Radioiodination and binding characteristics of a highly active opioid peptide

D-ala², N-Me-phe⁴, met (O)⁵-ol-enkephalin (Sandoz, FK 33-824) was radiolabeled to a specific activity of 1000 Ci/mmol using soluble lactoperoxidase and carrier-free Na¹²⁵l. The [¹²⁵l]-labeled enkephalin analog was purified by a combination of gel filtration and ion-exchange chromatography; it cochromatographed with its [¹²⁷l] labeled analog on thin layers. [¹²⁵l] D-ala², N-Me-phe⁴, met (O)⁵-ol-enkephalin binds stereospecifically to whole brain membrane preparations from rat with saturation at 5 nM at 25°C. Scatchard analysis reveals two classes of binding sites with apparent Kd's of 0.42 x 10^-9 M and 3.70 x 10^-9 M. The ratio of high affinity to low affinity binding sites is approximately 1:3. At 25°C, 40 mM sodium ion elicits half-maximal inhibition of stereospecifically bound counts, while 100 mM potassium inhibits binding by 25%. [¹²⁵l] FK 33-824 has been used in studies involving the purification of the mammalian opiate receptor. Preliminary characterization of a chemically cross-linked, solubilized [¹²⁵l] FK 33-824 enkephalin macromolecular complex has been carried out. The native complex has an apparent molecular weight of 380,000; SDS gel electrophoresis of the enkephalin-labeled receptor reveals a major labeled subunit of 35,000 daltons.