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 (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 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- 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 containing both 140,000 and 25,000) can be identified by successive molecular neurophysin and vasopressin may represent common biosyn- thetic 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 . Their associated 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 . 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 performed I15t '25I-Labeled neurophysin was always added as internal by the lactoperoxidase technique. Polyacrylamide gel elec- trfkker, and the entire corresponding radioactivity was eluted trophoresis was conducted on 15% acrylamide slab gels in the in tikr 10,000 volume. presence of 0.1% NaDodSO4 in 0.1 M Tris-HCl buffer (pH 8.6) Radioimmunoassays. All fractions from molecular sieve on 125I-labeled samples previously boiled in the detergent in filtration and affinity chromatography were tested for neuro- the absence or presence of a 1000-fold molar excess of di- physinand vasopressin immunoreactivities by a radioimmu- thiothreitol. At the end of the run, the gels were cut in 2-mm noassay procedure using various anti-neurophysin antisera slices for measurement of radioactivity. (S1Is and SI1O3 prepared in this laboratory and A5IV from J J. Legros, Liege, Belgium) (17) and one anti-AVP antiserum RESULTS (183. II-Labeled bovine neurophysin II and 125I-labeled AVP The radioimmunoassayable neurophysin activities in the eluate (125'7-AVP) were used as tracers. of the Sephadex G-75 molecular sieve fractionation (in 4 M 'The neurophysin radioimmunoassay was conducted in 0.5 urea) of neurohypophyseal extracts included materials larger M Tris-HCl buffer (pH 7.5) containing a protease inhibitor (130 than the Mr 10,000 neurophysin. Significant amounts of im- kallikrein inhibitor units of Trasylol per ml) and 3 mg of bovine munoreactive compounds with apparent Mr _45,000 (exclusion serum albumin per ml at 4VC for 36-48 hr. Separation of the volume of the Sephadex G-75 gel in 4 M urea) and 20,000- bound antibody from the free tracer was by precipitation with 25,000, together with the Mr 10,000 neurophysin, were oh. zirconyl phosphate at pH 5.5 (11). The radioactivity was assayed served (Fig. 1A). The elution profile of each peak of immu- on a Intertechnique (CG-2000) gamma counter. Under these noreactive material recovered in the first fractionation was not conditions, with a 1:4200 final dilution, both SII03 and SIlO4 affected by treatment with 8 M urea for 24 hr, in the cold or for gave a minimal detection of 1 ng and a 50% displacement of 4 hr at 60°C (Fig. 1B). Vasopressin-like immunoreactive ma- the tracer by 5 ng of bovine neurophysin II. The A5IV anti- terials coeluted with the neurophysin-like species both in the neurophysin was used at a dilution of 1:15,000 and gave a exclusion (Mr _70,000) and in the Mr 20,000 elution volumes minimal detection of 0.10 ng and a 50% displacement of tracer of the fractionation on Sephadex G-75 in the absence of urea by 0.7 ng of bovine neurophysin II. The SIlO3 and SII04 anti- (Fig. 1B). Antigenic similarities with the reference bovine era exhibited <5% crossreactivity with bovine neurophysin neurophysin on the one hand and with standard AVP on the I; the A5IV was not species specific (17). other were unambiguously established by competition exper- The vasopressin radioimmunoassay was performed as de- iments in the radioimmunoassay (Fig. 2). The two sets of ob- spribed (13, 18), using antiserum Bert at 1:45,000 final dilution. servations demonstrate that the higher Mr neurophysin-like and This allowed a minimal detection of 1-2 pg of AVP with 2-3 vasopressin-like forms