Functional studies of chloroplast glyceraldehyde-3-phosphate dehydrogenase subunits A and B expressed in Escherichia coli: Formation of highly active A(4) and B-4 homotetramers and evidence that aggregation of the B-4 complex is mediated by the B subunit carboxy terminus
Functional studies of chloroplast glyceraldehyde-3-phosphate dehydrogenase subunits A and B expressed in Escherichia coli: Formation of highly active A(4) and B-4 homotetramers and evidence that aggregation of the B-4 complex is mediated by the B subunit carboxy terminus
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DOI：
10.1007/bf00019102
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发表时间：
1996-11-01
影响因子：
5.1
通讯作者：
Martin, W
中科院分区：
文献类型：
--
作者：
Baalmann, E;Scheibe, R;Martin, W
高等植物叶绿体甘油醛-3-磷酸脱氢酶(磷酸化，E.C.1.2.1.13)(GAPDH)以A(2)B(2)异构体的形式存在，催化Calvin循环的还原步骤。在黑暗的叶绿体中，该酶显示出600 kDa的分子质量，而在有光的叶绿体中，分子质量改变为更活跃的150 kDa形式。我们已经在大肠杆菌中表达了与菠菜叶绿体GAPDH成熟的A和B亚基相对应的蛋白质(分别为GAPA和GapB)，以及缺乏GapB特异的C-末端延伸(CTE)的B亚基的衍生物。用常规方法将如此表达的三种蛋白质中的每一种都纯化一毫克，以获得电泳均一。从大肠杆菌中纯化的菠菜GAPA是一种高活性的同源四聚体(50-70U/mg)，在体外聚集条件下不与约600 kDa的高分子质量(HMM)相结合。由于Bq形式的酶还没有从任何来源描述，我们惊讶地发现从大肠杆菌中纯化的菠菜GapB具有活性(15-35U/mg)。从大肠杆菌中纯化的缺失CTE的菠菜GapB比含有CTE的GapB活性更高(130U/mg)。在聚集条件下，缺乏CTE的GapB是与HMM形式无关的四聚体，而具有CTE的GapB仅以聚集的HMM形式出现。这些数据表明，叶绿体GAPDH四聚体间的相互作用是通过GapB介导的蛋白质-蛋白质相互作用实现的。
Chloroplast glyceraldehyde-3-phosphate dehydrogenase (phosphorylating, E.C. 1.2.1.13) (GAPDH) of higher plants exists as an A(2)B(2) heterotetramer that catalyses the reductive step of the Calvin cycle. In dark chloroplasts the enzyme exhibits a molecular mass of 600 kDa, whereas in illuminated chloroplasts the molecular mass is altered in favor of the more active 150 kDa form. We have expressed in Escherichia coli proteins corresponding to the mature A and B subunits of spinach chloroplast GAPDH (GapA and GapB, respectively) in addition to a derivative of the B subunit lacking the GapB-specific C-terminal extension (CTE). One mg of each of the three proteins so expressed was purified to electrophoretic homogeneity with conventional methods. Spinach GapA purified from E. coli is shown to be a highly active homotetramer (50-70 U/mg) which does not associate under aggregating conditions in vitro to high-molecular-mass (HMM) forms of ca. 600 kDa. Since Bq forms of the enzyme have not been described from any source, we were surprised to find that spinach GapB purified from E. coli was active (15-35 U/mg). Spinach GapB lacking the CTE purified from E. coli is more highly active (130 U/mg) than GapB with the CTE. Under aggregating conditions, GapB lacking the CTE is a tetramer that does not associate to HMM forms whereas GapB with the CTE occurs exclusively as an aggregated HMM form. The data indicate that intertetramer association of chloroplast GAPDH in vitro occurs through GapB-mediated protein-protein interaction.