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Glucose deprivation activates a cAMP-independent protein kinase from Trypanosoma equiperdum

Published online by Cambridge University Press:  13 November 2018

Alberto Guevara ,
Cristina Lugo ,
Alejandro J. Montilla ,
Nelson A. Araujo ,
Maritza Calabokis  and
José Bubis Open the ORCID record for José Bubis [Opens in a new window]
Alberto Guevara
Affiliation:
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela Postgrado en Ciencias Biológicas, Universidad Simón Bolívar, Caracas, Venezuela
Cristina Lugo
Affiliation:
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela Postgrado en Ciencias Biológicas, Universidad Simón Bolívar, Caracas, Venezuela
Alejandro J. Montilla
Affiliation:
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela Postgrado en Ciencias Biológicas, Universidad Simón Bolívar, Caracas, Venezuela
Nelson A. Araujo
Affiliation:
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela
Maritza Calabokis
Affiliation:
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela
José Bubis*
Affiliation:
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela
*
Author for correspondence: José Bubis, E-mail: [email protected]
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Abstract

Kemptide (sequence: LRRASLG) is a synthetic peptide holding the consensus recognition site for the catalytic subunit of the cAMP-dependent protein kinase (PKA). cAMP-independent protein kinases that phosphorylate kemptide were stimulated in Trypanosoma equiperdum following glucose deprivation. An enriched kemptide kinase-containing fraction was isolated from glucose-starved parasites using sedimentation throughout a sucrose gradient, followed by sequential chromatography on diethylaminoethyl-Sepharose and Sephacryl S-300. The trypanosome protein possesses a molecular mass of 39.07–51.73 kDa, a Stokes radius of 27.4 Ǻ, a sedimentation coefficient of 4.06 S and a globular shape with a frictional ratio f/fo = 1.22–1.25. Optimal enzymatic activity was achieved at 37 °C and pH 8.0, and kinetic studies showed Km values for ATP and kemptide of 11.8 ± 4.1 and 24.7 ± 3.8 µm, respectively. The parasite enzyme uses ATP and Mg2+ and was inhibited by other nucleotides and/or analogues of ATP, such as cAMP, AMP, ADP, GMP, GDP, GTP, CTP, β,γ-imidoadenosine 5′-triphosphate and 5′-[p-(fluorosulfonyl)benzoyl] adenosine, and by other divalent cations, such as Zn2+, Mn2+, Co2+, Cu2+, Ca2+ and Fe2+. Additionally, the trypanosome kinase was inhibited by the PKA-specific heat-stable peptide inhibitor PKI-α. This study is the first biochemical and enzymatic characterization of a protein kinase from T. equiperdum.

Keywords

Dourineglucose deprivationkemptide kinase activityprotein kinasesprotein phosphorylationsignal transductionTrypanosoma equiperdum
Type
Research Article
Information
Parasitology , Volume 146 , Issue 5 , April 2019 , pp. 643 - 652
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Copyright
Copyright © Cambridge University Press 2018 

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