Effects of pH, salt and time on ligand binding properties of overexpressed melanocortin 4 receptor.
The G-protein coupled melanocortin 4 receptor (MC4r) plays an important role in the energy metabolism. We overexpressed the MC4r in CHO cells and performed characterisation studies on the cell membranes to determine functional stability and ligand binding properties of the receptor. The affinity for the ligands [Nle4, d-Phe7]-alphaMSH and MTII was lost below pH 6 but could be restored by returning to physiological pH. Increasing NaCl concentration up to 1 M had little influence on the binding of either ligand. At neutral pH, physiological salt concentration and 4 degrees C the ligand affinity of the receptor was stable for up to 6 days. These findings will facilitate design of purification methods for the receptor.
Dolby
Viveka
V
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-221 00, Lund, Sweden.
Lundqvist
Andreas
A
Fröberg
Thomas
T
Lüllau
Elke
E
Shaw
Judith
J
Tjerneld
Folke
F
Cronet
Philippe
P
D000818:Animals; D016588:Anticarcinogenic Agents / Q000737:chemistry* / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D044105:Receptor, Melanocortin, Type 4 / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000819:agonists / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012492:Salts / Q000737:chemistry; D013997:Time Factors; D000521:alpha-MSH / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism
D016588:Anticarcinogenic Agents; D044105:Receptor, Melanocortin, Type 4; D011994:Recombinant Proteins; D012492:Salts; C081720:alpha-MSH (4-10)amide, Ac-Nle(4)-cyclo(Asp(5)-Phe(7)-Lys(10))-; D000521:alpha-MSH; C027756:MSH, 4-Nle-7-Phe-alpha-