Purification of the opiate receptor of NG108-15 neuroblastoma-glioma hybrid cells.
Opiate receptors from NG108-15 neuroblastoma-glioma hybrid cell membranes were purified to apparent homogeneity in a form covalently labeled with the opiate affinity ligand 3-methylfentanylisothiocyanate (super-FIT). The purification procedure consists of five steps: quantitative labeling of opiate receptors in membranes with super-FIT; solubilization in a lubrol/3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate mixture; adsorption to and elution from a wheat germ agglutinin-Sepharose column; immunoaffinity chromatography on columns of immobilized anti-fentanyl; preparative gel electrophoresis in the presence of NaDodSO4. The protein was purified approximately 30,000-fold in 3% overall yield to a state in which there is 1 mol of super-FIT bound per mol of protein, corresponding to 21,000 pmol per mg of protein, the theoretically expected specific activity. The protein is glycosylated and migrates on NaDodSO4 gel electrophoresis with a Mr near 58,000. It has a strong tendency to dimerize, even in the presence of denaturing detergents, and it exists primarily as an oligomer in nondenaturing detergents.
Simonds
W F
WF
Burke
T R
TR
Rice
K C
KC
Jacobson
A E
AE
Klee
W A
WA
D000345:Affinity Labels; D000818:Animals; D002460:Cell Line; D005283:Fentanyl / Q000031:analogs & derivatives; D005910:Glioma; D006023:Glycoproteins / Q000302:isolation & purification; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000302:isolation & purification; D008970:Molecular Weight; D009447:Neuroblastoma; D011957:Receptors, Opioid / Q000302:isolation & purification*; D017465:Receptors, Opioid, delta
D000345:Affinity Labels; D006023:Glycoproteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011957:Receptors, Opioid; D017465:Receptors, Opioid, delta; C046222:3-methylfentanyl isothiocyanate; D005283:Fentanyl