Radiochemical assay for a NADP+-specific gamma-glutamate semialdehyde dehydrogenase extracted from mitochondrial membrane of rat intestinal epithelial cells

Journal Article · Mon Feb 01 00:00:00 EST 1988 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(88)90333-8· OSTI ID:7196215

Kramer, J J; Gooding, R C; Jones, M E

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 (/sup 3/H)pyrroline 5-carboxylate which is phosphorylated and oxidized by the enzyme to gamma-(/sup 3/H)glutamyl phosphate, a product that decomposes to (/sup 3/H)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 (/sup 14/C)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.

• Research Organization: Univ. of North Carolina, Chapel Hill (USA)
• OSTI ID: 7196215
• Journal Information: Anal. Biochem.; (United States), Vol. 168:2
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
OXIDOREDUCTASES
RADIOASSAY
CARBON 14 COMPOUNDS
CELL MEMBRANES
ENZYME ACTIVITY
EPITHELIUM
INTESTINES
MITOCHONDRIA
NADP
PHOSPHATES
PROLINE
PROTEINS
RATS
TRITIUM COMPOUNDS
AMINES
AMINO ACIDS
ANIMAL TISSUES
ANIMALS
AZOLES
BODY
CARBOXYLIC ACIDS
CELL CONSTITUENTS
COENZYMES
DIGESTIVE SYSTEM
ENZYMES
GASTROINTESTINAL TRACT
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
LABELLED COMPOUNDS
MAMMALS
MEMBRANES
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANOIDS
ORGANS
OXYGEN COMPOUNDS
PHOSPHORUS COMPOUNDS
PYRROLES
PYRROLIDINES
RODENTS
TISSUES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques