Warning: imagejpeg(C:\Inetpub\vhosts\kidney.de\httpdocs\phplern\24397525
.jpg): Failed to open stream: No such file or directory in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 117 Anal+Chem
2014 ; 86
(3
): 1583-91
Nephropedia Template TP
gab.com Text
Twit Text FOAVip
Twit Text #
English Wikipedia
Quantitation of cellular metabolic fluxes of methionine
#MMPMID24397525
Shlomi T
; Fan J
; Tang B
; Kruger WD
; Rabinowitz JD
Anal Chem
2014[Feb]; 86
(3
): 1583-91
PMID24397525
show ga
Methionine is an essential proteogenic amino acid. In addition, it is a methyl
donor for DNA and protein methylation and a propylamine donor for polyamine
biosynthesis. Both the methyl and propylamine donation pathways involve metabolic
cycles, and methods are needed to quantitate these cycles. Here, we describe an
analytical approach for quantifying methionine metabolic fluxes that accounts for
the mixing of intracellular and extracellular methionine pools. We observe that
such mixing prevents isotope tracing experiments from reaching the steady state
due to the large size of the media pools and hence precludes the use of standard
stationary metabolic flux analysis. Our approach is based on feeding cells with
(13)C methionine and measuring the isotope-labeling kinetics of both
intracellular and extracellular methionine by liquid chromatography-mass
spectrometry (LC-MS). We apply this method to quantify methionine metabolism in a
human fibrosarcoma cell line and study how methionine salvage pathway enzyme
methylthioadenosine phosphorylase (MTAP), frequently deleted in cancer, affects
methionine metabolism. We find that both transmethylation and propylamine
transfer fluxes amount to roughly 15% of the net methionine uptake, with no major
changes due to MTAP deletion. Our method further enables the quantification of
flux through the pro-tumorigenic enzyme ornithine decarboxylase, and this flux
increases 2-fold following MTAP deletion. The analytical approach used to
quantify methionine metabolic fluxes is applicable for other metabolic systems
affected by mixing of intracellular and extracellular metabolite pools.
free
free
free
