Evidence for the presence of pro-oxytocin/neurophysin-converting enzyme in the human ovary.

The activity of pro-oxytocin/neurophysin (pro-OT/Np)-processing enzymes was determined in human granulosa cells, follicular fluids and purified secretory granules of corpora lutea. We detected the presence of an endoprotease which releases OT-Gly10-Lys11-Arg12 on cleavage of the synthetic pro-OT/Np(1-20) peptide after the dibasic Lys11-Arg12 doublet. This endoprotease was inhibited by EDTA, but was not affected by phenylmethanesulfonyl fluoride and pepstatin. Enzymatic activity was markedly reduced by replacement of L-Arg by D-Arg in the basic amino acid doublet of the substrate. The molecular weight of this enzyme was estimated to be 60 kDa. These features closely resembled those of the endoprotease identified in the bovine pituitary. The endoprotease is a metalloenzyme, specific for the Lys-Arg doublet. We also detected a carboxypeptidasic activity, inhibited by guanidinoethane-mercaptosuccinic acid. In the light of our previous detection of the processing intermediates, OT-Gly-Lys-Arg, OT-Gly-Lys and OT-Gly, in human ovary, these observations are in favour of a pro-OT/Np-processing pathway in the human ovary comparable with that in the bovine ovary. Moreover, these results confirm that oxytocin post-translational processing occurs in the human ovary and strongly suggest that pro-OT/Np is synthesized locally.

Synthesis and processing of pro-ocytocin in bovine corpus luteum and granulosa cells.

Bovine corpus luteum is the site of intense production of pro-ocytocin-neurophysin mRNA at day 1 after estrus (Ivell et al. (1985) FEBS Lett. 190, 263-267) which is followed by apparent delayed production of ocytocin. Therefore it is a good model to study both the translational and post-translational production of this neuropeptide in non-hypothalamic tissues and its regulation. In order to assess if this mRNA is translated during the lag period we have analyzed the neurophysin-like species produced in this organ. As early as day 2 after estrus one neurophysin species (pI approximately 4.7) could be detected and was unequivocally identified as pro-ocytocin-neurophysin. In primary cultures of luteinizing granulosa cells, biosynthetic intermediates were characterized, i.e. ocytocin-Gly, ocytocin-Gly-Lys and ocytocin-Gly-Lys-Arg, whereas amidated, fully mature, ocytocin was undetectable. We conclude that translation of pro-ocytocin-neurophysin mRNA takes place soon after transcription and we propose that incomplete processing could be responsible for the low level of ocytocin in the early bovine corpus luteum.

C-terminally extended ocytocin and pro-ocytocin: neurophysin peptide converting enzyme in bovine corpus luteum.

Purified secretory granules from the corpus luteum of super ovulated and fecundated cows, at day 7-8 after the heat period, were used as a source of pro-ocytocin/neurophysin (pro OT/Np) processing enzymes. An endoprotease comparable to the previously described pituitary enzyme both by its catalytic properties and sensitivity to various inhibitors was characterized. This protease cleaves pro OT/Np (1-20) after the basic Lys11Arg12 doublet to release OT Gly10 Lys11 Arg12. Moreover C-terminally extended ocytocin, i.e. OTGlyLys and OTGlyLysArg together with ocytocin were identified in extracts from the corpus luteum. Together these data argue strongly in favor of pro OT/Np processing pathways in which cleavage of the precursor at the Arg12-Ala13 peptide bond is the primary event.

In vivo synthesis and processing of rat hypothalamic prosomatostatin.

The in vivo incorporation of [3H]phenylalanine into an apparent 15 kDa prosomatostatin was observed in the hypothalamus of rats injected with the labeled amino acid in the third ventricle. Precursor-product relationships were established between this newly synthesized material and both somatostatin-28 and -14.

An endopeptidase associated with bovine neurohypophysis secretory granules cleaves pro-ocytocin/neurophysin peptide at paired basic residues.

