NEUROPHYSIN BINDING AND NATRIURETIC PEPTIDES FROM THE POSTERIOR PITUITARY

A qualitative technique of gel filtration on Sephadex@ G-25 has been used to study binding of variously altered vasopressin (VP) and oxytocin (OT) peptide molecules to highly purified bovine neurophysin I1 (Np II).1*2 The basic technique (FIGURE 1) consists of using calibrated columns with known fractional elution positions of Np I1 alone and the small peptides alone. Binding experiments involved subjecting equimolar mixtures of Np I1 and the small peptides at a binding pH of 5.8 in pyridine-acetate buffer (selected so that subsequent freeze-drying of eluted fractions would eliminate solvent contamination) to gel filtration in the same buffer. Np I1 fractions and known fractions for the small peptide alone were pooled separately, and criteria of binding were the following : (1) ODmo to estimate peptide content in the two fraction pools; (2) dansylation analysis of the N-terminal residue in each fraction; (3) assay of biological activity (usually pressor, also uterotonic) in each fraction after freeze-drying and redissolving in water; (4) dissolving of the freeze-dried Np I1 fractions in 0.1 M HCOOH-dissociation conditions-followed by gel filtration on the same calibrated column, this time in 0.1 M HCOOH, and retesting of the same fractional positions as above to criteria 1-3. Of course, if the small peptide had a very low biological activity, criterion 3 was of little value. These experiments dealt with the following categories of structural alteration in the neurohypophyseal hormones: (a) hormonogen forms, in which the lysine-vasopressin (LVP) chain was extended by 1-3 additional residues (Leu, Phe Gly, Gly-Gly, Gly-Gly-Gly) a t the N-terminal, the new analogs all having an Nh-amino group present; (b) LVP shortened at the C-terminal: des-[9dycine amidel, des-[84ysine]des-[ g-glycine amidel, and pressinamide, or des-(Pro-Lys-Gly-NH2 ] with C-terminal Cys in amide form; (c) alterations in the disulfide bridge: l-monocarba (CH2 S), 6-monocarba (SCH21, dicarba (CH2CH2) and opening of the SS bond by conversion (with 13-mercaptoethanol and CH31) to SCH3 groups on both 1and 6-Cys; (d) variations in the p-position of the aromatic ring in residue 2: instead of the OH of Tyr, p-methoxy and pethoxy groups, and Phe with only a proton at this position. Sources, and details of the preparation of both the analogs and hormone fragments, have been presented e l ~ e w h e r e . ~ ~