Determination of serine hydroxymethyltransferase and reduced folate pools in tissue extracts.
Serine hydroxymethyltransferase (SHMT) from all sources tested catalyzes the slow exchange of the pro-2S proton of glycine with solvent protons. In the presence of tetrahydrofolate (H4PteGlun) this exchange rate is increased by about three orders of magnitude. This H4PteGlun-dependent exchange has been developed into a rapid and sensitive assay for both SHMT and H4PteGlun and the one-carbon derivatives of H4PteGlun. The procedure involves incubating [2-3H]glycine, H4PteGlun, and SHMT for 3 min followed by a separation of the exchanged protons in the solvent from the substrate glycine on a small Dowex-50 cation-exchange column at pH 2. In the presence of an excess of H4PteGlun the exchange rate is proportional to nanogram levels of SHMT. In the presence of an excess of SHMT the exchange rate is directly proportional to the concentration of H4PteGlun in the 0.1 to 1 pmol range. The concentration of one-carbon derivatives of H4PteGlun is determined by a preincubation of cell extracts with enzymes that convert each derivative into H4PteGlun. A complete reduced folate pool analysis of a tissue extract can be obtained in less than 2 h once a standard curve has been prepared for H4PteGlun. The method does not distinguish between mono- and polyglutamate forms of the coenzyme.
Kim
D W
DW
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond, Virginia, 23298-0614, USA.
Delle Fratte
S
S
Jeong
S S
SS
Schirch
V
V
D000818:Animals; D002411:Cation Exchange Resins; D012696:Glycine Hydroxymethyltransferase / Q000032:analysis*; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000201:enzymology; D008099:Liver / Q000737:chemistry / Q000201:enzymology; D011817:Rabbits; D051381:Rats; D012680:Sensitivity and Specificity; D012997:Solvents; D013763:Tetrahydrofolates / Q000032:analysis* / Q000378:metabolism; D014020:Tissue Extracts / Q000737:chemistry*
D002411:Cation Exchange Resins; D012997:Solvents; D013763:Tetrahydrofolates; D014020:Tissue Extracts; C030371:5,6,7,8-tetrahydrofolic acid; D012696:Glycine Hydroxymethyltransferase