The octacosapeptide sequence [Tyr18] pro-ocytocin/neurophysin (1-18)NH2 [pro-OT/Np(1-18)NH2] was synthesized and used as substrate to detect endoprotease(s) possibly involved in the processing of this precursor in bovine hypothalamo-neurohypophyseal tract. An endopeptidase (58 Kda) was detected in Lysates made from highly purified neurosecretory granules. This protease which cleaves the peptide bond on the carboxyl side of the Lys-Arg doublet, and no single basic residue, generates both OT-Gly10-Lys11-Arg12+Ala13-Val-Leu-Asp-Leu-Tyr18 (NH2) from the octacosapeptide substrate. In addition, a carboxypeptidase B-like activity converting OT-Gly10-Lys11-Arg12 into OT-Gly10 was detected in the same granule Lysates. It is hypothesized that a combination of these endoprotease and carboxypeptidase B-like activities together with the amidating enzyme of secretory granules might participate in the cleavage and processing of pro-OT/Np in vivo.

[Study of the peptide hormones of the hypothalamo-postpituitary system of the dromedary, Camelus dromedarius, (Camelidae, Mammalia) of Tunisia]. / Contribution à l'étude des hormones péptidiques du système hypothalamo-post-hypophysaire du dromadaire, Camel us dromedarius, (Camelidae, Mammalia) de Tunisie.

Ocytocin and vasopressin are two nonapeptides of the hypo thalamo-Post-hypophysary system. They are isolated and purified from dromedary post-hyphophysis, freshly collected in the meat centers of the south of Tunisia. The use of three chromatographies steps were found to be essential in obtaining high Pure hormones. These steps cousist in a sephadex G25 gel filtration and two successive High performance liquid chromatographies: H.P.L.C. The analysis of the two hormones by thin layer chromatography and Radioimmunoassays, and the identification of the amino-acid composition demonstrate that camel vasopressin is a 8 arginin vasopressin but ocytocin is identic to the other hormones isolated from different animal species.

Hypothalamic biosynthesis and transport of neurophysins and their precursors to the rat brain stem.

Twenty-four hours after the injection of [35S]cysteine near either the rat paraventricular nuclei or the supraoptic nuclei, the [35S]neurophysin-like proteins of the brain stem were extracted, immunoprecipitated with anti-bovine neurophysins antibodies and analyzed. They consisted essentially of species behaving as neurophysin on sodium dodecyl sulfate--polyacrylamide gel electrophoresis. There was a very low percentage of neurophysins precursors which could be characterized in the paraventricular nuclei. In the rats pretreated by colchicine, the [35S]neurophysins were not detected in the brain stem, while they appeared in the paraventricular nuclei indicating that the precursors have been processed and the transport inhibited. These results suggest that: (i) both the biosynthetic and transport events in the hypothalamo-brain stem pathway are comparable to those occurring in the hypothalamo-neurohypophyseal tract; (ii) this pathway originates both from the supraoptic and paraventricular nuclei. Moreover, they indicate that processing is essentially complete in the hypothalamus of colchicine-pretreated animals. This provides further support to a model associating enzymes with both the endoplasmic reticulum membranes and the derived corresponding secretory vesicles.

The Mr 80,000 common forms of neurophysin and vasopressin from bovine neurohypophysis have corticotropin- and beta-endorphin-like sequences and liberate by proteolysis biologically active corticotropin.

