The isolation of the native hormone-binding proteins from bovine pituitary posterior lobes. Crystallization of neurophysin-I and -II as complexes with [8-arginine]-vasopressin

Abstract

1. The native hormone-binding proteins, neurophysin-I and -II, have been isolated from acetone-desiccated bovine pituitary posterior lobes. 2. Neurophysin-I and -II are present in approximately equal quantities in the tissue and are localized in the neurosecretory granules. 3. The apparent molecular weight, determined by equilibrium sedimentation of neurophysin-I, was 19000 and that of neurophysin-II was 21000; their sedimentation coefficients, S(20,w), were 1.66 and 2.02s respectively. 4. Neurophysin-I and -II are similar in amino acid composition. Neurophysin-II was distinguished from neurophysin-I by the absence of histidine. 5. The proteins form complexes with oxytocin as well as with vasopressin. Complexes of both proteins with [8-arginine]-vasopressin have been crystallized. 6. Bioassay of the pressor and oxytocic activities of the crystals shows that neurophysin-I binds three molecules of either vasopressin or oxytocin whereas neurophysin-II binds only two molecules of each hormone per molecule of protein. Complexes containing two molecules of oxytocin and one molecule of [8-arginine]-vasopressin per molecule of protein are formed by neurophysin-I and -II; both proteins appear to possess three polypeptide-binding sites/molecule.

Isolation, assay, and secretion of individual human neurophysins.

“…The pH of the supernate was adjusted to 3.9 with 10 N HCl, and 80 g of NaCl was added to the extract which was constantly stirred at 4C for 24 h. A precipitate formed which was isolated by centrifugation at 31,000 g for 30 min at 40C. This has been referred to as neurophysin-hormone complex (14). The precipitate was dialyzed against multiple changes of distilled water at 4VC over 2 days and then lyophilized.…”

“…Posterior pituitaries were separated from these glands by blunt dissection and air-dried. The glands were extracted by the method of Hollenberg and Hope (14). 10 g of dried posterior pituitaries were crushed in 0.1 N HCl using a mortar and pestle and extracted overnight at 4VC in 400 ml 0.1 N HCl (final pH 1.5).…”

“…It is understandable that neurophysin is present in high concentration in extracts of whole pituitaries because of the high percentage of the weight of the posterior pituitary that is neurophysin, 5%. The extraction procedure used here is a standard method (14), and 10% of the pituitary extract was isolated as crude neurophysin. As radioimmunoassay indicated that more than 50% of the acid extract was neurophysin, this method is only 20% efficient.…”

Mentors And The Butterfly Effect: Triggers For Discovering Signalling by Proteinases via Proteinase-Activated Receptors (PARs) And More

“…Upon completing my M.Sc., my subsequent doctoral project involved understanding the complementary interactions between peptide hormones (oxytocin and vasopressin) and their puri ed neurophysin binding proteins. at insight led to their crystallization as stoichiometric complexes [3] and fostered my interest in peptide structure-activity relationships. It was unforeseen that the same principles would eventually be relevant to the study of the structure-activity relationships in peptide-stimulated activation of proteinase-activated receptors, as outlined in sections that follow.…”

“…e idea was to isolate rat mast cell proteinases and monitor their in ammatory e ects via the PARs; however, the literature-based procedures for the chromatographic puri cation of rat mast cell proteinases did not meet the standards I had come to expect from my doctoral work [3]. us, our strategy switched from testing the in ammatory actions of the mast cell enzymes directly, to testing the potential in ammatory actions of the PAR1/2-activating peptides themselves.…”