Glucose Deprivation Activates a Camp-Independent Protein Kinase Fromtrypanosoma Equiperdum
Parasitology(2018)
摘要
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 A, a sedimentation coefficient of 4.06 S and a globular shape with a frictional ratio f/f(o) = 1.22-1.25. Optimal enzymatic activity was achieved at 37 degrees C and pH 8.0, and kinetic studies showed K-m values for ATP and kemptide of 11.8 +/- 4.1 and 24.7 +/- 3.8 mu m, 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, beta,gamma-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-alpha. This study is the first biochemical and enzymatic characterization of a protein kinase from T. equiperdum.
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关键词
Dourine,glucose deprivation,kemptide kinase activity,protein kinases,protein phosphorylation,signal transduction,Trypanosoma equiperdum
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