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A liquid chromatography-tandem mass spectrometry-based assay for indole-3-acetic acid-amido synthetase.
Anal Biochem. 2009 Jul 15; 390(2):149-54.AB

Abstract

Indole-3-acetic acid (IAA) amide conjugates play an important role in balancing levels of free IAA in plant cells. The GH3 family of proteins conjugates free IAA with various amino acids. For example, auxin levels modulate expression of the Oryza sativa (rice) GH3-8 protein, which acts to prevent IAA accumulation by coupling the hormone to aspartate. To examine the kinetic properties of the enzyme, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay system. Bacterially expressed OsGH3-8 was purified to homogeneity and used to establish the assay system. Monitoring of the reaction confirms the reaction product as IAA-Asp and demonstrates that production of the conjugate increases proportionally with both time and enzyme amount. Steady-state kinetic analysis using the LC-MS/MS-based assay yields the following parameters: V/E(t)(IAA)=20.3 min(-1), K(m)(IAA)=123 microM, V/E(t)(ATP)=14.1 min(-1), K(m)(ATP)=50 microM, V/E(t)(Asp)=28.8 min(-1), K(m)(Asp)=1580 microM. This is the first assignment of kinetic values for any IAA-amido synthetase from plants. Compared with previously described LC- and thin-layer chromatography (TLC)-based assays, this LC-MS/MS method provides a robust and sensitive means for performing direct kinetic studies on a range of IAA-conjugating enzymes.

Authors+Show Affiliations

National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Validation Study

Language

eng

PubMed ID

19393612

Citation

Chen, Qingfeng, et al. "A Liquid Chromatography-tandem Mass Spectrometry-based Assay for Indole-3-acetic Acid-amido Synthetase." Analytical Biochemistry, vol. 390, no. 2, 2009, pp. 149-54.
Chen Q, Zhang B, Hicks LM, et al. A liquid chromatography-tandem mass spectrometry-based assay for indole-3-acetic acid-amido synthetase. Anal Biochem. 2009;390(2):149-54.
Chen, Q., Zhang, B., Hicks, L. M., Wang, S., & Jez, J. M. (2009). A liquid chromatography-tandem mass spectrometry-based assay for indole-3-acetic acid-amido synthetase. Analytical Biochemistry, 390(2), 149-54. https://doi.org/10.1016/j.ab.2009.04.027
Chen Q, et al. A Liquid Chromatography-tandem Mass Spectrometry-based Assay for Indole-3-acetic Acid-amido Synthetase. Anal Biochem. 2009 Jul 15;390(2):149-54. PubMed PMID: 19393612.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A liquid chromatography-tandem mass spectrometry-based assay for indole-3-acetic acid-amido synthetase. AU - Chen,Qingfeng, AU - Zhang,Baichen, AU - Hicks,Leslie M, AU - Wang,Shiping, AU - Jez,Joseph M, Y1 - 2009/04/23/ PY - 2009/02/05/received PY - 2009/04/16/revised PY - 2009/04/16/accepted PY - 2009/4/28/entrez PY - 2009/4/28/pubmed PY - 2009/8/6/medline SP - 149 EP - 54 JF - Analytical biochemistry JO - Anal Biochem VL - 390 IS - 2 N2 - Indole-3-acetic acid (IAA) amide conjugates play an important role in balancing levels of free IAA in plant cells. The GH3 family of proteins conjugates free IAA with various amino acids. For example, auxin levels modulate expression of the Oryza sativa (rice) GH3-8 protein, which acts to prevent IAA accumulation by coupling the hormone to aspartate. To examine the kinetic properties of the enzyme, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay system. Bacterially expressed OsGH3-8 was purified to homogeneity and used to establish the assay system. Monitoring of the reaction confirms the reaction product as IAA-Asp and demonstrates that production of the conjugate increases proportionally with both time and enzyme amount. Steady-state kinetic analysis using the LC-MS/MS-based assay yields the following parameters: V/E(t)(IAA)=20.3 min(-1), K(m)(IAA)=123 microM, V/E(t)(ATP)=14.1 min(-1), K(m)(ATP)=50 microM, V/E(t)(Asp)=28.8 min(-1), K(m)(Asp)=1580 microM. This is the first assignment of kinetic values for any IAA-amido synthetase from plants. Compared with previously described LC- and thin-layer chromatography (TLC)-based assays, this LC-MS/MS method provides a robust and sensitive means for performing direct kinetic studies on a range of IAA-conjugating enzymes. SN - 1096-0309 UR - https://www.unboundmedicine.com/medline/citation/19393612/A_liquid_chromatography_tandem_mass_spectrometry_based_assay_for_indole_3_acetic_acid_amido_synthetase_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-2697(09)00275-9 DB - PRIME DP - Unbound Medicine ER -