Identification of human neurophysins: complete amino acid sequences of MSEL- and VLDV-neurophysins.

Abstract

Two human neurophysins have been purified from acetone-desiccated posterior pituitaries by acidic extraction, molecular sieving, and ion-exchange chromatography. The complete amino acid sequence of each protein has been determined by using a sequencer and characterizing two sets of overlapping enzymic peptides. The two neurophysins belong to two structural families previously defined as MSEL-and VLDV-neurophysins according to the nature of the residues in positions 2, 3, 6, and 7.(MSEL-neurophysins contain methionine-2, serine-3, glutamic acid-6, and leucine-7; VLDV-neurophysins contain valine-2, leucine-3, aspartic acid-6, and valine- Neurophysins are small proteins found associated with neurohypophysial hormones in stoichiometric and reversible complexes (1). Isolation of neurophysins and neurohypophysial hormones from several mammalian species has been carried out often through this complex procedure (2).Purification of human neurophysins has been attempted previously by using either by-products of the gonadotropin purification (3) or directly from acetone-desiccated posterior pituitary glands (4, 5). Proteins are usually extracted by 0.1 M HCI at 4°C; after fractionation by molecular sieving, the crude neurophysins of Mr 10,000 are subjected to ion-exchange chromatography in order to isolate the different types of neurophysins (4, 5). Purification also has been carried out by using continuous polyacrylamide gel electrophoresis (6-8). Neurophysins are often termed neurophysin I, II, III, etc. according to their migrations to the anode, but this identification is confused by the presence of truncated forms. Therefore, another nomenclature, based upon the nature of the amino acids in positions 2, 3, 6, and 7, has been proposed, and two chemical types of neurophysins, termed MSEL-and VLDV-neurophysins, have been distinguished (9). (MSEL-neurophysins contain methionine-2, serine-3, glutamic acid-6, and leucine-7; VLDV-neurophysins contain valine-2, leucine-3, aspartic acid-6, and valine-7.) In each mammalian species, one protein of each type has been discovered. Extraction was usually carried out with fractions of 1 g of material. Each sample was extracted with 0.1 M HCl (50 ml/ g) for 4 hr at 40C. After centrifugation, the supernatant solution was directly subjected to a molecular sieving by passage through a column (2.5 x 170 cm) of Sephadex G-75 equilibrated with 0.1 M formic acid. Fractions (3-ml) were collected, and absorbance at 280 nm was measured. Five peaks were detected; the fourth (D), containing proteins of Mr 11,000 as estimated by sodium dodecylsulfate/polyacrylamide gel electrophoresis (13), was used for further purification.This "crude neurophysin" fraction was freeze-dried, and the material (30-40 mg) was chromatographed onto a column (0.4 x 0.50 cm) of DEAE-Sephadex A-50 by using a discontinuous ionic strength gradient (0.15 M to 1 M) of pyridine acetate (pH 5.9). Fractions (1-ml) were collected, and proteins were estimated with Folin-Lowry reagent (14). In a typical experiment carried...

Crystal structure of a bovine neurophysin II dipeptide complex at 2.8 A determined from the single-wavelength anomalous scattering signal of an incorporated iodine atom.

“…In a similar context, the significance to solution data of the extra peptide that helps to stabilize both the monomer-monomer and the tetramer-tetramer interfaces will require evaluation, as will the presence of oligomers of higher order than dimer. The present results are strong arguments in support of a role for such higher oligomers in the storage of NP complexes in the precipitated phase within the NSG and for their potential role in the initial phase ofprecursor packaging (1), with the importance of Tyr49 to such oligomer formation helping to account for evolutionary conservation of this residue (1,6). However, since a tetramer-based array of higher oligomers appears to be particularly stabilized by the extra peptide, while NP and hormone are synthesized in a 1:1 ratio, formation of this type of array in vivo might depend on other peptides present in the NSG, while additionally providing a mechanism for the storage ofthese peptides (1).…”

“…The noncovalent interactions between hormone and NP in the complex that stabilize the hormones within the NSG are similar to those within the precursor (5) and have been extensively studied in solution as a model of protein-peptide recognition (1). However, while the primary structures (6) and disulfide-pairing of the NPs (7) have been established and preliminary conformational data have been obtained in solution (e.g., ref. 1), neither the complete three-dimensional structure of a NP nor that of a NP complex has been reported.…”

c-<i>fos</i> Expression in Vasopressin and Oxytocin Neurons Reveals Functional Heterogeneity within Magnocellular Neurons

“…However, general use of antibodies produced against neurophysins to study the neurohypophysis was often hampered by cross-reactivity between the individual hormone-specific neurophysins. Of the 95 amino acids in neurophysin, amino acids 10 through 74 display exten sive homology between the OT and AVP neurophysins, both within and between various species [18], Many of the antibodies produced against neurophysins were likely to be directed against epitopes in the common sequences of the molecules. To obtain hormone-specific antibodies to neurophysins, we immunized with cus tom-synthesized peptide sequences of the 14 carboxy terminal peptides of the AVP and OT neurophysins.…”

“…Localizing the neurophysins has advantages over localization of OT or AVP. Neurophysins, part of the prohormones for AVP and OT, are 95 amino acids in length [18], approximately 10 times that of the neurohypophyseal hormones, and be cause of this larger size are more antigenic when used for antibody production and more stable in tissue and plasma. For anatomic studies of the neurohypophysis, brain and other tissues, the neurophysins are more stable in a variety of fixation techniques used for immunohistology [19].…”

Effects of Diabetes Insipidus Mutations on Neurophysin Folding and Function

“…This takes advantage of the strong homology in structure and function between Oxy NP and VP NP within and among different mammalian species (2,19). Fig.…”