Gite S, Reddy G, Shankar V
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Biochem J. 1992 Jul 15;285 ( Pt 2)(Pt 2):489-94. doi: 10.1042/bj2850489.
A simple procedure, involving heat-treatment, DEAE-Sephadex, AMP-Sepharose and Bio-Gel P-60 chromatography, was developed for the purification of S1 nuclease to homogeneity from commercially available Takadiastase powder. Chemical modification of the amino groups of purified S1 nuclease revealed that lysine is essential for single-stranded DNAase, RNAase and phosphomonoesterase activities associated with the enzyme. The kinetics of inactivation suggested the involvement of a single lysine residue in the active site of the enzyme. Additionally, lysine modification was accompanied by a concomitant loss of all the activities of the enzyme, indicating the presence of a common catalytic site responsible for the hydrolysis of single-stranded DNA, RNA and 3'-AMP. Substrate-protection and inhibitor-binding studies on enzyme modified with 2,4,6-trinitrobenzenesulphonic acid showed that lysine may be involved in the substrate binding.
已开发出一种简单的方法，该方法包括热处理、二乙氨基乙基葡聚糖（DEAE - Sephadex）、AMP - 琼脂糖和生物凝胶P - 60色谱法，用于从市售的高峰淀粉酶粉末中纯化S1核酸酶至均一状态。对纯化的S1核酸酶的氨基进行化学修饰表明，赖氨酸对于与该酶相关的单链DNA酶、RNA酶和磷酸单酯酶活性至关重要。失活动力学表明在该酶的活性位点存在单个赖氨酸残基。此外，赖氨酸修饰伴随着该酶所有活性的同时丧失，表明存在一个负责水解单链DNA、RNA和3'-AMP的共同催化位点。对用2,4,6 - 三硝基苯磺酸修饰的酶进行的底物保护和抑制剂结合研究表明，赖氨酸可能参与底物结合。
