Immunological Identification of High Molecular Weight Forms Common To

Proc. Nati. Acad. Sci. USA Vol. 77, No. 5, pp. 2587-2591, May 1980 Biochemistry Immunological identification of high molecular weight forms common to bovine neurophysin and vasopressin (prohormones/radioimmunoassay/affinity chromatography/proteolytic enzymes/neurohypophysis) PIERRE NICOLAS*, MARYSE CAMIER*, MARC LAUBER*, MARIE-J. 0. MASSE*, JAN MOHRINGt*, AND PAUL COHEN* Groupe de Neurobiochimie Cellulaire et Mol6culaire, Universite Pierre et Marie Curie, 96 boulevard Raspail, 75006 Paris, France; and tCentre de Recherches Merell International, 67000 Strasbourg, France Communicated by I. Robert Lehman, February 19,1980 ABSTRACT Extracts of bovine neurohypophysis made in the same precursor or else that the biosynthesis of both com- acid/ethanol solution containing protease inhibitors were pounds might be controlled by the same genetic unit. fractionated by two successive filtrations on Sephadex G-75 columns equilibrated in the presence and then in the absence Although a wealth of genetic, cytochemical, and pharma- of 4 M urea. Analysis of the pattern of neurophysin-like immu- cological observations suggested close relationships between noreactivity in the eluate, with two different antibodies, indi- the biosynthetic pathWays of both compounds (for a discussion, cated the presence of high M, forms of neurophysin (apparent see ref. 8), this hypothesis was never proved or supported by any sizes, _70,000 and 20,000-25,000, respectively) besides the Mr direct chemical evidence. Pulse-chase experiments in the rat, 10,000 neurophysin. [8-Arginine]vasopressin-like immuno- reactivity was also detected, coeluting with the neurophysin-like together with the use of the vasopressin-deficient Brattleboro species, in the material recovered in the exclusion and Mr species (9, 10), provided further biosynthetic arguments in favor 20,000-25,000 elution volumes of the same molecular sieve of the biogenesis of neurophysins via 20,000-dalton precursor fractionation of neurohypophyseal extracts. Upon subsequent molecules. Immunochemical and biochemical analysis of mouse Sephadex G-150 filtration, the immunoreactive material re- and bovine hypothalamic extracts revealed the presence of high covered in the exclusion volume of the Sephadex G-75 filtration showed an apparent Mr of approximately 140,000. Both neu- molecular weight neurophysin-like and [8-arginine]vasopressin rophysin-like and vasopressin-ike immunoreactivities coeluted (AVP)-like species (11-13) with Mrs of _30,000 and t17,000, in the same volume. The elution profile of this Mr 140,000 ma- respectively. These forms were interpreted as precursors of the terial was unmodified when reanalyzed by the same molecular neurosecretory components. sieve filtration after exposure to 8 M urea. When these Mr Analysis of the translation products of hypothalamic mRNA, 140,000 immunoreactive forms of vasopressin and neurophysin were submitted to affinity chromatography on anti-neurophysin immunoprecipitated with anti-neurophysin antisera, indicated antibodies immobilized on Sepharose, both immunoreactivities the synthesis of neurophysin-like material with Mr estimated were selectively coadsorbed to the immunoadsorbent. Similarly, as 20,000-25,000 (14, 15) and 17,000 (16) in the case of bovine the neurophysin and vasopressin immunoreactivities associated and rat or mouse, respectively. Because similar higher molec- with Mr t25,000 were retained together on the same anti-neu- ular weight immunoreactive forms could also be detected in rophysin immunoadsorbent. The Mr 140,000 and Mr 25,000 the species having both neurophysin and [8-arginineivasopressin lysate of neurosecretory granules purified from bovine antigenic determinants generated the two neurosecretory neurohypophysis (ref. 12; unpublished data), preparation of components when exposed to proteolytic activities. This in vitro these putative pro-hormones was undertaken starting from processing was inhibited in acid medium, at low temperature, acetone-dried bovine pituitaries. and in the presence of a mixture of protease inhibitors. It is In the present report we show that two large forms (Mr concluded that these two large forms of proteins containing both 140,000 and 25,000) can be identified by successive molecular neurophysin and vasopressin may represent common biosynthetic precursors of these two neurohypophyseal compo- sieve fractionations and immunoadsorption steps. These large nents. species react with both anti-neurophysin and anti-vasopressin antibodies and, by proteolysis, can yield both neurophysin and The neurohypophyseal nonapeptide hormones ocytocin and AVP. They may represent common biosynthetic precursors of vasopressin are synthesized in the magnocellular system