Photoaffinity labeling of opiate receptors with 3H-etorphine: possible species differences in glycosylation.
Opiate receptors from whole rat brain (minus cerebellum) and cow striatum were labeled irreversibly using the intrinsic photolability of 3H-etorphine. After incubation with 2 nM 3H-etorphine and centrifugal washing, membranes were irradiated with light of 254 nm. Non-specific binding was determined by carrying out incubations in presence and absence of 10 microM levallorphan. Specific binding in photolabeled membranes was 75-80%, with a photo-incorporation yield of approximately 50%. Photolabeled membranes were extracted with CHAPS/Lubrol and unbound 3H-etorphine was removed by dialysis and passage over Sephadex G-25. Solubilized proteins were then subjected to chromatography on wheat germ agglutinin, and retained proteins were eluted with N-acetyl D-glucosamine (NAG). Protein profiles from rat brain and cow striatum were identical, with 89% of the total protein flowing through unretained and 11% eluted by NAG. However, the profile of radioactivity was markedly different in the two species. With rat, the specific activity (cpm/A280) was the same for flow-through and NAG-eluate. With cow, the specific activity of the NAG-eluate was 17 times greater than the flow-through. These results indicate that cow striatum and rat whole brain contain populations of opiate receptors which are glycosylated differently.
Bowen
W D
WD
Section of Biochemistry, Brown University, Providence, R.I. 02912.
Kooper
G
G
D000345:Affinity Labels; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D003342:Corpus Striatum / Q000378:metabolism; D005048:Etorphine / Q000378:metabolism*; D006031:Glycosylation; D008027:Light; D009019:Morphinans / Q000378:metabolism*; D051381:Rats; D011957:Receptors, Opioid / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D013045:Species Specificity; D014466:Ultraviolet Rays
D000345:Affinity Labels; D009019:Morphinans; D011957:Receptors, Opioid; D005048:Etorphine