| Full text | |
| Author(s): |
Magalhaes, Luana
[1]
;
Cavalcante de Oliveira, Arthur Henrique
[1]
;
Vasconcellos, Raphael de Souza
[2, 3]
;
Mariotini-Moura, Christiane
[2, 3]
;
Firmino, Rafaela de Cassia
[2]
;
Rangel Fietto, Juliana Lopes
[2, 3]
;
Cardoso, Carmen Lucia
[1]
Total Authors: 7
|
| Affiliation: | [1] Univ Sao Paulo, Dept Quim, Fac Filosofia Ciencias & Letras Ribeirao Preto, BR-14040901 Ribeirao Preto, SP - Brazil
[2] Univ Fed Vicosa, Dept Bioquim & Biol Mol, BR-36570000 Vicosa, MG - Brazil
[3] INBEQMeDI, Sao Carlos, SP - Brazil
Total Affiliations: 3
|
| Document type: | Journal article |
| Source: | JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES; v. 1008, p. 98-107, JAN 1 2016. |
| Web of Science Citations: | 3 |
| Abstract | |
Nucleoside triphosphate diphosphohydrolase (NTPDase) is an enzyme belonging to the apyrase family that participates in the hydrolysis of the nucleosides di- and triphosphate to the corresponding nucleoside monophosphate. This enzyme underlies the virulence of parasites such as Leishmania. Recently, an NTPDase from Leishmania infantum (LicNTPDase-2) was cloned and expressed and has been considered as a new drug target for the treatment of leishmaniasis. With the intent of developing label-free online screening methodologies, LicNTPDase-2 was covalently immobilized onto a fused silica capillary tube in the present study to create an immobilized capillary enzyme reactor (ICER) based on LicNTPDase-2 (LicNTPDase-2-ICER). To perform the activity assays, a multidimensional chromatographic method was developed employing the LicNTPDase-2-ICER in the first dimension, and an analytical Ascentis C8 column was used in the second dimension to provide analytical separation of the substrates and products. The validated LicNTPDase-2-ICER method provided the following kinetic parameters of the immobilized enzyme: K-M of 2.2 and 1.8 mmol L-1 for the ADP and ATP substrates, respectively. Suramin (1 mmol L-1) was also shown to inhibit 32.9% of the enzymatic activity. The developed method is applicable to kinetic studies and enables the recognition of the ligands. Furthermore, a comparison of the values of LicNTPDase-2-ICER with those obtained with an LC method using free enzyme in solution showed that LicNTPDase-2-ICER-LC/UV was an accurate and reproducible method that enabled automated measurements for the rapid screening of ligands. (C) 2015 Elsevier B.V. All rights reserved. (AU) | |
| FAPESP's process: | 13/01710-1 - Enzyme ligand: new models of screening |
| Grantee: | Quezia Bezerra Cass |
| Support Opportunities: | Research Projects - Thematic Grants |
| FAPESP's process: | 12/10749-6 - Development of capillary columns enzymatic to application of screening of selective inhibitors for the enzyme NTPDase- 2 of Leishmania infantun |
| Grantee: | Luana Magalhães |
| Support Opportunities: | Scholarships in Brazil - Doctorate |