Izsvák Z, Jobbágy Z, Duda E
Institute of Biochemistry, MTA Biological Research Center, Szeged, Hungary.
Z Naturforsch C J Biosci. 1992 Nov-Dec;47(11-12):830-4. doi: 10.1515/znc-1992-11-1208.
CeqI, a type II restriction endonuclease, an isoschizomer of EcoRV was purified to apparent homogeneity by a combination of salt precipitation, ion exchange, dye affinity and hydrophobic interaction chromatographies. The crude enzyme was present in the form of large aggregates that could be pelleted by high speed centrifugation. The enzyme was not associated with cellular membranes, though non-ionic detergents lowered the apparent size of the aggregates. The purified enzyme also showed a tendency to form large molecular mass (66-600 kDa) complexes under physiological conditions, in the absence of cleavable DNA. The enzyme formed smaller complexes in the presence of DNA and non-ionic detergents and dissociated into subunits (and undergoes reversible loss of activity) in the presence of high concentrations of salts. According to SDS gel electrophoresis and sedimentation analysis the molecular mass of the monomer 32 +/- 2 kDa. The enzyme had a rather broad PH optimum, extending into the alkaline range and lost specificity and activity in buffers below pH 6.
CeqI是一种II型限制性内切酶，是EcoRV的同裂酶，通过盐沉淀、离子交换、染料亲和和疏水相互作用色谱法相结合的方法纯化至表观均一。粗酶以大聚集体的形式存在，可通过高速离心沉淀。该酶不与细胞膜相关，尽管非离子去污剂降低了聚集体的表观大小。在没有可切割DNA的生理条件下，纯化后的酶也显示出形成大分子质量（66 - 600 kDa）复合物的趋势。在DNA和非离子去污剂存在的情况下，该酶形成较小的复合物，而在高浓度盐存在的情况下解离成亚基（并经历可逆的活性丧失）。根据SDS凝胶电泳和沉降分析，单体的分子量为32±2 kDa。该酶具有相当宽的最适pH值范围，延伸至碱性范围，在pH值低于6的缓冲液中失去特异性和活性。
