Studies of the enzymatic oxidation of proline and pyrroline-5-carboxylate in mammalian mitochondria
Thesis/Dissertation
·
OSTI ID:6968977
The two-step oxidation of proline to glutamate in vivo is catalyzed unidirectionally by the enzymes proline oxidase (E.C. 1.5.99.8) and pyrroline-5-carboxylate (P5C) dehydrogenase (E.C. 1.5.1.12). Attempts were made to purify both proteins from rat liver. In the case of proline oxidase, an integral protein of the inner mitochondrial membrane, a number of detergents were used in efforts to solubilize active free enzyme. Detergent-treated fractions were subjected to gel permeation and other chromatographic procedures, and proline oxidase was found as part of a large particle which could not be purified. Accurate kinetic analysis of the enzyme using spectrophotometric techniques was difficult due to interference from assay components. A radiochemical assay was developed which directly measured the production of radiolabeled glutamate from P5C. The compounds were separated by cation-exchange chromatography. This assay was used to screen rat tissues for activity. Western blot analysis utilizing a polyclonal rabbit anti-rat antiserum suggests the two proteins may immunocrossreact. Kinetic analysis of these enzymes using semialdehydes and other aldehydes as substrates show a similar general pattern of substrate specificity, but the rate enzyme appears to be more specific for P5C. Aliphatic semialdehydes, closely analogous in structure to P5C, are commercially unavailable. A colorimetric assay was developed to accurately quantitate semialdehydes after synthesis. This assay is dependent upon oxidation of a covalent adduct of semialdehyde with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole.
- Research Organization:
- North Carolina Univ., Chapel Hill, NC (USA). Inst. of Statistics
- OSTI ID:
- 6968977
- Country of Publication:
- United States
- Language:
- English
Similar Records
Factors influencing pyrroline 5-carboxylate synthesis from glutamate by rat intestinal mucosa mitochondria
Purification and characterization of pyrroline-5-carboxylate dehydrogenase from rat liver mitochondrial matrix
Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis
Journal Article
·
Sat Oct 15 00:00:00 EDT 1983
· Arch. Biochem. Biophys.; (United States)
·
OSTI ID:5022491
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
Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis
Journal Article
·
Wed Aug 02 00:00:00 EDT 2017
· Frontiers in Microbiology
·
OSTI ID:1400287
Related Subjects
550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINES
AMINO ACIDS
ANIMALS
AZOLES
BODY
CARBOXYLIC ACIDS
CATALYSIS
CELL CONSTITUENTS
DATA
DIGESTIVE SYSTEM
ENZYME ACTIVITY
ENZYMES
EXPERIMENTAL DATA
GLANDS
GLUTAMIC ACID
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
INFORMATION
ISOTOPE APPLICATIONS
LIVER
MAMMALS
MITOCHONDRIA
NUMERICAL DATA
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANOIDS
ORGANS
OXIDOREDUCTASES
PROLINE
PURIFICATION
PYRROLES
PYRROLIDINES
RADIOASSAY
RATS
RODENTS
TRACER TECHNIQUES
VERTEBRATES
59 BASIC BIOLOGICAL SCIENCES
AMINES
AMINO ACIDS
ANIMALS
AZOLES
BODY
CARBOXYLIC ACIDS
CATALYSIS
CELL CONSTITUENTS
DATA
DIGESTIVE SYSTEM
ENZYME ACTIVITY
ENZYMES
EXPERIMENTAL DATA
GLANDS
GLUTAMIC ACID
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
INFORMATION
ISOTOPE APPLICATIONS
LIVER
MAMMALS
MITOCHONDRIA
NUMERICAL DATA
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANOIDS
ORGANS
OXIDOREDUCTASES
PROLINE
PURIFICATION
PYRROLES
PYRROLIDINES
RADIOASSAY
RATS
RODENTS
TRACER TECHNIQUES
VERTEBRATES