of the the two neurohypophyseal components. supraoptic and paraventricular nuclei of the hypothalamus. Their associated protein components, the neurophysins (for EXPERIMENTAL recent reviews, see refs. 1-4), are produced together with these PROCEDURE peptides in the perikarya of the specialized neurons. Under Materials. Bovine serum albumin, phenylmethylsulfonyl normal physiological conditions, the neurosecretory compounds fluoride (PhMeSO2F), Trasylol (aprotinin), and chloroquine are axonally transported via membrane-limited granules to the were purchased from Sigma. Human serum albumin was from nerve endings situated in the posterior hypophysis where they Behring (Marburg, W. Germany), and the soybean trypsin in- are either stored or secreted. In 1964, Sachs and Takabatake (5, hibitor was from Calbiochem. Urea (recrystalized from ethanol, 6) reported in vivo/in vitro evidence that vasopressin might stored in the dark, and known to be free of cyanate), spectro- be synthesized in the guinea pig, or dog, hypothalamo-neu- scopic-grade ethanol, and analytical grade ether, acetone, and rohypophyseal tract as an inactive precursor that is later pro- N-benzoyl-DL-arginine 4-nitroanilide hydrochloride were from cessed into the biologically active hormone. The hypothesis was Merck. Sephadex G-75 and G-150 and Sephacryl S-300 were subsequently made (7) that neurophysin might also be part of from Pharmacia. AVP was a generous gift from Ferring Lab- oratories (Malms, Sweden). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: PheMeSO2F, phenylmethylsulfonyl fluoride; AVP, vertisement" in accordance with 18 U. S. C. §1734 solely to indicate [8-arginine]vasopressin. this fact. * Deceased. 2587 Downloaded by guest on September 27, 2021 2588 Biochemistry: Nicolas et al. Proc. Natl. Acad. Sc. USA 77 (1980) .Frationation of Bovine Neurohypophysis Extracts. All reactivity was not due to either degradation of the labeled tracer oertqons were run at 4VC unless otherwise indicated. Bovine or to its sticking to a protein component (13). Postertor hypophysis was collected immediately after sacrifice Affinity Chromatography by Immunoadsorption. The and tr4psported in cold peroxide-free acetone. The solvent was immunoadsorbent was prepared with 160 mg of SI1O3 previ- cPiigehl three times, and the vacuum-dried glands were ground ously precipitated by ammonium sulfate and covalently witt Sorvall Omnimixer. To 1 g of this acetone powder was crosslinked to CH-activated Sepharose 4B (2 g) by the proce- iIdded 10 ml of solvent (40% ethanol/0.2 M formic acid/5 mM dure recommended by the manufacturer (Pharmacia). The t~ihieO2F) and the mixture was stirred for 6 hr; then, the samples were adsorbed at 4VC for 1 hr in 0.1 M sodium phos- ilti~t~ was centrifuged for 15 min at 5000 X g. This extraction phate, pH 7.8/0.1 M NaCl containing 0.1 mg of human serum w.as.,reeated once on the pellet by overnight contact with 10 albumin per ml and a mixture of protease inhibitors (pepstatin ml ofsolvent. The pooled supernatants (20 ml) were made 82% at 1 mg/liter; 130 kallikrein inhibitor units of aprotinin per ml). ineUhtiol by slow addition of 100% ethanol at -20'C, stirred This was followed by washing the complex with 0.01 M sodium 30 mii, and then let stand overnight. The precipitate was phosphate buffer at the same pH and containing the same in- cenifiuged 30 min at 20,000 X g, and the pellet was washed hibitors. Desorption of the immunoadsorbed material was withi 400% ethanol and with ether and then vacuum-dried. Use achieved with 1 M acetic acid. of '1-labeled neurophysin as internal marker indicated that In all experiments iodinated neurophysin tracer was added 196% pf the 125I radioactivity remained in the supernatant as internal marker. Under these conditions, _90% of the total durinithe ethanol precipitation step. The pellet was redissolved neurophysin radioactivity was retained on the column. Controls irin .2 M formic acid/8 M urea/0.5 mM chloroquine/1 mM indicated that <1.5% of 125I-AVP and ;5% of AVP standard P "e§02F containing 50 Mig of soybean trypsin inhibitor per were adsorbed on the column. Measurements with a synthetic HI Aaad applied to a Sephadex G-75 column equilibrated in 0.2 substrate (benzoylarginine nitroanilide) indicated that both the M tofitic acid/4 M urea. The columns were calibrated with the antiserum (SIIO3), before or after ammonium sulfate precipi- follovking ' I-labeled markers: immunoglobulin (150,000), tation, and the immunoadsorbent, exhibited discrete but ovalbdnin (45,000), human growth hormone (22,000), and measurable proteolytic activity. 125I labeling of the bovine b6vi4e neurophysin (10,000). The molecular sizes are given neurophysin II, or of other protein materials, was

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