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Related Experiment Videos

Insulin binding monitored by fluorescence correlation spectroscopy.

Z H Zhong1, A Pramanik, K Ekberg

  • 1Department of Surgical Sciences, Karolinska Hospital, Stockholm, Sweden.

Diabetologia
|October 13, 2001
PubMed
Summary
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Fluorescence correlation spectroscopy reveals two distinct insulin binding sites on cells, differing in affinity. This advanced technique offers precise kinetic analysis of ligand-receptor interactions, even at low expression levels.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Biophysics

Background:

  • Insulin receptor binding is crucial for cellular signaling.
  • Radioligand binding assays have limitations in studying binding kinetics.
  • Novel techniques are needed for high-sensitivity analysis of ligand-receptor interactions.

Purpose of the Study:

  • To investigate insulin binding kinetics to its receptor using fluorescence correlation spectroscopy (FCS).
  • To determine the distribution of diffusion times and affinities of insulin-receptor interactions.
  • To provide novel data on the kinetics of insulin binding at the single-molecule level.

Main Methods:

  • Cultured human renal tubular cells (HRTC) were incubated with tetramethyl rhodamine-labeled insulin (Rh-Ins).
  • Fluorescence intensity fluctuations and autocorrelation functions were measured using FCS at single-molecule detection sensitivity.

Related Experiment Videos

  • Scatchard analysis was performed to determine binding affinities.

    • Main Results:

    • FCS identified at least two diffusion components for Rh-Ins binding at the cell membrane.
    • Specific binding was confirmed by the displacement of Rh-Ins with unlabeled insulin.
    • Scatchard analysis revealed two binding processes with distinct affinities (Kass ~2 x 10(10) M(-1) and ~1 x 10(9) M(-1)).

    Conclusions:

    • FCS demonstrates two classes of insulin binding sites with differing affinities or negative cooperativity.
    • These findings align with previous radioligand binding assay studies.
    • FCS offers high sensitivity for precise kinetic evaluation of ligand-receptor interactions in living cells, especially at low receptor expression levels.