J. Biol. Chem., Vol. 255, Issue 12, 5742-5746, Jun, 1980
Hormone-receptor studies with avidin and biotinylinsulin-avidin complexes
FM Finn, G Titus, JA Montibeller and K Hofmann
Avidin can be labeled to high specific radioactivity by introducing 3-
(p-hydroxyphenyl)-propionyl groups into the molecule (pHPP-avidin).
125I-pHPP-avidin binds avidly to rat liver plasma membranes and is not
displaced by unlabeled pHPP-avidin. Nonspecific binding of 125I-pHPP-
avidin can be substantially reduced by succinoylation of pHPP-avidin with
succinic anhydride (SpHPP-avidin). Spectral changes ensuing when the dye
4-hydroxyazobenzene-2'-carboxylic acid binds to avidin cannot be used to
assess the binding characteristics of the modified avidins since the
absorption coefficients of the complexes are markedly different; however,
the modified molecules bind theoretical amounts of [14C]biotin.
Biotinylinsulin and biotinylinsulinSpHPP-avidin complexes compete with
125I-insulin for binding to receptor sites on rat liver plasma membranes.
Biotinylinsulin complexes with unmodified avidin display anomalous binding
behavior attributable to formation of membrane aggregates. In light of this
finding, results obtained using unmodified avidin must be interpreted with
caution. Biotinylinsulin125I- SpHPP-avidin binds specifically and saturably
to rat liver plasma membranes. The biotinylhormoneSpHPP-avidin technique
has potential for labeling peptide hormones and other compounds that cannot
be iodinated by conventional procedures.
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