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A one-step assay for pyrroline-5-carboxylate synthase using a short AG1-X8 column |
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| Authors: | Y Wakabayashi R Yamada A Iwashima |
| Affiliation: | Department of Biochemistry, Kyoto Prefectural University of Medicine, Kawaramachi Kamikyo-ku, Kyoto, Japan 602 |
| Abstract: | 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. |
| Keywords: | pyrroline-5-carboxylate glutamate ornithine proline small intestine mitochondrion PC pyrroline-5-carboxylate PC reductase pyrroline-5-carboxylate reductase PC synthase pyrroline-5-carboxylate synthase Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid |
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