Soluble antibody affinity chromatography technique investigated with ultratrace [125I]thyroxine

Warden, Beverly A.; Giese, Roger W.

doi:10.1016/s0021-9673(01)97743-x

Cited by 2 publications

(1 citation statement)

References 16 publications

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“…The serum was prepared and the antibody titer was determined by double immunodiffusion. The specific IgG was partially purified by precipitation with ammonium sulfate as described previously [60]. The antibody was aliquot and maintained at −20 °C until use.…”

Section: Methodsmentioning

confidence: 99%

Effect of the N‐terminal sequence on the binding affinity of transthyretin for human retinol‐binding protein

Leelawatwattana

Praphanphoj²,

Prapunpoj

2011

The FEBS Journal

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During vertebrate evolution, the N-terminal region of transthyretin (TTR) subunit has undergone a change in both length and hydropathy. This was previously shown to change the binding affinity for thyroid hormones (THs). However, it was not known whether this change affects other functions of TTR. In the present study, the effect of these changes on the binding of TTR to retinol-binding protein (RBP) was determined. Two wild-type TTRs from human and Crocodylus porosus, and three chimeric TTRs, including a human chimeric TTR in which its N-terminal sequence was changed to that of C. porosus TTR (croc ⁄ huTTR) and two C. porosus chimeric TTRs (hu ⁄ crocTTR in which its N-terminal sequence was changed to that of human TTR and xeno ⁄ crocTTR in which its N-terminal sequence was changed to that of Xenopus laevis TTR), were analyzed for their binding to human RBP by native-PAGE followed by immunoblotting and a chemilluminescence assay. The K d of human TTR was 30.41 ± 2.03 lM, and was similar to that reported for the second binding site, whereas that of crocodile TTR was 2.19 ± 0.24 lM. The binding affinities increased in croc ⁄ huTTR (K d = 23.57 ± 3.54 lM) and xeno ⁄ crocTTR (K d = 0.61 ± 0.16 lM) in which their N-termini were longer and more hydrophobic, but decreased in hu ⁄ crocTTR (K d = 5.03 ± 0.68 lM) in which its N-terminal region was shorter and less hydrophobic. These results suggest an influence of the N-terminal primary structure of TTR on its function as a co-carrier for retinol with RBP.

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Section: Methodsmentioning

confidence: 99%

Effect of the N‐terminal sequence on the binding affinity of transthyretin for human retinol‐binding protein

Leelawatwattana

Praphanphoj²,

Prapunpoj

2011

The FEBS Journal

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Vitellogenin purification and development of assay for vitellogenin receptor in oocyte membranes of the tilapia (Oreochromis niloticus, linnaeus 1766)

et al. 1991

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An assay has been successfully developed for vitellogenin receptors in isolated oocyte membranes of the tilapia (Oreochromis niloticus). Vitellogenin produced by estrogen stimulation of the male tilapia was purified by anion-exchange chromatography on DEAE-Sephacel and identified by its incorporation of 32P-orthophosphate and 3H-leucine in vivo. Only one molecular form of vitellogenin was found to be induced by estrogen treatment. After iodination with Iodogen, the 1251-vitellogenin was shown to be immunologically similar to the unlabeled protein and was used to develop an assay for vitellogenin receptors. For such assays, the optimal conditions for binding were pH 7.4,6-h incubation at 23°C with 7.5 mM Mg2+ and 10 mM Ca2+. The magnitude of the specific binding was dependent on the amount of oocyte membranes and was saturable. The binding shows both tissue and protein specificity: the 1251-vitellogenin binds specifically to only oocyte membranes and not to those of the brain, muscles, liver, intestine, and testis; its binding was displaceable only by unlabeled vitellogenin and a yolk protein extract. Saturation studies and Scatchard analyses revealed only a single class of binding sites. These studies provide unequivocal evidence that the binding sites for vitellogenin in the tilapia oocytes are specific receptors. The number and affinity (KD) of these receptors increase from the previtellogenic (9.98 x lOg/oocyte) to the vitellogenic stage (3.65 X 1012/oocyte), and remained unchanged at the preovulatory stage (3.53 X 1012/oocyte), at which time the affinity of the receptors was also highest (0.3 FM).

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Soluble antibody affinity chromatography technique investigated with ultratrace [125I]thyroxine

Cited by 2 publications

References 16 publications

Effect of the N‐terminal sequence on the binding affinity of transthyretin for human retinol‐binding protein

Effect of the N‐terminal sequence on the binding affinity of transthyretin for human retinol‐binding protein

Vitellogenin purification and development of assay for vitellogenin receptor in oocyte membranes of the tilapia (Oreochromis niloticus, linnaeus 1766)

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