Characterization of mycoplasma arginine deiminase expressed in E. coli and its inhibitory regulation of nitric oxide synthesis.
We previously reported that a cytostatic protein that is found in ASC-17D Sertoli cell-conditioned media was Mycoplasma arginine deiminase (ADI), which hydrolyzes L-arginine into L-citrulline and ammonia. Here, we report the over-expression of recombinant ADI (rADI) in E. coli and the down-regulation of lipopolysaccharide (LPS) induced-nitric oxide (NO) production by rADI treatment. We cloned the ADI gene from Mycoplasma arginini genomic DNA by a polymerase chain reaction, and changed five TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The rADI was purified to apparent homogeneity by DEAE-Sepharose and arginine-affinity chromatography. The rADI expressed in E. coli was identified as 45 kDa on SDS-PAGE and 90 kDa on native PAGE, implying that it exists as a dimer like ADI of M. arginini. The Km for arginine of rADI was approximately 370+/-50 microM. Its optimal temperature and pH were 41 degrees C and pH 6.4, respectively, and enzyme activity remained > or = 50% for 5 d at physiological temperature and pH. Treatment of purified rADI suppressed NO production in macrophage-like RAW 264.7 and primary glial cells that were exposed to LPS. Furthermore, an intraperitoneal injection of rADI significantly suppressed the rise of blood nitrite/nitrate levels that were induced by the systemic administration of bacterial endotoxin LPS to mice, resulting in an improvement in their survival rate. These results suggest that the depletion of blood arginine with an arginine-metabolizing enzyme, such as ADI, could suppress excessive production of NO that is caused by inducible NOS (iNOS) during the endotoxemia. Also, rADI may be used as a new approach to control NO-related diseases, such as sepsis.
Noh
Eun Joo
EJ
Department of Pharmacology, College of Medicine, Korea University, Seoul.
Kang
Sang Wook
SW
Shin
Yong Jae
YJ
Kim
Dong Chung
DC
Park
In Sun
IS
Kim
Min Young
MY
Chun
Boe Gwun
BG
Min
Bon Hong
BH
D000818:Animals; D001120:Arginine / Q000097:blood; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D019446:Endotoxemia / Q000097:blood / Q000188:drug therapy; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000627:therapeutic use; D007700:Kinetics; D008070:Lipopolysaccharides / Q000633:toxicity; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009174:Mycoplasma / Q000201:enzymology* / Q000235:genetics*; D009569:Nitric Oxide / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use
D004269:DNA, Bacterial; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D009569:Nitric Oxide; D001120:Arginine; D006867:Hydrolases; C019723:arginine deiminase