Sainis J K, Merriam K, Harris G C
Department of Biological Sciences, Wellesley College, Wellesley, Massachusetts 02181.
Plant Physiol. 1989 Jan;89(1):368-74. doi: 10.1104/pp.89.1.368.
When Ribulose- 1,5-bisphosphate carboxylase/oxygenase was purified from spinach leaves (Spinacia oleracea) using precipitation with polyethylene glycol and MgCl(2) followed by DEAE cellulose chromatography, 75% of phosphoribulokinase and 7% of phosphoriboisomerase activities copurified with ribulose- 1,5-bisphosphate carboxylase/oxygenase. This enzyme preparation showed ribose-5-phosphate and ribulose-5-phosphate dependent carboxylase and oxygenase activities which were nearly equivalent to its corresponding ribulose- 1,5-bisphosphate dependent activity. The ribose-5-phosphate and ribulose-5-phosphate dependent reaction rates were stable and linear for much longer time periods than the ribulose- 1,5-bisphosphate dependent rates. When sucrose gradients were used to purify ribulose- 1,5-bisphosphate carboxylase/oxygenase from crude stromal extracts, phosphoribulokinase was found to cosediment with ribulose- 1,5-bisphosphate carboxylase. Under these conditions most of the phosphoriboisomerase activity remained with the slower sedimenting proteins. Ammonium sulfate precipitation resulted in separation of the ribulose- 1,5-bisphosphate carboxylase peak from phosphoribulokinase peak. Crude extracts of peas Pisum sativum and spinach contained 0.725 to 0.730 milligram of phosphoribulokinase per milligram of chlorophyll, respectively, based on an enzyme-linked immunosorbent assay.
当使用聚乙二醇和氯化镁沉淀，随后通过二乙氨基乙基纤维素色谱法从菠菜叶（菠菜）中纯化1,5 - 二磷酸核酮糖羧化酶/加氧酶时，75%的磷酸核酮糖激酶和7%的磷酸核糖异构酶活性与1,5 - 二磷酸核酮糖羧化酶共纯化。这种酶制剂显示出依赖于5 - 磷酸核糖和5 - 磷酸核酮糖的羧化酶和加氧酶活性，这些活性几乎等同于其相应的依赖于1,5 - 二磷酸核酮糖的活性。与依赖于1,5 - 二磷酸核酮糖的速率相比，依赖于5 - 磷酸核糖和5 - 磷酸核酮糖的反应速率在更长的时间段内是稳定且呈线性的。当使用蔗糖梯度从粗基质提取物中纯化1,5 - 二磷酸核酮糖羧化酶/加氧酶时，发现磷酸核酮糖激酶与1,5 - 二磷酸核酮糖羧化酶一起沉降。在这些条件下，大部分磷酸核糖异构酶活性仍与沉降较慢的蛋白质在一起。硫酸铵沉淀导致1,5 - 二磷酸核酮糖羧化酶峰与磷酸核酮糖激酶峰分离。基于酶联免疫吸附测定，豌豆（豌豆）和菠菜的粗提取物每毫克叶绿素分别含有0.725至0.730毫克的磷酸核酮糖激酶。
