Document Detail


Elevation of gamma-glutamyltransferase activity in 293 HEK cells constitutively expressing antisense glutaminase mRNA.
MedLine Citation:
PMID:  16162416     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Previous studies have shown that the use of dynamic nutrient feeding to maintain glutamine at low levels in fed-batch cultures reduced the overflow of glutamine metabolism. This strategy resulted in the shift of metabolism towards an energetically more efficient state signified by reduced lactate and ammonia production and thus achieving a higher cell density for enhanced productivity. In an effort to mimic the metabolic changes effected by this fed-batch strategy at the molecular level, 293 HEK cells were engineered via stable transfection with an antisense fragment of the rat phosphate-dependent glutaminase (PDG) gene. PDG is localized in the mitochondria and catalyzes the deamination of glutamine to glutamate with the release of ammonia. Stable single cell clones were isolated from the transfected populations. Characterization of these transfectants revealed indications of an altered glutamine metabolism affected by the antisense strategy. Contrary to our expectations, glutamine consumption and ammonia production in the antisense cells did not deviate significantly from that of untransfected cells. Glutamate was also observed to accumulate to high level extracellularly, as opposed to a consumption pattern normally observed in non-transfected cells. Subsequent analyses show that gamma-glutamyltransferase (gamma-GT) may be a significant pathway that resulted in the formation of glutamate and ammonia from glutamine catabolism extracellularly. gamma-GT has been widely investigated in renal glutamine metabolism, but has rarely been implicated in cultured cell metabolism. This study highlights the importance of this alternative glutamine metabolism pathway in cell culture.
Authors:
Kathy T K Wong; Yih Yean Lee; Vesna Brusic; Janice Tan; Miranda G S Yap; Peter Morin Nissom
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Publication Detail:
Type:  Journal Article     Date:  2005-09-12
Journal Detail:
Title:  Metabolic engineering     Volume:  7     ISSN:  1096-7176     ISO Abbreviation:  Metab. Eng.     Publication Date:    2005 Sep-Nov
Date Detail:
Created Date:  2005-12-19     Completed Date:  2006-02-09     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9815657     Medline TA:  Metab Eng     Country:  Belgium    
Other Details:
Languages:  eng     Pagination:  375-83     Citation Subset:  IM    
Affiliation:
Bioprocessing Technology Institute, 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore. kathy_wong@bti.a-star.edu.sg
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MeSH Terms
Descriptor/Qualifier:
Ammonia / metabolism
Cell Line
Enzyme Activation
Gene Expression Regulation, Enzymologic / physiology*
Gene Silencing
Glutamic Acid / metabolism
Glutaminase / genetics*,  metabolism*
Glutamine / metabolism
Humans
Kidney / embryology,  metabolism*
Protein Engineering / methods*
RNA, Antisense / genetics
RNA, Messenger / genetics
Recombinant Proteins / metabolism
gamma-Glutamyltransferase / genetics*,  metabolism*
Chemical
Reg. No./Substance:
0/RNA, Antisense; 0/RNA, Messenger; 0/Recombinant Proteins; 56-85-9/Glutamine; 56-86-0/Glutamic Acid; 7664-41-7/Ammonia; EC 2.3.2.2/gamma-Glutamyltransferase; EC 3.5.1.2/Glutaminase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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