Cancilla M R, Davidson B E, Hillier A J, Nguyen N Y, Thompson J
Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria, Australia.
Microbiology (Reading). 1995 Jan;141 ( Pt 1):229-38. doi: 10.1099/00221287-141-1-229.
Triosephosphate isomerase (EC 5.3.1.1) from Lactococcus lactis was purified to electrophoretic homogeneity. Approximately 3 mg purified enzyme (specific activity 3300 U mg-1) was obtained from 70 g (wet wt) cells. In solution, triosephosphate isomerase (pI 4.0-4.4) was observed to exist as a homodimer (M(r) 57,000) of noncovalently linked subunits. The sequence of the first 37 amino acid residues from the NH2-terminus were determined by step-wise Edman degradation. This sequence, and that of a region conserved in all known bacterial triosephosphate isomerases, was used to design oligonucleotide primers for the synthesis of a lactococcal tpi probe by PCR. The probe was used to isolate a molecular clone of tpi from a lambda GEM11 library of L. lactis LM0230 DNA. The nucleotide sequence of tpi predicted a protein of 252 amino acids with the same NH2-terminal sequence as that determined for the purified enzyme and a subunit M(r) of 26,802 after removal of the NH2-terminal methionine. Escherichia coli cells harbouring a plasmid containing tpi had 15-fold higher triosephosphate isomerase activity than isogenic plasmid-free cells, confirming the identity of the cloned gene. Northern analysis of L. lactis LM0230 RNA showed that a 900 base transcript hybridized with tpi. The 5' end of the transcript was determined by primer extension analysis to be a G located 65 bp upstream from the tpi start codon. These transcript analyses indicated that in L. lactis, tpi is expressed on a monocistronic transcript. Nucleotide sequencing indicated that the DNA adjacent to tpi did not encode another Embden-Meyerhoff-Parnas pathway enzyme. The location of tpi on the L. lactis DL11 chromosome map was determined to be between map coordinates 1.818 and 1.978.
乳酸乳球菌中的磷酸丙糖异构酶（EC 5.3.1.1）被纯化至电泳纯。从70克（湿重）细胞中获得了约3毫克纯化酶（比活性为3300 U mg-1）。在溶液中，观察到磷酸丙糖异构酶（pI 4.0 - 4.4）以非共价连接亚基的同型二聚体（M(r) 57,000）形式存在。通过逐步埃德曼降解法测定了从NH2末端起的前37个氨基酸残基的序列。该序列以及所有已知细菌磷酸丙糖异构酶中保守区域的序列被用于设计寡核苷酸引物，通过聚合酶链反应（PCR）合成乳酸乳球菌tpi探针。该探针用于从乳酸乳球菌LM0230 DNA的λGEM11文库中分离tpi的分子克隆。tpi的核苷酸序列预测有一个252个氨基酸的蛋白质，其NH2末端序列与纯化酶测定的序列相同，去除NH2末端甲硫氨酸后亚基M(r)为26,802。携带含tpi质粒的大肠杆菌细胞的磷酸丙糖异构酶活性比同基因无质粒细胞高15倍，证实了克隆基因的同一性。对乳酸乳球菌LM0230 RNA的Northern分析表明，一个900个碱基的转录本与tpi杂交。通过引物延伸分析确定转录本的5'端是一个位于tpi起始密码子上游65 bp处的G。这些转录本分析表明，在乳酸乳球菌中，tpi在单顺反子转录本上表达。核苷酸测序表明，与tpi相邻的DNA不编码另一种糖酵解途径的酶。tpi在乳酸乳球菌DL11染色体图谱上的位置被确定在图谱坐标1.818和1.978之间。
