Hormone-receptor studies with avidin and biotinylinsulin-avidin complexes.
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.
D000595:Amino Acid Sequence; D000818:Animals; D001360:Avidin / Q000031:analogs & derivatives*; D001665:Binding Sites; D001667:Binding, Competitive; D001710:Biotin / Q000031:analogs & derivatives*; D002462:Cell Membrane / Q000378:metabolism; D007328:Insulin / Q000031:analogs & derivatives*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D010047:Ovalbumin / Q000031:analogs & derivatives*; D010666:Phenylpropionates; D011485:Protein Binding; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D011955:Receptors, Drug / Q000378:metabolism*
D007328:Insulin; D010666:Phenylpropionates; D011955:Receptors, Drug; C025360:insulin, biotinyl-; D001360:Avidin; C008869:phloretic acid; D001710:Biotin; D010047:Ovalbumin; D011972:Receptor, Insulin