在金黄色葡萄球菌核酸酶测活体系中Ca^(2+)与底物DNA的结合

THE BINDING OF SUBSTRATE DNA AND CALCIUM IN STAPHYLOCOCCAL NUCLEASE ASSAY SYSTEM

将携带金黄色葡萄球菌核酸酶(SNase)基因的质粒pFOG405转化到大肠杆菌SE6004中,获得分泌表达的SNase,在该酶的测活体系中,观察到激活剂Ca^(2+)与底物DNA微弱结合,引起底物DNA的UV光谱在260nm附近明显下降。在Ca^(2+)浓度为10mM时,底物DNA紫外区CD光谱在270—280nm和210—230nm处〔θ〕值减少,〔θ〕值为零的点从260nm移至257nm。这一结果表明,Ca^(2+)与底物DNA在缺少SNase的情况下仍能结合,引起DNA分子构象变化。

Staphylococcal nuclease (SNase) has been purified from Escherichia coli harboring arecombinat plasm id PFOG 405 with the SNase gene.In the assay system of the enzyme Ca2+ is a stimulator. We observed that Ca2+ weakly bound to the substrate DNA even in the absence of SNase. As measured by UV and CD spectra, the effect of Ca2+ on the substrate DNA can be seen from that the UV absorption decreases at the peak of 260 nm, the [θ] value decreases at 270-280 nm and 210-230 nm,and the point of [θ] = 0 shifted from 260 nm to 257 nm. It suggests that a confer mational change of the substrate DNA may be involved in the stimulation of SNase activity.