Reversed-phase liquid chromatographic purification and isolation of a radio-iodinated selective probe for mu opioid receptors in the brain.
A Guard-PAK precolumn system was used for the reversed-phase liquid chromatography purification of a small, synthetic radiolabeled opioid peptide, FK 33-824 (D-Ala2, methyl-phe4, Met (O)ol5 enkephalin) (FK). This procedure involves trace enrichment of iodinated peptide onto the precolumn while iodination reagents are not retained. Radioactive contamination of high-performance liquid chromatography columns and injectors is thus avoided. Precolumn chromatography has sufficient resolving power to separate not only labeled from unlabeled peptide but also mono- from di-iodinated peptide. Purified 125I-labeled FK (estimated specific activity 85.9-153.7 Ci/mmol) showed high specific binding to mouse corpus striatum, neocortex, cingulate cortex, nucleus accumbens septi, diagonal band of Broca, nucleus medialis septi, area preopticus magnocellularis, and the nucleus of the caudate/putamen. Radioligand binding was inhibited by both antagonists (naloxone and naltrexone); and agonists D-Ala2, N-methyl-phe4, gly-ol5-enkephalin [DAGO]; FK; and beta-endorphin at all concentrations tested (1 x 10(-8) to 1 x 10(-4) M). Adrenocorticotropin hormone (ACTH) did not block ligand binding at any concentration tested. Distribution of mu opioid receptors was analyzed by light microscopic autoradiography. Sections incubated with 125I-labeled FK in the presence of agonists and antagonists demonstrated decreasing ligand binding with increasing doses of competitor. ACTH did not block ligand binding at any concentration tested. HPLC analyses of ligand which had been iodinated 1.5 half lives before the date of the experiment demonstrated a single peak similar to that of freshly iodinated ligand. Similar binding kinetics and autoradiographic labeling patterns were observed as compared to those obtained with freshly iodinated peptide.(ABSTRACT TRUNCATED AT 250 WORDS)
Miller
M M
MM
Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada.
Gould
B E
BE
Joshi
D
D
Bennett
H P
HP
James
S
S
Billiar
R B
RB
D000818:Animals; D001345:Autoradiography; D001921:Brain / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005405:D-Ala(2),MePhe(4),Met(0)-ol-enkephalin; D005260:Female; D007457:Iodine Radioisotopes; D051379:Mice; D008810:Mice, Inbred C57BL; D011957:Receptors, Opioid / Q000378:metabolism*; D017450:Receptors, Opioid, mu
D007457:Iodine Radioisotopes; D011957:Receptors, Opioid; D017450:Receptors, Opioid, mu; D005405:D-Ala(2),MePhe(4),Met(0)-ol-enkephalin