Requirement for reducing agents in deoxyribonucleic acid strand scisson by the purified chromophore of auromomycin.
Requirement for reducing agents in deoxyribonucleic acid strand scisson by the purified chromophore of auromomycin.
复制标题
纯化的金霉素发色团导致脱氧核糖核酸链断裂时需要还原剂。
DOI：
10.1021/bi00562a010
复制
发表时间：
1980
期刊：
影响因子：
2.9
通讯作者：
Samy,TS
中科院分区：
文献类型：
--
作者：
Kappen,LS;Napier,MA;Goldberg,IH;Samy,TS
材料和方法MCR和AUR由Dr. H. Umezawa从Kanegafuchi Chemical Industries Co.获得。通过丙烯酰胺凝胶电泳和等电聚焦（pJ 5.4），通过Sephadex G-100和deae -纤维素层析（T. S. A. Sarny，未发表的实验）进一步纯化至均匀性。纯化后的mcr在非离子型AmberliteXAD-7上进行层析，去除任何剩余的发色团。色谱条件与前面描述的NCS （Napier et al., 1980）相同。采用h2o洗脱馏分。pMB9 DNA的制备如前所述（Kappen & Goldberg, 1979）。AUR发色团的制备。用干燥、再蒸馏的甲醇（1ml）在0℃下提取AUR （2mg）。离心后得到的蛋白渣用甲醇（1ml）重新提取。将含有非蛋白发色团的组合上清液在70℃下保存，蛋白质残留物在蒸馏水中再溶解并冷冻保存。根据Lowry等人（1951）的方法测定，发色团的制备中最多含有5%的蛋白质污染物。高压液相色谱法。使用Waters Associates的A2C/GPC-204型色谱仪，配备660型溶剂流动编程器，MBondapak C18色谱柱（3.9 mm X 30 cm）， 254 nm吸光度检测器和Schoeffel SF 970型荧光检测器（360 nm激发，418 nm发射截止滤波器）。
Materials and Methods MCR and AUR were obtained from Kanegafuchi Chemical Industries Co. through the courtesy of Dr. H. Umezawa. They were further purified to homogeneity, as determined by acrylamide gel electrophoresisand isoelectric focusing (pJ 5.4), by chromatography on Sephadex G-100 and DEAE-cellulose (T. S. A. Sarny, unpublished experiments). PurifiedMCR was chromatographed on nonionic AmberliteXAD-7 to remove any remaining chromophore. The conditions of the chromatography were identical with those described earlier (Napier et al., 1980) for NCS. The H20-eluted fraction was used. Preparation of pMB9 DNA was as described earlier (Kappen & Goldberg, 1979).Preparation of Chromophore from AUR. AUR (2 mg) was extracted at 0 C with dry, redistilled methanol (1 mL). The protein residue obtained after centrifugation was reextracted with methanol (1 mL). The combined supernatants which contain the nonprotein chromophore were stored at-70 C. The protein residue was redissolved in distilled water and was stored frozen. The chromophore preparation contained a maximum of 5% protein contaminant as determined by the method of Lowry et al.(1951). High-Pressure Liquid Chromatography (HPLQ. A Model A2C/GPC-204 chromatograph from Waters Associates equipped with a Model 660 solvent flow programmer, a MBondapak C18 column (3.9 mm X 30 cm), a 254-nm absorbance detector, and a Schoeffel Model SF 970 fluorescence detector (360-nm excitation with 418-nm emission cutoff filter) was used.