Isolation and characterisation of cAMP-dependent protein kinase from Candida albicans -: Purification of the regulatory and catalytic subunits
Isolation and characterisation of cAMP-dependent protein kinase from Candida albicans -: Purification of the regulatory and catalytic subunits
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DOI：
10.1046/j.1432-1327.1998.2520245.x
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发表时间：
1998-03-01
期刊：
影响因子：
--
通讯作者：
Cantore, ML
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文献类型：
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作者：
Zelada, A;Passeron, S;Cantore, ML
从白色念珠菌酵母细胞中分离并表征了 cAMP 依赖性蛋白激酶 (PKA)。全酶及其亚基的结构参数表明，白色念珠菌 PKA 是一个 287 kDa 的四聚体，由两个 64 kDa 的调节 (R) 亚基和两个 78 kDa 的异常大分子量的催化 (C) 亚基组成。 ATP 和 Kemptide 的表观 K-m 分别为 30 μM 和 60 μM。 cAMP 激活的 [A](0.5) 为 150 nM，希尔系数为 1.6。全酶在 R 亚基上发生自磷酸化，这是 II 型 R 亚基的特征。酵母和菌丝细胞粗提物的 8-叠氮基-[P-32]cAMP 光亲和标记强烈表明真菌中只存在一种类型的 R 亚基。 R 亚基被纯化为 64 kDa 的蛋白质，具有明显的同质性。针对纯化蛋白产生的高度特异性多克隆抗血清从粗提物中免疫沉淀出 64-kDa 蛋白，表明纯化的 R 亚基很可能代表该蛋白的天然形式。在粗提物和具有异源抗 C Ig 的 Mono Q Sepharose 柱级分中检测到了 78 kDa 形式的 C 亚基。它可以通过用抗R血清对粗提物免疫沉淀的全酶进行cAMP处理来分离，但这种形式不能进一步纯化。相反，从酵母细胞的可溶性提取物中纯化出具有 C 亚基主要特征的 60 kDa 蛋白质，使其接近同质。有证据表明该蛋白质很可能通过蛋白水解降解衍生自 78-kDa 形式。
cAMP-dependent protein kinase (PKA) from Candida albicans yeast cells was isolated and characterised. Structural parameters of the holoenzyme and those of its subunits suggested that C. albicans PKA is a tetramer of 287 kDa composed of, two regulatory (R) subunits of 64 kDa and two catalytic (C) subunits of unusually large molecular mass of 78 kDa. The apparent K-m for ATP and Kemptide were 30 mu M and 60 mu M respectively. The [A](0.5) for cAMP activation was 150 nM with a Hill coefficient of 1.6. The holoenzyme undergoes autophosphorylation on the R subunit, a characteristic of the type-II R subunits. Photoaffinity labeling with 8-azido-[P-32]cAMP of crude extracts from yeast and mycelial cells strongly suggests that only one type of R subunit is present in the fungus. The R subunit was purified to apparent homogeneity as a protein of 64 kDa. A highly specific polyclonal antiserum raised against the purified protein immunoprecipitated a 64-kDa protein from crude extracts, indicating that the purified R subunit very probably represents the native form of the protein. The 78-kDa form of the C subunit was detected in crude extracts and in Mono Q Sepharose column fractions with heterologous anti-C Ig. It could be isolated by cAMP treatment of the holoenzyme immunoprecipitated from crude extracts with anti-R serum, but this form could not be purified further. Instead, a 60-kDa protein with the main characteristics of C subunit was purified to near homogeneity from soluble extracts of yeast cells. Evidence is presented that this protein very probably derives from the 78-kDa form by proteolytic degradation.