Walker R P, Leegood R C
Robert Hill Institute, University of Sheffield, UK.
FEBS Lett. 1995 Mar 27;362(1):70-4. doi: 10.1016/0014-5793(95)00212-r.
Phosphoenolpyruvate carboxykinase (PEPCK) with a subunit molecular mass of 74 kDa has been purified 450-fold to homogeneity from the cotyledons of cucumber (Cucumis sativus L.). This is the first purification of the native form of the enzyme from any plant tissue. Incubation of the purified enzyme with [gamma-32P]ATP and either phosphoenolpyruvate-carboxylase kinase or mammalian cAMP-dependent protein kinase led to labelling of the enzyme in a part of the molecule separate from the active site. This was reversed by incubation with protein phosphatase 2A. Cotyledons of cucumber seedlings were also supplied with 32Pi. Homogenates of such cotyledons contained a heavily labelled polypeptide which was confirmed as PEPCK by immunoprecipitation. Labelling of PEPCK by 32Pi in darkened cotyledons was reversed by illumination.
已从黄瓜（Cucumis sativus L.）子叶中纯化出分子量为74 kDa的亚基的磷酸烯醇丙酮酸羧激酶（PEPCK），纯化倍数达450倍，达到同质。这是首次从任何植物组织中纯化出该酶的天然形式。将纯化后的酶与[γ-32P]ATP以及磷酸烯醇丙酮酸羧化酶激酶或哺乳动物cAMP依赖性蛋白激酶一起孵育，会导致该酶分子中与活性位点分开的一部分被标记。用蛋白磷酸酶2A孵育可使这种标记逆转。黄瓜幼苗的子叶也被供应了32Pi。这种子叶的匀浆含有一种高度标记的多肽，通过免疫沉淀证实其为PEPCK。光照可逆转黑暗中子叶中32Pi对PEPCK的标记。
