In vivo 15N-enrichment of metabolites in suspension cultured cells and its application to metabolomics.
The incorporation of stable isotopes in suspension cultured cells is very simple and useful as a preliminary experimental method in the experimental scene of plant metabolomics to elucidate the metabolic profiles of mutants and transformants. Stable isotope methods would afford a dynamic explanation of turnover speed that would concern the metabolic flux. Utilization of suspension cultured cells allows genes to be easily induced or suppressed, culture conditions to be controlled, and samples to be easily prepared. Stable isotope tracing allows an index of metabolic flux to be obtained. Here we present an experiment feeding (15)N-labeled inorganic salts to Arabidopsis (cell line T87) and Coptis cultured cells. Results of a comparison of (15)N labeling ratios of amino acids derived from T87 cells cultured under light with those cultured in the dark corresponded to transcriptional expressions revealed by microarray experiments published previously, demonstrating the validity of this procedure. Furthermore, (15)N labeling ratios of Coptis cultured cells revealed arginine and lysine metabolism inhibition, which should result in inhibition of polyamine biosynthesis and cell division. This very simple experiment allowed us to uncover metabolic dynamic features of the plant cell. Therefore this method is very useful for forming working hypotheses and experimental design.
Harada
Kazuo
K
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Japan.
Fukusaki
Eiichiro
E
Bamba
Takeshi
T
Sato
Fumihiko
F
Kobayashi
Akio
A
D000596:Amino Acids / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D017360:Arabidopsis / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division; D002460:Cell Line; D002826:Chorismate Mutase / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D039323:Coptis / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D009566:Nitrates / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D009587:Nitrogen Isotopes; D040081:Protein Array Analysis / Q000379:methods; D000644:Quaternary Ammonium Compounds / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D012680:Sensitivity and Specificity
D000596:Amino Acids; D009566:Nitrates; D009587:Nitrogen Isotopes; D000644:Quaternary Ammonium Compounds; D002826:Chorismate Mutase