Molecular Cloning, Characterization, and Expression in Escherichia coli of Full-length cDNAs of Three Human Glutathione S -Transferase Pi Gene Variants EVIDENCE FOR DIFFERENTIAL CATALYTIC ACTIVITY OF THE ENCODED PROTEINS*
Molecular Cloning, Characterization, and Expression in Escherichia coli of Full-length cDNAs of Three Human Glutathione S -Transferase Pi Gene Variants EVIDENCE FOR DIFFERENTIAL CATALYTIC ACTIVITY OF THE ENCODED PROTEINS*
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发表时间：
1997
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通讯作者：
F. Ali-Osman;O. Akande;G. Antoun;Jialei Mao;J. Buolamwini
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作者：
F. Ali-Osman;O. Akande;G. Antoun;Jialei Mao;J. Buolamwini
我们报道了三个全长cDNA的分离，对应于密切相关的谷胱甘肽S -转移酶（GST）Pi基因的mRNA，命名为hGSTP 1 *A，hGSTP 1 *B和hGSTP 1 *C，在正常细胞和恶性胶质瘤中表达。变异cDNA分别由核苷酸（cid：49）313和（cid：49）341处的A3 G和C3 T转换产生。hGSTP 1 *A的104位密码子由ATC（Ile）变为hGSTP 1 *B和hGSTP 1 *C的GTC（瓦尔），hGSTP 1 *C的113位密码子由GCG（Ala）变为GTG（瓦尔）。两个氨基变化都在GST Pi肽的亲电结合活性位点。推导的编码肽的晶体结构的计算机建模显示，作为氨基酸变化的结果，关键亲电结合活性位点氨基酸的原子间距离的显着偏差。在大肠杆菌中表达并经GSH亲和层析纯化的编码蛋白显示，hGSTP 1 *A编码蛋白的Km（CDNB）比hGSTP 1 *B和hGSTP 1 *C编码蛋白低3倍，Kcat/Km高3-4倍。75例分析显示hGSTP 1 ~*C的相对频率为4倍。变体GST Pi蛋白的热稳定性-为了进一步确定氨基酸变化对变体GST Pi蛋白的影响，我们部分基于预期的差异（cid：97）-螺旋稳定性，并且先前的研究（41）表明，通过定点诱变产生的对应于GSTP 1b-1b的重组GSTPi酶，在功能上比亲本酶更耐热。将每种变体GST Pi蛋白以约0.1单位/ml在45 °C下在水浴中的磷酸盐缓冲盐水（pH 7.2）中孵育。在1小时内，每15分钟从每个孵育物中取出50-（cid：109）μ l等分试样，并如前所述使用CDNB作为底物测定总GST活性。相对于保持在25 °C的对照的活性计算残余GST活性，并相对于时间作图。如前所述进行SDS-PAGE和蛋白质印迹以确定在孵育期间是否发生GST Pi肽的降解。GSTPi基因变异体中核苷酸转换引起的限制性内切酶位点的改变为检测细胞中表达的GSTPi基因变异体提供了一种简单、快速和特异的技术。该方法适用于筛选Southern或北方的大量GSTPi表型/基因型。
We report the isolation of three full-length cDNAs cor- responding to the mRNAs of closely related glutathione S -transferase (GST) Pi genes, designated hGSTP1*A , hGSTP1*B , and hGSTP1*C , expressed in normal cells and malignant gliomas. The variant cDNAs result from A 3 G and C 3 T transitions at nucleotides (cid:49) 313 and (cid:49) 341, respectively. The transitions changed codon 104 from ATC (Ile) in hGSTP1*A to GTC (Val) in hGSTP1*B and hGSTP1*C and changed codon 113 from GCG (Ala) to GTG (Val) in hGSTP1*C . Both amino changes are in the electrophile-binding active site of the GST Pi peptide. Computer modeling of the deduced crystal structures of the encoded peptides showed significant deviations in the interatomic distances of critical electrophile-bind-ing active site amino acids as a consequence of the amino acid changes. The encoded proteins expressed in Escherichia coli and purified by GSH affinity chroma- tography showed a 3-fold lower K m (CDNB) and a 3–4-fold higher K cat / K m for the hGSTP1*A encoded protein than the proteins encoded by hGSTP1*B and hGSTP1*C . Analysis of 75 cases showed the relative frequency of hGSTP1*C to be 4-fold Hybridization bands were quantitated by densitometry and plotted against time. Thermostability of Variant GST Pi Proteins— To further determine the effects of the amino acid changes on the variant GST Pi proteins, we compared the thermal stabilities of the enzymatic function of the three variant GST Pi proteins based in part on the expected differences in (cid:97) -helix stability of the region of the GST Pi peptides containing the amino acid changes and on a previous study (41) that showed that a recombinant GST Pi enzyme corresponding to GSTP1b-1b, created by site-directed mutagenesis, was functionally more heat-stable than the parent enzyme. Each variant GST Pi protein was incubated at approx- imately 0.1 unit/ml at 45 °C in phosphate-buffered saline (pH 7.2) in a water bath. Every 15 min, over 1 h, a 50- (cid:109) l aliquot was removed from each incubate, and total GST activity was determined as described previously using CDNB as substrate. Residual GST activity was com- puted relative to the activity of controls maintained at 25 °C and plotted against time. SDS-PAGE and Western blotting were performed as described earlier to determine if degradation of the GST Pi peptides had occurred during the incubation. , the alterations in restriction endonuclease sites caused by the nucleotide transitions in the GST Pi gene variants provide a simple, rapid, and specific technique for determining the GST Pi gene variant(s) expressed in cells The method is suitable for screening large of GST Pi phenotype/genotype be Southern or Northern