Extracti-Gel D chromatography is a simple, efficient method for removing digitonin during receptor purification: application to the kappa 1 opioid receptor.
Digitonin is widely used for extracting active neurotransmitter receptors from membranes. However, its low critical micellar concentration has made its removal from samples problematic. Here we report that digitonin can be efficiently removed (> 90%) from solution using Extracti-Gel D, a detergent-absorbing matrix. Active kappa 1 opioid receptors solubilized from brain survive Extracti-Gel D chromatography with a recovery of 50-55% and 25% dilution by added volume. The loss of receptor and the dilution, however, are compensated for to a large extent by the disinhibition of binding that results from the removal of digitonin. Extracti-Gel D chromatography had little or no effect on the apparent equilibrium dissociation constant for [3H]U-69,593 binding to the kappa 1 receptor. We conclude that Extracti-Gel D column chromatography is a simple, highly efficient and practical method for markedly reducing the concentration of digitonin in biological samples. Application of the procedure should allow characterization of digitonin-solubilized receptors with minimal complications from bound digitonin and extend the usefulness of digitonin to studies going beyond the initial stages of receptor purification.
Nock
B
B
Washington University School of Medicine, Department of Psychiatry, St. Louis, MO 63110.
Wich
M M
MM
Moore
B W
BW
D000042:Absorption; D000700:Analgesics / Q000493:pharmacokinetics; D000818:Animals; D044482:Benzeneacetamides; D001923:Brain Chemistry; D002852:Chromatography, Ion Exchange; D004072:Digitonin / Q000302:isolation & purification*; D006168:Guinea Pigs; D007700:Kinetics; D008566:Membranes / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000032:analysis; D011759:Pyrrolidines / Q000493:pharmacokinetics; D017473:Receptors, Opioid, kappa / Q000302:isolation & purification*; D012997:Solvents
D000700:Analgesics; D044482:Benzeneacetamides; D009419:Nerve Tissue Proteins; D011759:Pyrrolidines; D017473:Receptors, Opioid, kappa; D012997:Solvents; C045444:U 69593; D004072:Digitonin