RESOLUTION AND CHARACTERIZATION OF THE GLYCINE-CLEAVAGE REACTION IN PEA LEAF MITOCHONDRIA - PROPERTIES OF THE FORWARD REACTION CATALYZED BY GLYCINE DECARBOXYLASE AND SERINE HYDROXYMETHYLTRANSFERASE
RESOLUTION AND CHARACTERIZATION OF THE GLYCINE-CLEAVAGE REACTION IN PEA LEAF MITOCHONDRIA - PROPERTIES OF THE FORWARD REACTION CATALYZED BY GLYCINE DECARBOXYLASE AND SERINE HYDROXYMETHYLTRANSFERASE
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DOI：
10.1042/bj2550169
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发表时间：
1988-10-01
影响因子：
4.1
通讯作者：
DOUCE, R
中科院分区：
文献类型：
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作者：
BOURGUIGNON, J;NEUBURGER, M;DOUCE, R
保留在 XM-300 Diaflo 膜上的来自豌豆 (Pisum sativum) 线粒体基质的高分子量蛋白质（“基质提取物”）在 NAD+ 和四氢蝶酰-L-谷氨酸（H4叶酸）存在的情况下，只要介质表现出低离子强度，就会表现出高甘氨酸氧化率。丝氨酸羟甲基转移酶 (SHMT) (4 × 53 kDa) 和甘氨酸裂解系统的四种蛋白，包括含磷酸吡哆醛的酶（“P-蛋白”；2 × 97 kDa）、含有共价结合硫辛酸的载体蛋白（“H-蛋白”；15.5 kDa）、一种表现出硫辛酰胺脱氢酶活性的蛋白(“L-蛋白”；2×61 kDa)和H4叶酸依赖性酶(“T-蛋白”；45 kDa)已通过使用凝胶过滤、离子交换和苯基-Superose快速蛋白液相色谱从基质提取物中纯化至表观同质性。在 50 mM-KCl 存在下，Sephacryl S-300 的凝胶过滤被证明是破坏该复合物的关键步骤。在基质提取物催化的甘氨酸氧化过程中，5,10-亚甲基-H4叶酸的产生和利用达到了稳态平衡，这表明甘氨酸裂解和SHMT通过可溶性H4叶酸池连接在一起。基质提取物催化的甘氨酸氧化速率对 NADH/NAD+ 摩尔比敏感，因为 NADH 竞争性抑制硫辛酰胺脱氢酶催化的反应。
High-molecular-mass proteins from pea (Pisum sativum) mitochondrial matrix retained on an XM-300 Diaflo membrane (''matrix extract'') exhibited high rates of glycine oxidation in the presence of NAD+ and tetrahydropteroyl-L-glutamic acid (H4folate) as long as the medium exhibited a low ionic strength. Serine hydroxymethyltransferase (SHMT) (4 .times. 53 kDa) and the four proteins of the glycine-cleavage system, including a pyridoxal phosphate-containing enzyme (''P-protein''; 2 .times. 97 kDa), a carrier protein containing covalently bound lipoic acid (''H-protein''; 15.5 kDa), a protein exhibiting lipoamide dehydrogenase activity (''L-protein''; 2 .times. 61 kDa) and an H4folate-dependent enxyme (''T-protein''; 45 kDa) have been purified to apparent homogeneity from the matrix extract by using gel filtration, ion-exchange and phenyl-Superose fast protein liquid chromatography. Gel filtration of Sephacryl S-300 in the presence of 50 mM-KCl proved to be the key step in disrupting this complex. During the course of glycine oxidation catalysed by the matrix extract a steady-state equilibrium in the production and utilization of 5,10-methylene-H4folate was reached, suggesting that glycine cleavage and SHMT are linked together via a soluble pool of H4folate. The rate of glycine oxidation catalysed by the matrix extract was sensitive to the NADH/NAD+ molar ratios, because NADH competitively inhibited the reaction catalysed by lipoamide dehydrogenase.