Identification of a domain in cytochrome C that displaces [3H]TCP binding from rat brain membrane receptors: Synthesis of β-neuroprotectin

Abstract 1. 1. A stereospecific radioreceptor binding assay for the phencyclidine analogue, [ 3 H]TCP, was utilized to screen for inhibition of binding in extracts of rat brain. 2. 2. Extracts were prepared from rat cerebral cortex and hippocampus by methods employing aqueous acid. The extracts were fractionated by reversed phase-HPLC (RP-HPLC) and tested for activity in the radioreceptor assay. Three zones of activity were detected. The middle zone was further purified by high performance-size exclusion chromatography (HP-SEC). 3. 3. Size exclusion chromatography revealed a single zone of activity corresponding to mol. wts of ca 12,000–31,000 daltons. A fraction from this zone was digested with trypsin, and the resulting enzyme fragments, isolated by a combination of HP-SEC and RP-HPLC, were identified as fragments of rat cytochrome C . 4. 4. Horse cytochrome C was digested with trypsin and the fragments were similarly purified on the basis of the [ 3 H]TCP binding displacement assay. The fragments were sequenced and found to be trypsin cleavage products of a single largely invariant domain of the cytochrome C molecule: Lys-Lys-Lys-Asp-Glu-Arg-Ala-Asp-Leu-Ile-Ala-Tyr-Leu-Lys-Lys. 5. 5. β-neuroprotectin ( d )-Ala-Asp-Leu-Ile-Ala-Tyr-Leu-NH 2 , inhibits [ 3 H]TCP binding and provides protection against NMDA mediated neuronal cell death at low concentrations.