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Optimized Assay of Glycine Cleavage Activity in Mammalian Cells
Author(s) -
Steiner Joshua Dylan,
Gregory Jesse
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a644-d
Subject(s) - chemistry , tris , glycine , nad+ kinase , chromatography , cleavage (geology) , incubation , biochemistry , glycine cleavage system , amino acid , enzyme , biology , paleontology , fracture (geology)
Studies regarding assay development and optimization were conducted using HepG2 cells. The glycine cleavage system is a multiprotein complex in the inner mitochondrial membrane of the mitochondria and is responsible for the catabolism of glycine into CO 2, NH 3 , and NADH. HepG2 cells were resuspended and homogenized in a buffer containing 20mM Tris‐HCl pH 8.0, 1mM DTT, 1mM PLP and 10ug/ml Leupeptin.. The standard assay mixture was comprised of 1mM tetrahydrofolate, 60mM Tris‐HCl pH 8.0, 1mM NAD, and 2.5 μCi [1‐ 14 C] glycine, specific activity 57mCi/mmol in a volume of 500 μL. Reactions were stopped by addition of 200μl 30% w/v TCA. Optimized conditions for trapping released 14 CO 2 involved use of filter paper saturated with 2M KOH attached inside the cap of the reaction tube, which was then quantified by liquid scintillation counting. The assay was linear up to 60 min incubation with 350μg crude protein homogenate, with rate proportional to cellular protein added. Preliminary results also indicate that this assay is applicable to measuring glycine cleavage activity in human peripheral blood lymphocytes. Supported by NIH DK072398.