Molecular cloning and characterization of lactate dehydrogenase gene 1 in the silkworm, Bombyx mori.
Lactate dehydrogenase (LDH) catalyzes the reduction of pyruvate into lactate and constitutes a major checkpoint of anaerobic glycolysis. Recently, LDH draws a great deal of attention for its potential to be used as a novel diagnostic and therapeutic target for various diseases, including cancer and malaria. Insect LDHs have been mainly identified from fruit fly and mosquitoes, but not from silkworm. In this study, a novel LDH homologue, designated as BmLDH1, was firstly identified and characterized from the silkworm, Bombyx mori. The BmLDH1 cDNA contains an open reading frame of 996 bp, and encodes a protein of 331 amino acid residues with calculated molecular mass of 36 kDa. Sequence comparison showed BmLDH1 is a highly conserved protein. RT-PCR revealed BmLDH1 is transcripted in all tissues and in all developmental stages tested, indicating its essential roles for silkworm physiology and development. The BmLDH1 gene was subcloned and expressed in E. coli, and was further characterized by Western blot and Mass Spectrometry. The expressed protein contained the LDH activity, and could be inhibited by reduced glutathione in vitro. Immunofluoresence showed that the BmLDH1 was located in the cytoplasm. The cloned BmLDH1 sequence was deposited in the GenBank (accession number EU334850).
Xia
Hengchuan
H
Institute of Life Sciences, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, People's Republic of China.
Wu
Chao
C
Xu
Qinggang
Q
Shi
Jing
J
Feng
Fan
F
Chen
Keping
K
Yao
Qin
Q
Wang
Yong
Y
Wang
Lin
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D019295:Computational Biology; D057075:Enzyme Assays; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017344:Genes, Insect / Q000235:genetics*; D005978:Glutathione / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D021381:Protein Transport / Q000187:drug effects; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D007527:Isoenzymes; D011994:Recombinant Proteins; D007770:L-Lactate Dehydrogenase; C409667:lactate dehydrogenase 1; D005978:Glutathione