A radiochemical assay for a NADP+-specific gamma-glutamate semialdehyde dehydrogenase extracted from mitochondrial membrane of rat intestinal epithelial cells.
A radiochemical assay has been developed for a NADP+-specific gamma-glutamate semialdehyde dehydrogenase from rat intestinal epithelial cells. The spectrophotometric assay utilized to measure the enzyme in bacterial cell homogenates is not sensitive enough for homogenates from rat mitochondria, which require an assay that can measure as little as 0.5 nmol NADPH formed/min/ml extract. The assay described here is sensitive to 0.1 nmol product formed/min/ml of extract and employs the use of [3H]pyrroline 5-carboxylate which is phosphorylated and oxidized by the enzyme to gamma-[3H]glutamyl phosphate, a product that decomposes to [3H]pyrrolidone 5-carboxylate. The latter product is separated from the substrate by ion-exchange chromatography. In order to correct for any product loss during separation by ion-exchange [14C]pyrrolidone 5-carboxylate is added as an internal standard to the deproteinized assay mixture. Under the assay conditions described mammalian gamma-glutamate semialdehyde dehydrogenase activity is linear with respect to time and protein concentration. Comparison between the kinetic parameters reported for the bacterial enzyme and those reported here for the mammalian enzyme indicate similarities in the pH optima as well as a requirement for phosphate. Kinetic studies on mammalian enzyme yield apparent Km values of 1.8 mM for pyrroline 5-carboxylate, 0.2 mM for NADP+, and 11.3 mM for phosphate.
Kramer
J J
JJ
University of North Carolina, Department of Biochemistry and Nutrition, Chapel Hill 27514.
Gooding
R C
RC
Jones
M E
ME
D000445:Aldehyde Oxidoreductases / Q000032:analysis*; D000818:Animals; D002852:Chromatography, Ion Exchange; D004848:Epithelium / Q000201:enzymology; D050698:Glutamate-5-Semialdehyde Dehydrogenase; D007422:Intestines / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology*; D009249:NADP / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D011392:Proline / Q000031:analogs & derivatives / Q000378:metabolism; D011506:Proteins / Q000494:pharmacology; D011761:Pyrrolidonecarboxylic Acid / Q000378:metabolism; D011854:Radiochemistry; D051381:Rats; D013696:Temperature; D013997:Time Factors
D010710:Phosphates; D011506:Proteins; C009141:3,4-dihydro-2H-pyrrole-5-carboxylic acid; D009249:NADP; D011392:Proline; D000445:Aldehyde Oxidoreductases; D050698:Glutamate-5-Semialdehyde Dehydrogenase; D011761:Pyrrolidonecarboxylic Acid