Vellekoop P, Lugones L, van Brederode J
University of Utrecht, Department of Plant Ecology and Evolutionary Biology, The Netherlands.
FEBS Lett. 1993 Sep 6;330(1):36-40. doi: 10.1016/0014-5793(93)80914-g.
An UDP-glucose:flavonoid, 7-O-glucosyltransferase, from Silene latifolia was isolated from petals and purified 450-fold using a combination of gel-filtration, affinity chromatography and anion-exchange chromatography. Affinity chromatography on a phenyl-Sepharose CL-4B column in combination with elution with the substrate, isovitexin (6-C-glucosylapigenin), was an especially effective purification step. A purification factor between 10 and 20 could be reached using this column. A possible mechanism for the specific interaction of the enzyme with the phenyl-Sepharose will be discussed. This method of purification may also be applicable to other enzymes which use aromatic compounds as substrates. On a SDS-PAGE gel a band of 54 kDa, which co-purified with enzyme activity, could be detected in the purest fraction.
从宽叶蝇子草花瓣中分离出一种UDP-葡萄糖：类黄酮7-O-葡萄糖基转移酶，并通过凝胶过滤、亲和色谱和阴离子交换色谱相结合的方法将其纯化了450倍。在苯基琼脂糖CL-4B柱上进行亲和色谱，并与底物异荭草素（6-C-葡萄糖基芹菜素）洗脱相结合，是一个特别有效的纯化步骤。使用该柱可达到10至20的纯化因子。将讨论该酶与苯基琼脂糖特异性相互作用的可能机制。这种纯化方法也可能适用于其他以芳香族化合物为底物的酶。在SDS-PAGE凝胶上，在最纯的组分中可以检测到一条与酶活性共纯化的54 kDa条带。
