Distinct sites for deoxyguanosine and deoxyadenosine phosphorylation on a monomeric kinase from Lactobacillus acidophilus.
Distinct sites for deoxyguanosine and deoxyadenosine phosphorylation on a monomeric kinase from Lactobacillus acidophilus.
复制标题
嗜酸乳杆菌单体激酶上脱氧鸟苷和脱氧腺苷磷酸化的不同位点。
DOI：
10.1021/bi00320a053
复制
发表时间：
1984
期刊：
影响因子：
2.9
通讯作者：
D. H. Ives
中科院分区：
文献类型：
--
作者：
R. Chakravarty;S. Ikeda;D. H. Ives
基线分离两对脱氧核苷激酶活性（脱氧胞苷/脱氧腺苷和脱氧鸟苷/脱氧腺苷激酶），以前解决了蓝色琼脂糖重叠峰，现在已经实现。通过用双底物混合物洗脱Blue Sepharose实现相对小体积的改进的分离和回收：0.5mM dCyd加ImM ATP释放的dCyd/dAdo激酶，和ImM dGuo加5 mM ATP洗脱的dGuo/dAdo激酶。后一对活性通过UDP-Sepharose亲和层析和HPLC阴离子交换层析显示共纯化。HPLC制备似乎是均匀的，在几个凝胶浓度和pH值的非变性聚丙烯酰胺凝胶电泳的基础上。dGuo和dAdo激酶活性与蛋白条带一致。十二烷基硫酸钠凝胶电泳也观察到单一蛋白条带。变性蛋白质的估计分子量（56 000）与通过沉降平衡或凝胶渗透色谱法获得的天然活性值密切一致。发现dAdo磷酸化的速率被dGuo的存在刺激超过3倍，并且dGuo激酶也被dAdo的存在轻微激活。这种相互激活表明dGuo和dAdo激酶活性不共享共同位点。通过5 '-[对-（氟磺酰基）苯甲酰基]腺苷选择性化学灭活dGuo激酶消除了dGuo刺激dAdo激酶的能力，同时dGuo激酶活性丧失。这些证据有力地表明，dGuo和dAdo激酶活性是单体多肽上不同位点的功能，并且这些位点可能处于变构通信中。
Base-line separation of two paired deoxynucleoside kinase activities (deoxycytidine/deoxyadenosine and deoxyguanosine/deoxyadenosine kinase), previously resolved as overlapping peaks from Blue Sepharose, has now been achieved. The improved separation and recovery in relatively small volumes were accomplished by eluting Blue Sepharose with a bisubstrate mixture: 0.5 mM dCyd plus 1 mM ATP released dCyd/dAdo kinase, and 1 mM dGuo plus 5 mM ATP eluted dGuo/dAdo kinase. The latter pair of activities showed copurification through UDP-Sepharose affinity chromatography and HPLC anion-exchange chromatography. The HPLC preparation appeared to be homogeneous, on the basis of nondenaturing polyacrylamide gel electrophoresis at several gel concentrations and pH values. Both dGuo and dAdo kinase activities coincided with the protein band. A single band of protein was also observed upon sodium dodecyl sulfate gel electrophoresis. The estimated molecular weight of the denatured protein (56 000) agrees closely with values obtained for native activity by sedimentation equilibrium or gel permeation chromatography. The rate of dAdo phosphorylation was found to be stimulated more than 3-fold by the presence of dGuo, and dGuo kinase was also slightly activated by the presence of dAdo. This mutual activation indicates that dGuo and dAdo kinase activities do not share a common site. Selective chemical inactivation of dGuo kinase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine eliminated the ability of dGuo to stimulate dAdo kinase in parallel with the loss of dGuo kinase activity. These lines of evidence strongly suggest that dGuo and dAdo kinase activities are functions of separate sites on a monomeric polypeptide and that these sites may be in allosteric communication.