ENZYME MECHNISM OF LYSINE-2,3-AMINOMUTASE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 人Fhit和突变的Fhit-H96 G酶从E.根据公开的方法（B）从pSGA 02-Fhit转化大肠杆菌菌株SG 100。将过夜细胞培养物稀释100倍至16升含有卡那霉素和氨苄青霉素的LB培养基。细胞培养物在37 ° C下生长，直到OD 600达到0.6。加入异丙基硫代-b-D-半乳糖苷（IPTG）至1 mM并在37 ℃诱导6小时后，收获细胞（~ 17 g），悬浮于100 ml含有0.5 mM苯甲基磺酰氟（PMSF）的缓冲液A（50 mM HEPES，pH 6.8，10%体积/体积甘油）中。将细胞超声处理5分钟，并以100，000 g离心15分钟以收集上清液。将上清液稀释至150 ml，并如（B）所述，上400 ml DEAE-Sephacel柱。合并来自DEAE-Sephacel柱的含有Ap 3A水解酶活性的级分，并通过PM 10 memberane浓缩至40 ml，将其稀释至100 ml，并以2 ml/min的流速通过200 ml Q-Sepharose柱，用1000 ml缓冲液A至0.2 M NaCl的缓冲液A溶液梯度洗脱。PAGE凝胶电泳。Fhit的亚基浓度根据公开的方法（c）通过使用从Fhit的氨基酸序列计算的e280 = 8310 M-1cm-1来确定。

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).