Purification and characterization of pyrroline-5-carboxylate dehydrogenase from rat liver mitochondrial matrix

Conference · Fri May 01 00:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6374280

Small, W C; Jones, M E

Pyrroline-5-carboxylate (P5C) dehydrogenase catalyzes the second step of the irreversible two-step oxidation of proline to glutamate or the oxidative second step of the two-step conversion of ornithine to glutamate in mitochondria. Activity was assayed by monitoring directly the conversion of (/sup 3/H) L-P5C to (/sup 3/H) L-glutamate. Using this assay, the authors find P5C dehydrogenase most prevalent in liver in rat, with kidney having 71%, heart 51%, and and spleen 15% of the specific activity of liver. Starting with a subcellular fraction enriched for mitochondria, they have isolated a protein fraction enriched in this activity. The soluble protein fraction of the mitochondrial isolate was subjected to (NH/sub 4/)/sub 2/SO/sub 4/ precipitation and successive chromatography on DE 52 anion exchange and Brown 10 dye ligand affinity resins. This procedure yielded a fraction purified more than 500-fold over whole liver homogenate. HPLC and 5'-AMP agarose fractionation experiments now in progress to achieve further purification show promise. Physical studies show a M/sub r/ of 105,000 upon sucrose density gradient centrifugation and 94,000 on molecular sieve HPLC for the activity. Flat bed gel isoelectric focusing of the protein indicates a pI of 5.7. The purified protein exhibits an apparent K/sub m/ of 0.1 mM for L-P5C.

• Research Organization: Univ. of North Carolina, Chapel Hill
• OSTI ID: 6374280
• Report Number(s): CONF-870644-; TRN: 87-033997
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
OXIDOREDUCTASES
CHEMICAL COMPOSITION
ENZYME ACTIVITY
FRACTIONATION
PROLINE
METABOLISM
GLUTAMIC ACID
ION EXCHANGE CHROMATOGRAPHY
LIGANDS
LIQUID COLUMN CHROMATOGRAPHY
LIVER
MITOCHONDRIA
RATS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
AMINES
AMINO ACIDS
ANIMALS
AZOLES
BODY
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CHROMATOGRAPHY
DIGESTIVE SYSTEM
ENZYMES
GLANDS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MAMMALS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANOIDS
ORGANS
PYRROLES
PYRROLIDINES
RODENTS
SEPARATION PROCESSES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques