A naturally occurring delta-EEG enhancing nonapeptide in rabbits - X. Final isolation, characterization and activity test

Abstract

By extracorporeal dialysis of blood from the sagittal venous sinus in rabbit donors during electrical stimulation of the ventromedial intralaminary thalamus, a hemodialysate was obtained. This dialysate, or its purified fractions, infused into the mesodiencephalic ventricular system of recipient rabbits, induced behavioral and electroencephalographic (EEG) changes, i.e., enhanced delta-EEG. These effects of the dialysate and of its subsequent fractions were quantified by EEG analysis of the cortical delta activity (2-3 Hz), using a calibrated automatic wave analyzer. The activity values of the last pure fractions were furthermore processed on an 1108 computer system. It was thus possible to express the delta enhancement in RMS μV and the corresponding time integral referred to the preinfusion values as well as to the values of the control rabbits. Ultrafiltration through a UM-05 Diaflo membrane and gel-filtration over Sephadex G-15 yielded an active fraction showing a symmetrical ninhydrinpositive peak, with a corresponding absorption peak at 280 nm and a mol.wt. between 355 and 1000. Preparative TLC and high voltage paper electrophoresis, followed by a second gel-filtration over Sephadex G-15 and quantitative TL-electrophoresis, yielded a progressively purified ninhydrin-positive EEG active compound, with an absorption maximum at 280 nm. A final compound was analyzed and quantified by amino acid analysis, UV and fluorescence spectroscopy. The delta-EEG enhancing compound was shown to be a small peptide, comprising 9 amino acid residues: 1 Trp-(2 Ala, 1 Asp, 1 Glu, 3 Gly, 1 Ser), with a mol.wt. of 848.98. The minimal effective dose for delta-EEG enhancement has been estimated at 6×10-9 mol/kg B.W. when dissolved in 0.05 ml c.s.f.-like solution and infused intraventricularly over a period of 3.5 min. The specific delta activity due to the peptide was compared to that in control rabbits infused not only with a c.s.f.-like solution, but also with a c.s.f. solution to which a similar synthetic nonapeptide (mol. wt. 996.14) had been added. The purpose of this comparison was to test the effect of the specific delta-EEG enhancing peptide against a corresponding unspecific nonapeptide-"analogue". RMS μV and time integrals of the delta values of the two control groups showed no difference. By contrast, the RMS μV and time integral of the delta-EEG enhancement due to the "delta-sleep-inducing-peptide" (DSIP), when compared to the values of one of the two or both control groups, were significant: The nonapeptide increased the delta-EEG-activity by +45.8±6.65 RMS μV or 43.1±6.25% as compared to the controls. © 1977 Springer-Verlag.