J. Biol. Chem., Vol. 260, Issue 3, 1386-1389, Feb, 1985
Interaction of S-acyl fatty acid synthase thioester hydrolase with fatty acid synthase. Direct measurement of binding by fluorescence anisotropy
RJ Foster, RF Bonsall, AJ Poulose and PE Kolattukudy
Treatment of S-acyl fatty acid synthase thioester hydrolase from the
uropygial gland of Peking duck with pyrenebutylmethanephosphonofluoridate
resulted in inactivation of the enzyme with covalent attachment of the
pyrene derivative to the enzyme. One mole of the derivative was
attached/mol of protein, most probably at the active serine. When avian
fatty acid synthase was added to the modified thioesterase, the
fluorescence anisotropy of the pyrene derivative increased dramatically.
That this increase represented the functionally significant binding between
the two proteins was suggested by the fact that increasing salt
concentration resulted in concomitant loss in enzyme activity and
fluorescence anisotropy. As the synthase concentration increased,
anisotropy increased giving a saturation pattern. From a Scatchard plot
analysis the association constant for the binding of the two proteins was
calculated to be 10(6) M-1 and one- to-one stoichiometry was shown for this
association. These results show that fluorescence anisotropy of the pyrene
derivative attached to the thioesterase can be used to directly measure the
binding of this enzyme to fatty acid synthase.
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