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克氏锥虫中与NADP⁺相关的谷氨酸脱氢酶：序列、基因组组织及表达

The NADP+-linked glutamate dehydrogenase from Trypanosoma cruzi: sequence, genomic organization and expression.

作者信息

Barderi P, Campetella O, Frasch A C, Santomé J A, Hellman U, Pettersson U, Cazzulo J J

机构信息

Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martín. Av. General Paz y Albarellos, Casilla de Correo 30, 1650 San Martín, Prov. de Buenos Aires, Argentina.

出版信息

Biochem J. 1998 Mar 1;330 ( Pt 2)(Pt 2):951-8. doi: 10.1042/bj3300951.

DOI:10.1042/bj3300951
PMID:9480915
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1219230/
Abstract

NADP-linked glutamate dehydrogenase (NADP+-GluDH, EC 1.4.1.4) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr have been obtained. Using oligonucleotide primers synthesized according to the amino acid sequence of the N-terminus of the mature enzyme and to the nucleotide sequence of a clone corresponding to the C-terminus, obtained by immunological screening of an expression library, two complete open reading frames (TcGluDH1 and TcGluDH2) were isolated and sequenced. The sequences obtained are most similar to that of the NADP+-GluDH of Escherichia coli (70-72% identity), and less similar (50-56%) to those of lower eukaryotes. Using TcGluDH1 as a probe, evidence for the presence of several genes and developmental regulation of the expression of NADP+-GluDH in different parasite stages was obtained. TcGluDH1 encodes an enzymically active protein, since its expression in E. coli resulted in the production of a GluDH activity with kinetic parameters similar to those of the natural enzyme.

摘要

已通过改进的方法从克氏锥虫的无鞭毛体中纯化出烟酰胺腺嘌呤二核苷酸磷酸（NADP）连接的谷氨酸脱氢酶（NADP + -GluDH，EC 1.4.1.4）至同质状态，并获得了用胰蛋白酶、赖氨酸内切肽酶、精氨酸内切肽酶或溴化氰消化得到的11个内部肽段的氨基酸序列。根据成熟酶N端的氨基酸序列以及通过对表达文库进行免疫筛选获得的对应于C端的克隆的核苷酸序列合成寡核苷酸引物，分离并测序了两个完整的开放阅读框（TcGluDH1和TcGluDH2）。所得序列与大肠杆菌的NADP + -GluDH序列最为相似（同一性为70 - 72%），与低等真核生物的序列相似性较低（50 - 56%）。以TcGluDH1为探针，获得了不同寄生虫阶段存在多个基因以及NADP + -GluDH表达的发育调控的证据。TcGluDH1编码一种具有酶活性的蛋白质，因为其在大肠杆菌中的表达导致产生了一种谷氨酸脱氢酶活性，其动力学参数与天然酶相似。