Purification and characterization of glutaryl-CoA dehydrogenase from porcine and human liver

Thesis/Dissertation · Tue Jan 01 04:00:00 EST 1985

OSTI ID:6655546

Lenich, A C

Glutaryl-CoA dehydrogenase (GCDH) was purified from porcine liver mitochondria by pH and ammonium sulfate fractionations followed by a series of column chromatographies. The purified porcine enzyme was found by sodium dodecyl-sulfate polyacrylamide gel electrophoresis to have a subunit molecular weight of 47,800 and by gradient polyacrylamide gel electrophoresis (PAGE) to have a native molecular weight of approximately 186,000. The product of the GCDH reaction with its primary substrate, glutaryl-CoA, was investigated by radio-gas chromatography and found to be crotonyl-CoA. Alternate substrates as well as crotonyl-CoA, the glutaryl-CoA reaction end product, demonstrated competitive inhibition when incubated with (1,5-/sup 14/C)-glutaryl-CoA in the presence of porcine GCDH. Kinetic parameters for the interaction of both ETF and glutaryl-CoA with porcine GCDH were determined. Purified porcine GCDH was used to produce an antiserum which cross-reacted with human liver GCDH with a reaction of partial identity, but proved too insensitive to detect GCDH in control human fibroblasts. As a result of these negative findings, GCDH was purified by a series of column chromatographies from human liver. The purified human enzyme was found by SDS-PAGE and gel filtration to have subunit and native molecular weights of 58,800 and 256,000 respectively.

• Research Organization: Colorado Univ., Denver (USA). Health Sciences Center
• OSTI ID: 6655546
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
BIOCHEMICAL REACTION KINETICS
BODY
CARBON 14 COMPOUNDS
CELL CONSTITUENTS
CHROMATOGRAPHY
DIGESTIVE SYSTEM
DOMESTIC ANIMALS
ELECTROPHORESIS
ENZYMES
GAS CHROMATOGRAPHY
GLANDS
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIQUID COLUMN CHROMATOGRAPHY
LIVER
MAMMALS
MAN
MITOCHONDRIA
ORGANOIDS
ORGANS
OXIDOREDUCTASES
PRIMATES
PURIFICATION
REACTION KINETICS
SEPARATION PROCESSES
SWINE
TRACER TECHNIQUES
VERTEBRATES