detected in the acid/ethanol extracts of pg of--25I-AVP as tracer (specific activity, 1600-1800 mCi/mg; neurohypophysis are neither aggregates of neurophysin or 1 Ci = 3.7 X 10'0 becquerels). Under these conditions, 15% and vasopressin nor immunoreactive molecules noncovalently at- 0.3% crossreactivity with [Lys8]vasopressin and ocytocin, re- tached to other protein components. spectively, was observed. Crossreactivity with neurophysin was The size of the immunoreactive species recovered in the void <0.05%. Binding of both the labeled and the unlabeled AVP volume (vo, Fig. 1B) of the Sephadex G-75 fractionation was on the Bert antiserum were identical, and a single apparent assessed by further filtration on either Sephadex G-150 or equilibrium association constant, 0.77 X 101" M'1, was calcu- Sephacryl S-300. This material was found to elute in a volume lated both from Scatchard and Lineweaver-Burk plots. This indicative of a Mr of -140,000 (Fig. IC). Here again, after demonstrates that antiserum Bert possesses a homogeneous exposure of the Mr 140,000 species to 8 M urea followed by population of AVP binding sites. In both neurophysin and va- filtration on either molecular sieve (G-150 or Sephacryl S-300), sopressin radioimmunoassays, controls were systematically the elution patterns of both neurophysin and vasopressin im- performed in order to establish that the measured immuno- munoreactivities were unmodified and remained undissociable Downloaded by guest on September 27, 2021 Biochemistry: Nicolas et al. Proc. Natl. Acad. Sci. USA 77 (1980) 2589 Neurophysin, ng AVP, pg 0.39 6.25 100

6

4

2

-A 0 ZP ._ (A 2 100 10 1 256 64 16 4 1 0 Reciprocal of dilution factor FIG. 2. Immunological characterization of the higher Mr mate- a) rials recovered in the Sephadex G-75 fractionation (experiments of '0 0 Fig. 1 B and C) and of the neurophysin-like and vasopressin-like a_ -o x components generated by processing of the Mr 140,000 species (from Fig. 3B). (A) Cross-reactivity of reference neurophysin (0) with the Mr 25,000 (-) or Mr 140,000 (-) species recovered from Fig. 1 B or c)5 .0 C experiments and the neurophysin-like product (Mr 10,000) (A) a .n generated in the Fig. 3B experiment. (B) Cross-reactivity of the ref- erence AVP (0) with the Mr 25,000 (0) or Mr 140,000 (-) species recovered from Fig. 1 B or C experiments and the vasopressin-like product (Mr 1000) (A) generated in the Fig. 3B experiment. (Fig. 3A). Several control experiments were conducted in order to verify this large apparent Mr.§ Extraction of the neurohy- pophysis was conducted under various conditions with aqueous solutions in the absence of organic solvents. Both the Mr 140,000 species and the intermediary-size materials were observed after Sephadex G-150 filtration as in the case of the acid/alcohol extracts (not shown). Also, 125I-labeled neurophysin was added to the starting material of the routine acid/alcohol extraction steps as an internal marker. Less than 0.005% of the radioac- tivity was recovered in the Mr 140,000 elution volume of the gel fractionation. Finally, analysis of lysates obtained by osmotic shock of neurosecretory granules purified from the neurohy- Fractions pophysis confirmed the presence of high Mr neurophysin-like species comparable to those observed under more drastic ex- FIG. 1. Fractionation, by molecular sieve filtration, of higher Mr traction conditions (unpublished data). forms of neurophysin and vasopressin from neurohypophysis extracts. Demonstration that both the AVP and neurophysin antigenic (A) Extracts were fractionated on a Sephadex G-75 column (150 X determinants are part of the same molecule was achieved by 1.2 cm) in 0.2 M formic acid/4 M urea. Elution (3.5 ml/hr) of the a series of experiments. First, when applied on a covalent neurophysin-like material was monitored by radioimmunoassay (0) conjugate of antineurophysin antibodies to CH-Sepharose 4B, on an aliquot of each 1.3 ml fraction. 125I-Labeled neurophysin the Mr 140,000 immunoreactive material was found to be (- * - - -) was added as internal marker. (B) Filtration on a Sephadex G-75 column (95 X 1.5 cm) in 0.2 M formic acid containing 1 mg of quantitatively adsorbed but the standard 12'I-labeled or unla- pepstatin per liter (flow rate, 3.5 ml/hr) of each type of higher Mr form beled AVP was not (Fig. 4). The adsorption-desorption profile recovered in A, after 24-hr exposure to 8 M urea. Each fraction (1.1 indicated unequivocally that both AVP and neurophysin im- ml) was tested both for its neurophysin (0 or *) and vasopressin (O munoreactivities were selectively retained on the immunoad- or o) immunoreactivities. 1251-Labeled neurophysin (- -- -) was sorbent and then desorbed a drastic Second, the added as internal marker. (C) Filtration on a Sephadex G-150 column by pH change. (95 X 1.5 cm) in 0.2 M formic acid containing 1 mg of pepstatin per material recovered after irrigation of the affinity column with liter and 130 kallikrein inhibitor units of Trasylol per ml (flow rate, 1 M acetic acid was found to behave as Mr 140,000 immuno- 2.5 ml/hr) of the larger higher Mr species recovered from either A or reactive species sharing both AVP and neurophysin determi- B experiment. Both neurophysin (-) and vasopressin (0) immuno- nants when refiltered, after exposure to 8 M urea, on a Sephadex reactivities were measured on each 1.2-ml fraction. - - - - -, Elution G-150 (Fig. 4, Inset) or else on a Sephacryl S-300 column (not profile of 1251-labeled neurophysin internal standard. The columns were precalibrated with the following markers: a, rabbit IgG (150,000); shown). The same method applied to the intermediary size b, ovalbumin (45,000); c, monomeric human growth hormone (22,000); immunoreactive material (Mr 25,000) showed that these species d, 125I-labeled bovine neurophysin II; e, human serum albumin (68,000). The recovery was 85% of the total immunoreactivity applied § This apparent Mr was evaluated by reference to the behavior of rabbit on the columns. The Mr 140,000 and 25,000 immunoreactive peaks IgG. Since these immunoreactive species may carry carbohydrate represented about 0.6 and 4%, respectively, of the total amount of moieties (11, 13), this evaluation may be largely impaired and should neurophysin recovered by the usual extraction procedure. be taken as tentative. Downloaded by guest on September 27, 2021 2590 Biochemistry: Nicolas et al. Proc. Natl. Acad. Sci. USA 77 (1980)

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la0 v) 0 PQ Fraction FIG. 4. Affinity chromatography (immunoadsorption) of the neurophysin and vasopressin immunoreactivities associated with the Mr 140,000 species. Step A. The component recovered from the Sephadex G-150 column (Fig. 1C) was adsorbed together with 1251_ labeled neurophysin on the affinity column (6 ml) for 1 hr at 40C in 0.1 M sodium-phosphate buffer (pH 7.5) containing 1 mg of pepstatin per liter and 130 kallikrein inhibitor units of Trasylol per ml. The complex was washed with the same buffer solution in the cold. Step B. Desorption was by 1 M acetic acid. Each 1-ml fraction was lyoph- ilized and then subjected to radioimmunoassay for neurophysin (0) 40 60 and vasopressin (0) activities. The elution profiles of 125I-labeled Fraction neurophysin (- - - - -) and 1251-AVP (- - - -) were monitored. The Elution profile of FIG. 3. Processing of the higher Mr neurophysin-like and vaso- recovery of immunoreactivity was _75%. (Inset): coad- pressin-like form (Mr 140,000). (A) Molecular sieve filtration, on the the neurophysin (0) and vasopressin (0) immunoreactivities same G-150 column as in Fig. 1C, of the starting material sorbed on the affinity column (see above) and subsequently applied Sephadex the recovered from Fig. 1C experiment after exposure to 8 M urea for 24 on a Sephadex G-150 column under same experimental conditions as in Fg. 1C. d, Elution volume of 125I-labeled neurophysin. hr. - - - -, 1251-AVP standard, - - -, 125I-labeled neurophysin standard. (B) Same filtration as in A of the compounds obtained after exposure of the Mr 140,000 species to rabbit antiserum immobilized of products obtained by proteolytic conversion of the Mr on Sepharose-4B (The reaction was for 30 hr in 0.1 M Na phosphate 140,000 species were labeled with 125I and then fractionated buffer at pH 7.9 in the absence of protease inhibitors and at 300C). on a Sephadex G-75 column (not shown). The immunoreactive The neurophysin (0) and vasopressin (0) immunoreactivities were materials in each fraction were precipitated with a double- determined on aliquots of each 1.36-ml fraction. d and f, Peaks of antibody technique using anti-bovine neurophysin antibodies elution of the 1251-labeled neurophysin and 1251-AVP standards. and then goat anti-rabbit immunoglobulins. The immu- noprecipitated materials were analyzed by polyacrylamide gel also share the antigenic determinants of both the nonapeptide electrophoresis on calibrated gels (15% acrylamide) in 0.1% hormone and its associated protein, neurophysin. NaDodSO4 before and after reduction of the disulfide links with Finally, when exposed to proteolytic activities, both the large a large excess of dithiothreitol. The '25I label in both cases was (Mr 140,000) and intermediate-size (Mr 25,000) materials were found associated with species of apparent Mr 140,000, 60,000, found to generate components immunologically related to, or 25,000, and, mainly, 10,000, a pattern equivalent to the dis- identified as, neurophysin and vasopressin. Preliminary ob- tribution of immunoreactive forms observed in the Sephadex servations suggested that, during contact of the high Mr species G-150 fractionation (Fig. 3B). with the immunoadsorbent in the absence of protease inhibitors The Mr 10,000 neurophysin-like component generated by at neutral pH, significant amounts of smaller immunoreactive processing of the Mr 140,000 form (from Fig. 3B) was further components were generated. This proteolysis, which was identified by its ability to compete with standard 125I-labeled minimized by acid or the presence of serine protease inhibitors, neurophysin for binding to the antibodies (Fig. 2A). Similarly, might be due to the serum proteases as indicated by controls the smaller vasopressin-like peak recovered after Sephadex performed with a synthetic substrate. Upon exposure of the Mr G-150 fractionation of the products of processing (from Fig. 140,000 species to antiserum immobilized on Sepharose-4B at 3B) was found to compete with the standard 125I-AVP for neutral pH and at 300C, proteolysis of the larger molecule was binding to the anti-AVP antibodies (Fig. 2B). intermediate-size neurophysin-like observed (Fig. 3B). Some DISCUSSION components were generated together with a large proportion of Mr 10,000 immunoreactive species. Also, vasopressin-like An increasing number of examples indicate that the biosyn- components were apparent, with elution profiles suggesting that thetic pathways of secretory peptides or proteins, including intermediate-size vasopressin-like fragments were produced hormonal components, involve the posttranslational cleavage together with the hormone. of larger precursor molecules. This seems to apply to different Further analysis of these materials on Sephadex G-50 col- hormones such as insulin (19), nerve growth factor (20), epi- umns indicated the