Journal of Lipid Research, Vol 28, 599-604, Copyright © 1987 by Lipid Research, Inc.
Determination of the quantity of acetyl CoA carboxylase by [14C]methyl avidin binding
CR Roman-Lopez, J Goodson and JB Allred
Conditions are described under which monomeric [14C]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 [14C]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.
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