Guranowski A, Starzyńska E, Rataj-Guranowska M, Günther Sillero M A
Katedra Biochemii, Akademia Rolnicza, Poznań, Poland.
Protein Expr Purif. 1991 Aug;2(4):235-9. doi: 10.1016/1046-5928(91)90078-w.
Neutralized 1 M perchloric acid (PCA) extracts of yellow lupin (Lupinus luteus) seedling cotyledons contain considerable amounts of apyrase (EC 3.6.1.5). Investigators who use PCA extraction for the estimation of nucleotide levels, particularly in plant tissues, should be aware of this danger. Only when the material is treated with 1.8-2 M PCA are the extracts obtained free of apyrase activity. Chromatography of neutralized 1 M extracts obtained from 7-day-old seedling cotyledons on DEAE-Sephacel and Sephadex G-100 yields almost homogeneous apyrase that shows a band of M(r) 51,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. The molecular weight of the native enzyme is also about 51,000. The apyrase preparation is free of nonspecific phosphatases, nucleotidases, and adenosine nucleosidase, as well as dinucleoside polyphosphate-degrading enzymes. The apyrase exhibits a broad pH optimum between 6 and 8. Mg2+ and Ca2+ are required for maximum activity; Zn2+ and Mn2+ are less effective and Co2+, Ni2+, and Cd2+ are without effect. The Km values for ATP and ADP are about 20 microM. All common 5'-nucleoside tri- and diphosphates as well as adenosine 5'-tetraphosphate are substrates.
黄羽扇豆（Lupinus luteus）幼苗子叶的中和1 M高氯酸（PCA）提取物含有大量的腺苷三磷酸双磷酸酶（EC 3.6.1.5）。使用PCA提取法来估算核苷酸水平的研究人员，尤其是在植物组织中，应该意识到这种风险。只有当材料用1.8 - 2 M PCA处理时，所获得的提取物才没有腺苷三磷酸双磷酸酶活性。对7日龄幼苗子叶的中和1 M提取物在DEAE - Sephacel和Sephadex G - 100上进行色谱分析，得到几乎均一的腺苷三磷酸双磷酸酶，其在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳凝胶上显示出一条分子量为51,000的条带。天然酶的分子量也约为51,000。该腺苷三磷酸双磷酸酶制剂不含非特异性磷酸酶、核苷酸酶、腺苷核苷酶以及二核苷多磷酸降解酶。该腺苷三磷酸双磷酸酶在6至8之间表现出较宽的最适pH值。Mg2 +和Ca2 +是最大活性所必需的；Zn2 +和Mn2 +的效果较差，而Co2 +、Ni2 +和Cd2 +则无作用。ATP和ADP的Km值约为20 microM。所有常见的5'-核苷三磷酸和二磷酸以及腺苷5'-四磷酸都是底物。
