A one-step assay for pyrroline-5-carboxylate synthase using a short AG1-X8 column.
A rapid and simple method for assay of pyrroline-5-carboxylate synthase is presented. In this method, the incubation is terminated by raising the pH of incubation mixture to 10, and [14C]pyrroline 5-carboxylate produced from the substrate, [14C]glutamate, is first converted quantitatively to [14C]proline by reduction with NaBH4 at pH 10 and then the proline is allowed to pass through column of AG1-X8 anion exchanger under the conditions where the glutamate is completely retained by the column. Radioactive counting of the eluate gives the synthase activity. The entire procedure takes only one hour.
D000818:Animals; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005971:Glutamates; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007421:Intestine, Small / Q000201:enzymology; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D009953:Ornithine-Oxo-Acid Transaminase / Q000032:analysis*; D011392:Proline; D051381:Rats; D011919:Rats, Inbred Strains; D000637:Transaminases / Q000032:analysis*
D005971:Glutamates; D018698:Glutamic Acid; D011392:Proline; D000637:Transaminases; D009953:Ornithine-Oxo-Acid Transaminase