An enriched fraction of an inhibitor of both the catalytic subunit of the cAMP-dependent protein kinase (PKA) from pig heart and a Trypanosoma equiperdum PKA catalytic subunit-like protein (TeqC-like) was obtained from the soluble fraction of T. equiperdum parasites after three consecutive purification steps: sedimentation through a linear 5-20% sucrose gradient, diethylaminoethyl-Sepharose anion-exchange chromatography, and Bio-Sil Sec-400-S size-exclusion high-performance liquid chromatography. The inhibitor was identified as the T. equiperdum PKA regulatory subunit-like protein (TeqR-like) on the basis of Western blot and mass spectrometry analyses, and behaved as an uncompetitive or anti-competitive inhibitor of the parasite TeqC-like protein, with respect to a fluorescently labeled substrate (kemptide, sequence: LRRASLG), showing a Ki of 1.17 μM. The isolated protein possesses a molecular mass of 57.54 kDa, a Stokes radius of 3.64 nm, and a slightly asymmetric shape with a frictional ratio f/fo = 1.43. As revealed during the purification steps and by immunoprecipitation experiments, the TeqR-like and TeqC-like proteins were not associated forming a heterooligomeric complex, differing from traditional PKA subunits. Co-immunoprecipitation results followed by mass spectrometry sequencing identified two isoforms of the parasite heat-shock protein 70, α-tubulin, and β-tubulin as candidates that interact with the TeqR-like protein in T. equiperdum.

中文翻译：

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来自马齿锥虫的cAMP依赖性蛋白激酶调节亚基样蛋白的生化特征，其抑制活性的检测以及潜在相互作用蛋白的鉴定。

从马齿T寄生虫的可溶性级分中获得了来自猪心脏的cAMP依赖性蛋白激酶（PKA）催化亚基和马氏锥虫PKA催化亚基样蛋白（TeqC-like）的抑制剂的富集级分。经过三个连续的纯化步骤：通过线性5-20％蔗糖梯度沉降，二乙氨基乙基-Sepharose阴离子交换色谱和Bio-Sil Sec-400-S尺寸排阻高效液相色谱。在Western印迹和质谱分析的基础上，该抑制剂被鉴定为马兜铃虫PKA调节亚基样蛋白（TeqR-like），并作为寄生虫TeqC-样蛋白的无竞争性或反竞争性抑制剂，与相对于荧光标记底物（kemptide，序列：LRRASLG），Ki为1。17微米 分离出的蛋白质的分子量为57.54 kDa，斯托克斯半径为3.64 nm，呈稍不对称的形状，摩擦比f / fo = 1.43。正如纯化步骤和免疫沉淀实验所揭示的，TeqR样和TeqC样蛋白没有缔合，形成了异源寡聚复合物，与传统的PKA亚基不同。共免疫沉淀结果，随后进行质谱测序，确定了寄生虫热休克蛋白70的两种亚型，即α-微管蛋白和β-微管蛋白，它们与马鞭草中的TeqR-样蛋白相互作用。与传统的PKA亚基不同，TeqR样和TeqC样蛋白没有结合形成异寡聚复合物。共免疫沉淀结果，随后进行质谱测序，确定了寄生虫热休克蛋白70的两种亚型，即α-微管蛋白和β-微管蛋白，它们与马鞭草中的TeqR-样蛋白相互作用。与传统的PKA亚基不同，TeqR样和TeqC样蛋白没有结合形成异寡聚复合物。共免疫沉淀结果，随后进行质谱测序，确定了寄生虫热休克蛋白70的两种亚型，即α-微管蛋白和β-微管蛋白，它们与马鞭草中的TeqR-样蛋白相互作用。