ISOLATION AND CHARACTERIZATION OF THE CYTOSOLIC AND CHLOROPLASTIC 3-PHOSPHOGLYCERATE KINASE FROM SPINACH LEAVES
ISOLATION AND CHARACTERIZATION OF THE CYTOSOLIC AND CHLOROPLASTIC 3-PHOSPHOGLYCERATE KINASE FROM SPINACH LEAVES
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DOI：
10.1104/pp.93.1.40
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发表时间：
1990-05-01
期刊：
影响因子：
7.4
通讯作者：
SCHNARRENBERGER, C
中科院分区：
文献类型：
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作者：
KOPKESECUNDO, E;MOLNAR, I;SCHNARRENBERGER, C
对菠菜胞浆和叶绿体3-磷酸甘油酸激酶（3-PGK）进行了纯化，达到明显的均匀性。该方法包括在deae -纤维素上进行常规阴离子交换色谱，主要是一系列高效液相色谱柱。两种同工酶的电荷差异非常小，因此只有在HPLC SynChropak AX 300柱上进行阴离子交换色谱才能成功分离。两种同工酶在叶片组织中的含量分别为5%和95%。主要的3-PGK与分离的叶绿体相关，而其他3-PGK仅存在于可溶性细胞部分。纯化酶的比活性约为800单位（每毫克蛋白质）。两种3-PGK在非变性（尺寸排除色谱）和变性（SDS-PAGE）条件下的分子量均在40千道尔左右，胞浆型3-PGK略小于叶绿体型3-PGK。抗绿质体3-PGK血清与胞浆3-PGK的交叉反应仅为4.6%。甘油-3-磷酸和MgATP2-的动力学是双相的。Na2SO4的存在改变了MgATP2-的线性依赖性，但没有改变甘油-3-磷酸的线性依赖性。
The cytosol and chloroplast 3-phosphoglycerate kinases (3-PGK) from spinach (Spinacia oleracea L.) were purified to apparent homogeneity. The procedure included a conventional anion-exchange chromatography on DEAE-cellulose and mainly a series of HPLC columns. The charge differences of the two isoenzymes were so small that separation was only successful by anion-exchange chromatography on a HPLC SynChropak AX 300 column. The portion of the two isoenzymes in leaf tissue was estimated as 5% and 95%. The major 3-PGK was associated with isolated chloroplasts while the other 3-PGK was only found in the soluble cell fraction. The specific activity of the purified enzymes were in the order of the 800 units (per milligram of protein). The molecular weight for the two 3-PGDs under nondenaturing (size exclusion chromatography) and denaturing (SDS-PAGE) conditions were in the order of 40 kilodaltons, with the cytosolic 3-PGK being slightly smaller than the chloroplastic 3-PGK. An antiserum against the chloroplastic 3-PGK showed only 4.6% cross-reaction of the chloroplastic 3-PGK with the cytosolic 3-PGK. The kinetics for glycerate-3-phosphate and MgATP2- were biphasic. The presence of Na2SO4 changed the MgATP2- dependence to linearity but not the glycerate-3-phosphate dependence.