ENZYME MECHNISM OF LYSINE-2,3-AMINOMUTASE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 根据已发表的方法(B)，从pSGA02-FHIT转化的大肠杆菌SG100菌株中分离到人FHIT和突变的FHIT-H96G酶。将过夜的细胞培养物稀释100倍至16升含卡那霉素和氨苄西林的LB液。细胞培养温度为37℃，OD600达到0.6。将异丙基硫代-b-D-半乳糖苷(IPTG)加入1 mM，37℃诱导6h后，收集细胞(~17g)，悬浮在含有0.5 mM苯甲基磺酰氟(PMSF)的100ml缓冲液A(50 mM HEPES，pH 6.8，10%VOL/VOL/甘油)中。细胞超声处理5分钟，100000克离心15分钟收集上清液。将上清液稀释至150毫升，然后用400毫升DEAE-Sephacel色谱柱进行分析(B)。从DEAE-Sephacel柱中得到的含有Ap3A水解酶活性的组分，用PM10膜片结合并浓缩到40ml，稀释到100ml，然后在200ml的Q-Sepharose柱上，缓冲液A中以2ml/min的流速洗脱，缓冲液A中含有1000ml梯度的缓冲液和0.2M的氯化钠。收集显示FHIT活性的两个峰，并检查其纯度，用于SDS-PAGE凝胶电泳法。根据已发表的方法(C)，根据FHIT的氨基酸序列计算出E280=8310M-1 cm-1，从而确定FHIT的亚基浓度。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Human Fhit and mutated Fhit-H96G enzymes were isolated from E. coli strain SG100 sells transformed from pSGA02-Fhit according the published method (b). An overnight cell culture was diluted by 100 folds to 16 litters of LB medium containing kanamycin and ampicillin. The cell culture was grown at 37 ¿C untill OD600 reaches 0.6. After addition of isopropyl thio-b-D-galactoside (IPTG) to 1 mM and induction at 37 ¿C for 6 hours, cells were harvested (~ 17 g), suspended in 100 ml buffer A (50 mM HEPES, pH 6.8, 10% vol/vol glycerol) containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF). The cells were sonicated 5 minutes and centrifuged at 100,000 g for 15 minutes to collect the supernatant. The supernatant was diluted to 150 ml and subjected to a 400 ml DEAE-Sephacel column as described (b). The fractions containing Ap3A hydrolase activity from the DEAE-Sephacel column were combined and concentrated by PM10 memberane to 40 ml, which was diluted to 100ml and subjected to a 200 ml Q-Sepharose column eluted with 1000 ml gradient of buffer A to 0.2 M NaCl in buffer A at a flow rate of 2 ml/min. Two peaks showing Fhit activity were collected and checked for purity for SDS-PAGE gel electrophoresis. The subunit concentration of Fhit was determined by use of e280 = 8310 M-1cm-1 calculated from the amino acid sequence of Fhit according to a published method ( c).