heterogeneity of the immunoreactive dermal growth factor (21), adrenocorticotropin, melanotropin components and the presence of a Mr -8,000 vasopressin-like (22-24), parathyrin (25), and somatostatin (26). In only one case, species with no neurophysin immunoreactivity. The mixture that of pro-opiocortin, was the prohormone demonstrated to Downloaded by guest on September 27, 2021 Biochemistry: Nicolas et al. Proc. Natl. Acad. Sci. USA 77 (1980) 2591 be plurifunctional-i.e., to include in its structure the sequences The excellent technical assistance of Christine Fahy is appreciated. of several known peptide hormones (22-24). The present data This work was supported in part by grants from the Universite Pierre provide immunological and biochemical evidence for the et Marie Curie, Centre National de la Recherche Scientifique (Equipe presence of two types of higher molecular weight immuno- de Recherches Associee 693), the Fondation pour la Recherche MMl- icale Francaise, and the Delegation a la Recherche Scientifique et reactive forms of both neurophysin and vasopressin in the Technique (contract 79-7-0788). The support of the Departement de storage and excretion organ of the hypothalamo-neurohypo- Biologie du Centre d'Etudes Nucleaires, Saclay, for the supply of ra- physeal tract. The demonstration that the Mr 140,000 as well diochemicals and a doctoral fellowship to M.L. is greatly appre- as the Mr 25,000 form can provide, by proteolytic cleavage, the ciated. neurosecretory components vasopressin and neurophysin strongly suggests that these forms are common biosynthetic precursors. This finding is of particular interest because the 1. Walter, R. (1975) Ann. N.Y. Acad. Sci. 248, 1-512. exact function of neurophysin has not yet been entirely defined 2. Pickering, B. T. & Jones, C. W. (1978) in Hormonal Proteins and (for a discussion, see ref. 4). The neurohypophyseal hormone- Peptides, ed. Li, C. H. (Academic, New York), Vol. 5, 103- 158. binding protein, which possesses duplicated hormones binding 3. Breslow, E. (1979) Annu. Rev. Biochem. 48,251-274. sites in connection with the internal duplication of amino acid 4. Cohen, P., Nicolas, P. & Camier, M. (1979) Curr Top. Cell. Reg. sequences, exhibits half-of-the-sites reactivity and allosteric 15,263-318. properties (4, 27). The present observations could be taken as 5. Sachs, H. & Takabatake, Y. (1964) Endocrinology 75, 943- supporting the idea that, besides its role in the intragranular 948. 6. Takabatake, Y. & Sachs, H. (1964) Endocrinology 75, 934- stabilization of the hormones ocytocin and vasopressin, neu- 942. rophysin can be considered as directly related to the biosynthesis 7. Fawcett, C. P., Powell, A. E. & Sachs, H. (1968) Endocrinology of vasopressin. 83, 1299-1310. The fact that relatively large amounts of the higher Mr forms 8. Gainer, H., Peng Loh, Y. & Same, Y. (1977) in Peptides in can be detected in the neurohypophysis [in the granules as well Neurobiology, ed. Gainer, H. (Plenum, New York), pp. 183- as in the hypothalamus (11-13)] supports the conclusion by 219. 9. Gainer, H., Same, Y. & Brownstein, M. J. (1977) Science 195, others (8, 9) that processing of the prohormones may occur 1354-1356. during axoplasmic transport. 