Asai M, Matamoros-Trejo G, Linares G, Agustín P

Language: Spanish
References: 18
Page: 13-19
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ABSTRACT

Several protein hormones and neuropeptides are derived by nzymatic cleavage from a large and inactive peptide precursor. The enkephalins’ precursor, the proenkephalin A (PA) has been deduced by sequencing cloned DNA. The PA contains four copies of Met-Enkephalin and one copy each of Leu-Enkephalin, heptapeptide and octapeptide. Proenkephalin (amino acids 1-70)does not contain any opioid peptide sequence and has been named synenkephalin (Syn). Initial evidences suggested that Syn is produced and secreted as an intact molecule or as part of the precursors in the adult brain and adrenal medulla, respectively. However, under specific physiological conditions, i.e., proliferating immune cells, Syn (8.6 kDa) it is processed to low molecular weight peptides (1.0 kDa). Until now there is no evidence of the production or release of a 1.0 kDa peptide derived from synenkephalin in the adult rat brain. In this work we studied the postranslational processing of synenkephalin in rat striatum, either in whole tissue or in vitro release conditions.
Male Wistar rats were used throughout this experiment. The rats were sacrificad by decapitation and the striatum removed. Samples were divided into three groups: 1) Thes striatum (n=8) was homogenized and centrifuged; the supernatant was lyophilized and resuspended in 2 ml water. 2) Striatum was dissected and sliced in two directions at 90° at 300 mm intervals with a tissue chopper. The slices were used for the assessment of the in vitro release of synenkephalin and Met-Enkephalin, using potassium as depolarized stimulus. 3) The synaptosomal fraction was obtained from striatum; samples were depolarized with potassium 55 mM, and the perfusates were lyophilized and resuspended in 2 ml water. Samples were individually applied to a Sephadex G-50 column. Eluates were lyophilized and resuspended in RIA buffer. Synenkephalin and Met-Enkephalin were measured by RIA using a C-terminally directed antiserum. Gel filtration chromatography (Sephadex G-50) showed that in the extracts of adult rat brain 45% of the IR-Synenkephalin eluted in the position of the authentic peptide (8.6 kDa), and the rest of the immunoreactivity corresponded to partially processed peptides of 4.8 and 2.5 kDa. After depolarized stimulus the chromatogram showed a differential pattern. In both, slices and synaptosomal samples, synenkephalin was processed to a single peak with a molecular weight of 1.0 kDa. This last peptide, which was further characterized by HPLC, resembles the same retention time when compared to the standard reagent (YEESHLLA). IR-Met-Enkephalin in whole tissue (analyzed before and after enzymatic digestion with trypsin and carboxypeptidase B) corresponded mainly to non-processed products. These results indicate that the non-opioid portion of proenkephalin (synenkephalin-derived peptides) rather than the opioid portion (Met-Enkephalin-containing peptides) is fully cleaved to the 1.0 kDa peptide in the depolarized samples. These data suggest that Synenkephalin (1-70) is not processed in basal conditions in the adult brain. However, when the neuronal activity is increased, i.e., after depolarized stimulus, synenkephalin is fully processed to a low molecular weight peptide. This last peptide has been related to the physiology of the synenkephalinderived peptides.

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