Solubilization and purification of melatonin receptors from lizard brain.
Melatonin receptors in lizard brain were identified and characterized using 125I-labeled melatonin ([125I]MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resulted in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.
Rivkees
S A
SA
Laboratory of Developmental Chronobiology, Massachusetts General Hospital, Boston 02114.
Conron
R W
RW
Reppert
S M
SM
D000818:Animals; D001923:Brain Chemistry; D002462:Cell Membrane / Q000032:analysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004072:Digitonin / Q000008:administration & dosage / Q000494:pharmacology; D005615:Freezing; D019204:GTP-Binding Proteins / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006358:Hot Temperature; D007457:Iodine Radioisotopes; D007700:Kinetics; D008116:Lizards / Q000378:metabolism*; D008550:Melatonin / Q000378:metabolism; D015394:Molecular Structure; D044094:Receptors, Melatonin; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013873:Thionucleotides / Q000494:pharmacology
D007457:Iodine Radioisotopes; D044094:Receptors, Melatonin; D017981:Receptors, Neurotransmitter; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D008550:Melatonin; D004072:Digitonin