Analyzing Binding Data

Measuring the rate and extent of radioligand binding provides information on the number of binding sites, and their affinity and accessibility of these binding sites for various drugs. This unit explains how to design and analyze such experiments. Curr. Protoc. Neurosci. 52:7.5.1‐7.5.65. © 2010 by John Wiley & Sons, Inc.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143761/1/cpns0705.pd

Solubilization and chromatographic separation of gonadotrop in receptor from adenylate cyclase in ovarian preparations

The distribution of gonadotropin receptor and adenylate cyclase activities has been monitored in Sepharose 6B chromatographic eluates of a Lubrol solubilized fraction of 25 day old rat ovarian tissue. The receptor was resolved from the adenylate cyclase activity. Chromatography of a gonadotropin desensitized rat ovarian preparation showed that the desensitized preparation contained normal amounts of the adenylate cyclase activity when compared with the control ovarian preparation, but there was a marked reduction in the receptor activity in the desensitized ovaries. This study demonstrates that in ovarian tissue adenylate cyclase is a separate entity distinct from the receptor molecule and that the desensitization causes a reduction in the receptor activity with no detectable change in the catalytic and chromatographic properties of the adenylate cyclase.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23312/1/0000251.pd

Rapid kinetics of G protein subunit association: A rate-limiting conformational change?

G protein subunit association and dissociation are thought to play an important role in signal transduction. We measured [alpha][beta][gamma] heterocomplex formation using resonance energy transfer. Fluorescein-labelled [alpha](F-[alpha]) emission was quenched ~ 10% on mixing with eosin-labelled [beta][gamma](E-[beta][gamma]). Unlabelled [beta][gamma] did not quench F-[alpha] fluorescence. Stopped-flow kinetics showed a t1/2 ranging from 2.5 s to 0.25 s for 50 nM to 1200 nM E-[beta][gamma]. The rate saturated at high E-[beta][gamma] concentrations consistent with a two-step mechanism. We report the first rapid-mix studies of G protein subunit association kinetics which suggest that [alpha] and [beta][gamma] combine by a two-step process with a maximal rate of 4.1 +/- 0.4 s-1.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31136/1/0000033.pd

Formyl peptide chemotaxis receptors on the rat neutrophil: Experimental evidence for negative cooperativity

To examine the existence of negative cooperativity among formyl peptide chemotaxis receptors, steady-state binding of f Met-Leu-[ 3 H]Phe to viable rat neutrophils and their purified plasma membranes was measured and the data were subjected to statistical analysis and to computer curve fitting using the NONLIN computer program. Curvilinear, concave upward Scatchard plots were obtained. NONLIN and statistical analyses of the binding data indicated that a two-saturable-sites model was preferable to a one-saturable-site model and statistically valid by the F-test (P < 0.1). In addition, Hill coefficients of 0.80 ± 0.02 were obtained. Kinetic dissociation experiments using purified plasma membranes showed evidence of site-site interactions of the destabilizing type (negative cooperativity). Thus, unlabeled f Met-Leu-Phe accelerated the dissociation of f Met-Leu-[ 3 H]Phe under conditions where no rebinding of radioligand occurred. The rate of dissociation of f Met-Leu-[ 3 H]Phe from the plasma membranes was dependent on the fold excess of unlabeled f Met-Leu-Phe used in the dilution medium; at the highest concentration tested (10,000-fold excess), the dissociation rate was more than double the dissociation rate seen with dilution alone, In addition, occupancy-dependent affinity was ascertained directly by studying the effect of increasing fractional receptor saturation with labeled ligand on the dissociation rate of the receptor-bound labeled ligand. These data showed that the f Met-Leu-[ 3 H]Phe dissociation rate was dependent on the degree of binding site occupancy over the entire biologically relevant range of formyl peptide concentrations. Furthermore, monitoring of the time course of dissociation of the receptor/f Met-Leu-[ 3 H]Phe complex as a function of receptor occupancy revealed that receptor affinity for f Met-Leu-Phe remained occupancy-dependent during the entire time of dissociation examined (up to 10 min). Finally, the average affinity profile of the equilibrium binding data demonstrated a 60% decrease in receptor affinity in changing from the high affinity to the low affinity conformation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38446/1/240270406_ftp.pd