Determination of the quantity of acetyl CoA carboxylase by (/sup 14/C)methyl avidin binding
Conditions are described under which monomeric (/sup 14/C)methyl avidin binds to SDS-denatured biotin enzymes and remains bound through polyacrylamide gel electrophoresis. The location of radioactive proteins on the dried gel was determined by fluorography and their identity was established by subunit molecular weight. The relative quantity of bound radioactive avidin, stoichiometrically equivalent to the molar quantity of biotin protein, can be determined by scanning the fluorograph with a soft laser densitometer. To determine the absolute quantity of biotin protein, the radioactive areas of the dried gel were cut out, resolubilized, and assayed for radioactivity. Since the specific radioactivity of the (/sup 14/C)methyl avidin was known, the quantity of avidin bound and therefore the quantity of biotin enzyme could be calculated. The method is illustrated by the analysis of purified acetyl CoA carboxylase and is applied to the analysis of biotin enzymes in isolated rat liver mitochondria.
- Research Organization:
- Ohio State Univ., Columbus
- OSTI ID:
- 6025640
- Journal Information:
- J. Lipid Res.; (United States), Journal Name: J. Lipid Res.; (United States) Vol. 28:5; ISSN JLPRA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
AUTORADIOGRAPHY
AZOLES
BIOCHEMICAL REACTION KINETICS
BIOTIN
BODY
CARBON 14 COMPOUNDS
CARBON-CARBON LYASES
CARBOXY-LYASES
CARBOXYLASE
CARBOXYLIC ACIDS
CHEMICAL ANALYSIS
DENSITOMETERS
DIGESTIVE SYSTEM
ELECTROPHORESIS
ENZYMES
GLANDS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
IMIDAZOLES
KINETICS
LABELLED COMPOUNDS
LIVER
LYASES
MAMMALS
MEASURING INSTRUMENTS
MOLECULAR WEIGHT
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANS
PHOTOMETERS
PROTEIN DENATURATION
PROTEINS
QUANTITATIVE CHEMICAL ANALYSIS
RATS
REACTION KINETICS
RODENTS
VERTEBRATES
VITAMIN B GROUP
VITAMINS