Mass spectrometry assay for studying kinetic properties of dipeptidases: characterization of human and yeast dipeptidases.
IR@IMTECH: CSIR-Institute of Microbial Technology, Chandigarh
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| Title |
Mass spectrometry assay for studying kinetic properties of dipeptidases: characterization of human and yeast dipeptidases.
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| Creator |
Pandya, Vaibhav
Ekka, Mary Krishna Dutta, Rajesh Kumar Kumaran, Sangaralingam |
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| Subject |
QD Chemistry
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| Description |
Chemical modifications of substrate peptides are often necessary to monitor the hydrolysis of small bioactive peptides. We developed an electrospray ionization mass spectrometry (ESI-MS) assay for studying substrate distributions in reaction mixtures and determined steady-state kinetic parameters, the Michaelis-Menten constant (K(m)), and catalytic turnover rate (V(max)/[E](t)) for three metallodipeptidases: two carnosinases (CN1 and CN2) from human and Dug1p from yeast. The turnover rate (V(max)/[E](t)) of CN1 and CN2 determined at pH 8.0 (112.3 and 19.5s(-1), respectively) suggested that CN1 is approximately 6-fold more efficient. The turnover rate of Dug1p for Cys-Gly dipeptide at pH 8.0 was found to be slightly lower (73.8s(-1)). In addition, we determined kinetic parameters of CN2 at pH 9.2 and found that the turnover rate was increased by 4-fold with no significant change in the K(m). Kinetic parameters obtained by the ESI-MS method are consistent with results of a reverse-phase high-performance liquid chromatography (RP-HPLC)-based assay. Furthermore, we used tandem MS (MS/MS) analyses to characterize carnosine and measured its levels in CHO cell lines in a time-dependent manner. The ESI-MS method developed here obviates the need for substrate modification and provides a less laborious, accurate, and rapid assay for studying kinetic properties of dipeptidases in vitro as well as in vivo.
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| Publisher |
Elsevier Science
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| Date |
2011-11-01
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| Type |
Article
PeerReviewed |
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| Relation |
http://www.sciencedirect.com/science/article/pii/S000326971100426X
http://crdd.osdd.net/open/458/ |
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| Identifier |
Pandya, Vaibhav and Ekka, Mary Krishna and Dutta, Rajesh Kumar and Kumaran, Sangaralingam (2011) Mass spectrometry assay for studying kinetic properties of dipeptidases: characterization of human and yeast dipeptidases. Analytical biochemistry, 418 (1). pp. 134-42. ISSN 1096-0309
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