 1.03 | 60 |
Benzyl isothiocyanate (BITC) | 39.0 ± 5.2 | 63 |
Allyl isothiocyanate (AITC) | 1.94 ± 0.60 | 38 |
Propyl isothiocyanate (PITC) | 2.55 ± 0.20 | 49 |
Cyclohexyl isothiocyanate (cHITC) | 1.27 ± 0.08 | 11 |
Ethacrynic acid (EA) | 2.05 ± 0.13 | 2.0 |
trans-2-Pentenal | 0.100 ± 0.002 | N/A |
trans-2-Nonenal | 0.124 ± 0.005 | N/A |
trans-2-Decenal | 0.111 ± 0.005 | N/A |
trans-2-Dodecenal | 0.046 ± 0.006 | N/A |
Δ5-androstene-3,17-dione (AD) | 0.014 ± 0.005 | 0.01 |
Inhibitor | IC50 (µM) |
---|---|
Ethacrynic acid | 4.38 ± 1.96 |
Triethyltin bromide | 83.6 ± 1.9 |
Triphenyltin chloride | 3.11 ± 0.38 |
Tributyltin acetate | 0.85 ± 0.13 |
Cibacron Blue | 0.043 ± 0.007 |
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Ismail, A.; Lewis, E.; Sjödin, B.; Mannervik, B. Characterization of Dog Glutathione Transferase P1-1, an Enzyme Relevant to Veterinary Medicine. Int. J. Mol. Sci. 2021, 22, 4079. https://doi.org/10.3390/ijms22084079
Ismail A, Lewis E, Sjödin B, Mannervik B. Characterization of Dog Glutathione Transferase P1-1, an Enzyme Relevant to Veterinary Medicine. International Journal of Molecular Sciences. 2021; 22(8):4079. https://doi.org/10.3390/ijms22084079
Chicago/Turabian StyleIsmail, Aram, Elizabeth Lewis, Birgitta Sjödin, and Bengt Mannervik. 2021. "Characterization of Dog Glutathione Transferase P1-1, an Enzyme Relevant to Veterinary Medicine" International Journal of Molecular Sciences 22, no. 8: 4079. https://doi.org/10.3390/ijms22084079