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Ascorbate Destruction of Opiate Stereospecific Binding in Guinea Pig Brain Homogenate

1979; American Society for Pharmacology and Experimental Therapeutics; Volume: 16; Issue: 1 Linguagem: Inglês

10.1016/s0026-895x(25)11376-x

1521-0111

Claud E. Dunlap, F.M. Leslie, M Radó, Brian M. Cox,

Alcoholism and Thiamine Deficiency

Ascorbic acid was found to destroy opioid stereospecific binding to guinea pig brain homogenate irreversibly. This inactivation of stereospecific binding (SSB) displayed two components: a rapid phase that destroyed up to 50% of the binding within one minute, and a slower phase that proceeded by pseudo-first order kinetics. The ascorbate destruction of opioid binding was pH dependent, and was manifested primarily as a reduction in the number of binding sites, as demonstrated by Scatchard analysis. The slow phase of inactivation was subject to autoinhibition by high concentrations of ascorbate. Destruction of stereospecific binding by direct chemical reduction was ruled out, since several other reducing agents were found to be without effect. The stereoisomer of ascorbic acid, D-isoascorbic acid, produced destruction of SSB similar to that caused by ascorbate. Dehydroascorbic acid produced only slight loss of SSB, and protected against further destruction by ascorbic acid. All other analogues tested produced neither destruction of SSB nor protection against destruction of SSB by ascorbate. Destruction of stereospecific binding was prevented in homogenates incubated with ascorbate in the absence of oxygen. Reagents that inhibit ascorbate catalyzed lipid peroxide formation were shown to inhibit ascorbate destruction of SSB also, and dose-response curves for ascorbate destruction of SSB paralleled those for ascorbate-induced lipid peroxidation. Stereospecific binding could be partially but significantly protected by preincubation with phosphatidyl serine, but not with phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, cholesterol, cerebroside sulfates, sphingomyelin, arachidonic acid, or docosahexaenoic acid. It is suggested that a membrane lipid, sensitive to ascorbate induced peroxidation, plays a critical role in the structural integrity of the opioid stereospecfiic binding site.