Analysis of the mechanism of ATP stimulation of calf thymus DNA alpha-polymerase.
Analysis of the mechanism of ATP stimulation of calf thymus DNA alpha-polymerase.
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ATP刺激小牛胸腺DNA α聚合酶的机制分析。
DOI：
10.1021/bi00314a007
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发表时间：
1984
期刊：
影响因子：
2.9
通讯作者：
Bambara,RA
中科院分区：
文献类型：
--
作者：
Lawton,KG;Wierowski,JV;Schechter,S;Hilf,R;Bambara,RA
凯西·G Lawton，** James V. Wierowski，Steven Schechter，1 Russell Hilf，and Robert A. Bambara §摘要：ATP刺激小牛DNA α-聚合酶A2型的生化动力学分析表明，当DNA或引物末端为可变底物时，最大反应速度与ATP浓度无关。当dNTP浓度为可变底物时，表观Km不随ATP变化。这些结果表明，ATP引起的合成速率的增加是由于引物末端合成起始的改善。用柱层析法测定了ATP对α-A2-聚合酶DNA结合亲和力的影响。聚合酶在70 mM离子强度下通过天然DNA-纤维素导致40%的酶结合。在4 mM ATP存在下，结合增加到80%。在这两种情况下，结合的聚合酶可以通过370 mM离子强度洗涤洗脱。用0.1 mM ATP、4 mM GTP或各4 mM不可水解的ATP类似物腺苷-5 '-基亚氨基二磷酸或腺苷5'-O-亚氨基二磷酸，获得了与在不存在ATP时观察到的类似的洗脱曲线。
Kathy G. Lawton,** James V. Wierowski, Steven Schechter, 1 Russell Hilf, and Robert A. Bambara § abstract: Biochemical kinetic analyses of the ATP stimu-lation of the A2 form of calf DNA a-polymerase show that when DNA or primer termini are the variable substrates, maximum reaction velocity is independent of ATP concen-tration. When dNTP concentration is the variable substrate, the apparent Km is invariant with ATP. Such results indicate that theincrease in the synthetic rate caused by ATP results from an improvement in synthesis initiation at primer termini. The effect ofATP on the DNA binding affinity of a-A2-polymerase was examined by using column chromatography. Passage of the polymerase through native DNA-cellulose at 70 mM ionic strength resulted in40% binding of theenzyme. In the presence of 4 mM ATP, binding increased to80%. In both cases, the bound polymerase could be eluted by a 370 mM ionicstrength wash. An elution profile similar to that observed in the absence of ATP was obtained with 0.1 mM ATP, 4 mM GTP, or 4 mM each of the nonhydrolyzable ATP analogues adenyl-5'-yl imidodiphosphate or adenosine 5'-O-