10. Brownstein, M. J. & Gainer, H. (1977) Proc. Natl. Acad. Sci. USA The possible relationship between the Mr 140,000 and 25,000 74,4046-4049. forms of neurophysin and vasopressin is not as yet clear. The 11. Lauber, M., Camier, M. & Cohen, P. (1979) FEBS Lett. 97, intermediate-size immunoreactive form described above, or 343-347. previously reported (11, 13), can be compared with the Mr 12. Lauber, M., Camier, M., Masse, M. J. 0. & Cohen, P. (1979) Biol. 20,000 35S-pulse-labeled molecules found in the rat (8-10) or Cell. 36, 111-118. to the Mr 17,000 (16) or Mr 25,000 (14, 15) neurophysin-con- 13. Camier, M., Lauber, M., M6hring, J. & Cohen, P. (1979) Fed. Eur. Biochem. Soc. Lett. 108,369-373. taining components immunoprecipitated from the products 14. Giudice, L. C. & Chaiken, I. M. (1979) Proc. Natl. Acad. Sci. USA of cell-free translation of bovine and mouse or rat hypothalamus 76,3800-3804. mRNA.1 The observation that in vitro proteolytic cleavage of 15. Schmale, H., Leipold, B. & Richter, D. (1979) FEBS Lett. 108, the Mr 140,000 species can generate some of the intermedi- 311-316. ate-size immunoreactive components suggests that the latter 16. Lin, C., Joseph-Bravo, P., Sherman, T., Chan, L. & MacKelvy, may represent intermediates in the posttranslational events J. F. (1979) Biochem. Biophys. Res. Commun. 89,943-950. leading from the large precursors to the "final" secretory 17. Legros, J. J. & Franchimont, P. (1974) Ann. Endocrinol. (Paris) compounds. However, the possibility that the two large im- 35, 189-194. munoreactive forms are the products of expression of two dis- 18. Mohring, B. & Mohring, J. (1975) Life Sci. 17, 1307-1314. 19. Steiner, D. F. & Oyer, P. E. (1967) Proc. Natl. Acad. Sci. USA tinct genes must be considered. Alternatively, they might cor- 57,473-480. respond to a different translation of the same polycistronic 20. Berger, E. A. & Shooter, E. M. (1977) Proc. Natl. Acad. Sci. USA mRNA. 74,3647-3651. At the present time the biological significance of such a large 21. Frey, P., Forand, R., Maciag, T. & Shooter, E. M. (1979) Proc. molecule is not clear. It may represent the redundant form of Natl. Acad. Sci. USA 76,6294-6298. a smaller basic module containing the sequences of neuro- 22. Mains, R. E., Eipper, B. A. & Ling, N. (1977) Proc. Natl. Acad. physins I and II, vasopressin, and possibly ocytocin. The alter- Sci. USA 74,3014-3018. native possibility that it might include in its structure the se- 23. Roberts, J. L. & Herbert, E. (1977) Proc. Natl. Acad. Sci. USA quences of a number of other hormonal peptides remains open. 74,5300-5304. Large immunoreactive forms of several pituitary factors in- 24. Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A. C. Y., Cohen, S. N. & Numa, S. (1979) Nature (London) 278, cluding luteotropin (28), thyrotropin (29), growth hormone (30), 423-427. and folliculotropin (31) have been reported. 25. Kemper, B., Habener, J. F., Potts, J. T. & Rich, A. (1972) Proc. Natl. Acad. Sci. USA 69,643-647. 26. Lauber, M., Camier, M. & Cohen, P. (1979) Proc. Natl. Acad. Sci. Note Added in Proof. After this manuscript was submitted, others USA 76, 6004-6008. reported (32) that trypsin liberates neurophysin and "vasopressin" from 27. Nicolas, P., Camier, M., Dessen, P. & Cohen, P. (1976) J. Biol. a 20,000- to 25,000-daltons material pulse-labeled with [35S]cysteine. Chem. 251, 3965-3971. Although this observation may be taken as consistent with our reported 28. Liu, T. C., Ax, R. L. & Jackson, G. L. (1979) Endocrinology 105, Mr 25,000 molecules sharing both neurophysin and vasopressin anti- 10-15. genic determinants, the products of processing were not demonstrated 29. Klug, T. L. & Adelman, R. C. (1977) Biochem. Biophys. Res. to be released from a homogeneous population of molecules. Commun. 77, 1431-1437. 30. Stachura, M. E. & Frohman, L. A. (1975) Science 187, 447- 449. I Careful examination of the data reported by these authors (8,9, 16, 31. Reichert, L. E. & Ramsey, R. B. (1977) J. Clin. Endocrinol. 32) does not allow us to exclude the presence of labeled, or immu- Metab. 44,545-550. noprecipitated, species with a larger size, although their relationships 32. Russell, J. T., Brownstein, M. J. & Gainer, H. (1979) Proc. Natl: to neurophysin remain to be established. Acad. Sci. USA 76,6086-6090. Downloaded by guest on September 27, 2021