Interaction of diazosulfanilic acid and the opiate receptor. Inhibition of specific binding to synaptic membranes and labeling of a membrane lipid stereospecifically protected by opioids

P. Y. Law, T. J. Ostwald, E. L. Way, H. H. Loh

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2 Scopus citations

Abstract

3,5-Diiodo-4-diazosulfanilic acid (DIDSA) inhibited the opiate specific binding to the rat brain synaptic membrane. DIDSA inhibited [3H]dihydromorphine binding more rapidly and to a greater extent than [3H]naloxone binding. That the inhibition of the opiate binding resulted from the derivatization of the opiate receptor sites by DIDSA was demonstrated by Scatchard analysis of the binding data and by the ability of 0.1 μM concentrations of various opiate ligands and not 0.1 μM dextrorphan to attenuate the DIDSA inhibition. DIDSA was probably reacting with a population of sites distinct from those which reacted with N-ethylmaleimide (NEM). This was demonstrated by the ability of 100 mM Na+ to decrease NEM inhibition of [3]naloxone binding and not that of DIDSA inhibition. The difference in NEM and DIDSA reactive sites was further demonstrated by the ability of hypoosmotic lysis of synaptosomes to enhance DIDSA but not NEM inhibition of naloxone binding. When synaptic membranes were derivatized with a combination of 0.4 mM DIDSA and 0.1 mM NEM in the presence of levorphanol and subsequently labeled with [125I]diazosulfanilic acid (DISA), there was a levorphanol concentration-dependent increase in radioactivity incorporated into the chloroform:methanol (2:1) extractable membrane lipids previously protected from the reagents with levorphanol but not those lipids protected with dextrorphan. There was a parallel increase in the amount of opiate receptor binding activity protected by the levorphanol. Moreover, the 2-fold increase in the radiolabeling of the protected membrane polar lipids could be eliminated by the addition of 0.1 μM levorphanol but not by the addition of dextrorphan in the reaction mixtures containing [125I]DISA. The identity of the polar lipid selectively protected by levorphanol was determined by thin-layer chromatography in silica gel G plates in three different solvent mixtures. The chromatographic patterns of the membrane polar lipids extract in these solvent mixtures compared favorably with those of the standard phosphatidylethanolamine. Separation of membranous protein with Sephadex column chromatography and polyacrylamide gel isoelectric focusing did not resolve any proteins which were significantly protected by levorphanol.

Original languageEnglish (US)
Pages (from-to)355-366
Number of pages12
JournalMolecular Pharmacology
Volume19
Issue number3
StatePublished - 1981

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