Purification of adenylosuccinate lyase from rat skeletal muscle by a novel affinity column. Stabilization of the enzyme, and effects of anions and fluoro analogues of the substrate.
Purification of adenylosuccinate lyase from rat skeletal muscle by a novel affinity column. Stabilization of the enzyme, and effects of anions and fluoro analogues of the substrate.
复制标题
通过新型亲和柱从大鼠骨骼肌中纯化腺苷琥珀酸裂合酶。
DOI：
10.1042/bj2460263
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
John M. Lowenstein
中科院分区：
文献类型：
--
作者：
Patrick J. Casey;John M. Lowenstein
采用离子交换层析和亲和层析相结合的方法，对含有共价结合的腺苷磷酸丙酸琼脂糖纯化大鼠骨骼肌中腺苷琥珀酸裂解酶，使其具有明显的同质性。纯化后的酶在-70℃下储存在20%甘油中是稳定的，并且可以以最小的活性损失进行解冻和再冷冻。在25℃条件下，腺苷琥珀酸裂解酶的比活性为每mg蛋白质11 μ mol/min，经SDS/聚丙烯酰胺凝胶电泳，其亚基Mr为52,000，表观天然Mr约为。20万，凝胶过滤。纯化后的酶在Hepes/KOH缓冲液（pH 7.4）中对琥珀酸腺苷酸和4-（n -琥珀酸）-5-氨基咪唑-4-羧酰胺核糖核苷酸（SAICAR）的Km值分别为1.5微米和约1微米。几个单阴离子和阴离子在低浓度下激活酶；其中一些在高浓度时抑制酶。采用高纯度琥珀酸腺苷合酶和SAICAR合酶分别合成琥珀酸腺苷和SAICAR的氟类似物，并用红- β -氟天冬氨酸代替天冬氨酸。在两种酶催化的反应中，这两种类似物都是腺苷琥珀酸裂解酶的竞争性抑制剂，Ki值远低于两种底物的Km值。
Adenylosuccinate lyase from rat skeletal muscle was purified to apparent homogeneity by a combination of ion-exchange chromatography and affinity chromatography on agarose containing covalently bound adenylophosphonopropionate. The purified enzyme is stable when stored in 20% glycerol at -70 degrees C, and can be thawed and re-frozen with minimal loss of activity. Adenylosuccinate lyase has a specific activity of 11 mumol/min per mg of protein at 25 degrees C. Its subunit Mr is 52,000, by SDS/polyacrylamide-gel electrophoresis, and its apparent native Mr is approx. 200,000, by gel filtration. The purified enzyme has Km values for adenylosuccinate and 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) of 1.5 microM and approximately 1 microM respectively, in Hepes/KOH buffer, pH 7.4. Several monoanions and dianions activate the enzyme at low concentration; several of these inhibit the enzyme at high concentrations. Fluoro analogues of adenylosuccinate and SAICAR were synthesized by using highly purified adenylosuccinate synthase and SAICAR synthase respectively, and erythro-beta-fluoroaspartate in place of aspartate. Both analogues are competitive inhibitors of adenylosuccinate lyase in both of the reactions catalysed by the enzyme, with Ki values well below the Km values for the two substrates.