Characterization of leptin binding in bovine kidney membranes.
The interactions of leptin with its receptor and other leptin binding sites is not well described or understood. We have used Scatchard analysis of saturation binding data to characterize the affinity of leptin for binding sites in bovine kidney membranes. 125I-Leptin was used in saturation studies, over a range of concentrations from 50 pM to 9 nM. 125I-Leptin differentiated a high affinity binding site from an abundant low affinity site. The high affinity/low density binding site (putative leptin receptor) had K(d)=0.098 nM and B(max)=46.2f mol/mg protein. An additional class of low affinity, highly abundant sites with an apparent K(d)=175 nM, and B(max)=574 fmol/mg protein was characterized. The association and dissociation kinetics for 125I-leptin binding were also studied. Dissociation of the leptin-receptor complex was very rapid, and this necessitated the use of a specially developed separation method for radioligand binding studies (precipitation with PEG and filtration). Competitive displacement of 125I-leptin by mouse and human leptin and polyclonal anti-bovine leptin antibodies was dose-dependent. Specificity of binding was shown as bound 125I-leptin was not displaced by insulin or control antibodies. These data indicate that leptin binds the bovine leptin receptor with high affinity and that a pool of leptin is bound to abundant cell membrane-associated proteins. These observations are consistent with the plasma concentration range for leptin and imply that free leptin concentration in the tissues may be partially buffered by cell-associated and bound forms in plasma. Thus, acute changes in leptin secretion may have little effect at the leptin receptor. The development of leptin agonists/antagonists should facilitate further characterization of leptin binding and clarify the role of abundant low affinity binding sites at the leptin axis.
School of Chemical and Biomedical Sciences, Central Queensland University, Box 5545, 4702, Qld, Rockhampton, Australia.
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Margetic
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Gazzola
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Hill
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006207:Half-Life; D007457:Iodine Radioisotopes; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D020738:Leptin / Q000378:metabolism*; D008297:Male
D007457:Iodine Radioisotopes; D020738:Leptin