We have tested the hypothesis that the high M(r) forms common to both neurophysin and vasopressin detected in bovine neurohypophysis extracts (Nicolas, P., Camier, M., Lauber, M., Masse, M.-J. O., Möhring, J. & Cohen, P. (1980) Proc. Natl. Acad. Sci. USA 77, 2587-2591) might also contain the sequences of other known neuropeptides. The following evidence indicates that corticotropin- and beta-endorphin-like sequences are associated with similar high M(r) forms and are included in these M(r) 80,000 molecules. During the fractionation steps of high M(r) material, both corticotropin and beta-endorphin immunoreactive species were found to coelute with the neurophysin and vasopressin ones, either under M(r) 140,000 (in 0.1 M formic acid) or M(r) 70,000-80,000 (in 6 M guanidine) elution volumes. Corticotropin immunoreactivity was found to cofocus at pIs 6.05 and 5.8 with the M(r) 80,000 neurophysin-containing species. This material was submitted to affinity chromatography on purified anti-neurophysin antibodies covalently attached to Sepharose 4B. Both the corticotropin and beta-endorphin immunoreactivities, together with the neurophysin and vasopressin immunoreactivities, were retained on the immunoadsorbent and codesorbed by either a drastic pH change or by selective displacement with an excess of neurophysin. Comparison of the tryptic-digest maps of either the M(r) 68,000 fragment immunoprecipitated by anti-corticotropin antibodies or the M(r) 68,000 fragment released after precipitation of the M(r) 80,000 species by anti-neurophysin antibodies indicated large sequence homologies. Exposure of either the M(r) 80,000 or 68,000 components to mild proteolytic activities resulted in the formation of lower-size fragments. The resulting corticotropin-like immunoreactive material, recovered under the elution volume of standard (125)I-labeled corticotropin-(1-24), was tested for its ability to activate glucocorticoid biogenesis by the amphibian interrenal tissue (adrenal) in perifusion. It was found to exhibit a noticeable activity qualitatively undistinguishable from the one of the reference human corticotropin-(1-39). The name neurohypophyseal "coenophorin" (from the Greek word for common) is proposed for this class of M(r) 80,000 polypeptides that might represent the common precursor store-house for a set of neuropeptides produced in the hypothalamo-neurohypophyseal tract.

[Biosynthesis of some hypothalamic neuropeptides. Identification of precursor forms (author's transl)]. / Mécanismes de biosynthèse de quelque neuropeptides hypothalamiques. Identification de formes précurseurs.

The current knowledge on the biosynthetic mechanisms of three hypothalamic neuropeptides, neurophysins, vasopressin and somatostatin, is reviewed. Both neurophysin and vasopressin appear to be first synthesized as higher molecular weight precursors. The methodology elaborated allows to characterize essentially two main forms with apparent Mr similar to or approximately 25 000 and 80 000 respectively. It can be demonstrated that both neurophysin and vasopressin are derived from common precursors.

Immunological identification of high molecular weight forms common to bovine neurophysin and vasopressin.

Extracts of bovine neurohypophysis made in acid/ethanol solution containing protease inhibitors were fractionated by two successive filtrations on Sephadex G-75 columns equilibrated in the presence and then in the absence of 4 M urea. Analysis of the pattern of neurophysin-like immunoreactivity in the eluate, with two different antibodies, indicated the presence of high M(r) forms of neurophysin (apparent sizes, [unk]70,000 and 20,000-25,000, respectively) besides the M(r) 10,000 neurophysin. [8-Arginine]vasopressin-like immunoreactivity was also detected, coeluting with the neurophysin-like species, in the material recovered in the exclusion and M(r) 20,000-25,000 elution volumes of the same molecular sieve fractionation of neurohypophyseal extracts. Upon subsequent Sephadex G-150 filtration, the immunoreactive material recovered in the exclusion volume of the Sephadex G-75 filtration showed an apparent M(r) of approximately 140,000. Both neurophysin-like and vasopressin-like immunoreactivities coeluted in the same volume. The elution profile of this M(r) 140,000 material was unmodified when reanalyzed by the same molecular sieve filtration after exposure to 8 M urea. When these M(r) 140,000 immunoreactive forms of vasopressin and neurophysin were submitted to affinity chromatography on anti-neurophysin antibodies immobilized on Sepharose, both immunoreactivities were selectively coadsorbed to the immunoadsorbent. Similarly, the neurophysin and vasopressin immunoreactivities associated with M(r) approximately 25,000 were retained together on the same anti-neurophysin immunoadsorbent. The M(r) 140,000 and M(r) 25,000 species having both neurophysin and [8-arginine]vasopressin antigenic determinants generated the two neurosecretory components when exposed to proteolytic activities. This in vitro processing was inhibited in acid medium, at low temperature, and in the presence of a mixture of protease inhibitors. It is concluded that these two large forms of proteins containing both neurophysin and vasopressin may represent common biosynthetic precursors of these two neurohypophyseal components.