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. 125 I -Leptin was used in saturation studie...
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Published in | Domestic animal endocrinology Vol. 23; no. 3; pp. 411 - 424 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
United States
Elsevier Inc
01.10.2002
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Subjects | |
Online Access | Get full text |
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Summary: | 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.
125
I
-Leptin was used in saturation studies, over a range of concentrations from 50
pM to 9
nM.
125
I
-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=46.2
fmol/mg protein. An additional class of low affinity, highly abundant sites with an apparent
K
d=175
nM, and
B
max=5740
fmol/mg protein was characterized. The association and dissociation kinetics for
125
I
-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
125
I
-leptin by mouse and human leptin and polyclonal anti-bovine leptin antibodies was dose-dependent. Specificity of binding was shown as bound
125
I
-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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0739-7240 1879-0054 |
DOI: | 10.1016/S0739-7240(02)00180-7 |