Acute effects of benzo[a]pyrene on digestive gland enzymatic biomarkers and DNA damage on mussel Mytilus galloprovincialis.

In the present study, mussel (Mytilus galloprovincialis) digestive gland biotransformation and detoxification responses to acute exposure to the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) were investigated. Mussels were exposed to a sublethal dose of B[a]P (75 nM; 19 microg/l per animal) for 24, 48 and 72h. The following biological responses were measured in the digestive gland tissues: (1) B[a]P hydroxylase (BPH) activity, as phase I biotransformation parameter; (2) glutathione S-transferase (GST) activity as a phase II conjugation enzyme, (3) catalase (CAT) activity as potential biomarker of oxidative stress, (4) acetylcholinesterase (AChE) activity as an indication of possible neurotoxicity response. DNA damage was assessed over time using the single cell gel electrophoresis comet assay and the micronuclei test. BPH and GST activities showed an increasing trend over exposure period. CAT activity showed a symmetrical bell shape response with a maximum at 48h. AChE activity was significantly depressed after 48 and 72h exposure to B[a]P. Comet assay and micronuclei test in digestive gland cells suggest that B[a]P exposure induced significant DNA damage with a maximum response after 72h exposure.

Metallothionein and metal levels in liver, gills and kidney of Sparus aurata exposed to sublethal doses of cadmium and copper.

The levels of metallothionein (MT), a biomarker of metal exposure, and of Cd and Cu, known as MT inducers, were investigated in Sparus aurata intraperitoneally injected with 500 microg/kg of Cu and Cd for 2 days. MT and metal concentrations (Cd and Cu) were determined in liver, gills and kidney. MT levels were significantly increased in all investigated tissues, with the highest value in liver of Cu as Cd-treated fishes (3.56-fold and 3.3- fold, respectively). Metal concentrations were statistically different between all tissues. Highest metal concentrations were in the liver. The higher metal concentrations and MT induction levels support the main role of MT in metal homeostasis and detoxification.

Mixture toxicity assessment of cadmium and benzo[a]pyrene in the sea worm Hediste diversicolor.

In the present study, Hediste diversicolor biotransformation and anti-oxidant responses to acute exposure to cadmium (Cd) and to the polycyclic aromatic hydrocarbons benzo[a]pyrene (B[a]P) were investigated. Worms were submitted to 0.2, 0.4 and 1 microM of each contaminant and to their mixture during a period of test of 48h. Following biological responses were measured: (1) NADPH cytochrome c reductase (NADPH cyt c) activity, as phase I biotransformation parameter; (2) gluthathione-S-transferase (GST) activity as a phase II conjugation enzyme, (3) catalase activity as anti-oxidant response and (4) malondialdehyde accumulation (MDA) as lipid peroxydation marker. The cholinergic system was evaluated using the acetylcholinesterase activity (AChE). Exposure to the mixture resulted in low dose level additive effects on the investigated biomarkers. However, worms exposed to 1 microM of the single compounds and to their mixture exhibited the highest MDA accumulation and the lowest enzymatic biomarkers activities suggesting severe toxicological effects. These data should be carefully considered in view of the biological effects of mixture pollutants and particularly in marine sediment ecosystems.

Acute effects of benzo[a]pyrene on liver phase I and II enzymes, and DNA damage on sea bream Sparus aurata.

In the present study biotransformation and detoxification responses to acute exposure to the polycyclic aromatic hydrocarbons benzo[a]pyrene (B[a]P) were investigated in the liver of Sparus aurata (sea bream). Sexually immature gilthead sea bream were treated by intraperitoneal injection of B[a]P (20 mg kg(-1)) for 6, 12, 24, and 48 h. B[a]P accumulation was quantified in sea bream liver by mean of gas phase chromatography (GPC-MS) after the various exposure periods. The following biological responses were measured: (1) ethoxyresorufin-O-deethylase (EROD) activity, as a phase I biotransformation parameter; (2) liver glutathione S-transferase (GST) activity as a phase II conjugation enzyme. DNA damage was assessed over time using the single-cell gel electrophoresis comet assay. B[a]P bioaccumulation in the liver resulted in a biphasic curve with an increasing uptake up to 5.55 +/- 0.67 microg g(-1) dry weight after only 6 h exposure and 4.67 +/- 0.68 microg g(-1) dry weight after 48 h exposure. EROD activity showed a nonsymmetrical bell-shaped kinetic with a maximum at 24 h and lower but significant activities at 12 and 48 h with respect to control animals. Hepatic GST activities were only significant after 48 h exposure. Comet assay showed an increase in liver cells DNA damage with a maximum after 48 h exposure reaching up to 12.17 % DNA in the tail.

Acute effects of cadmium on liver phase I and phase II enzymes and metallothionein accumulation on sea bream Sparus aurata.

This research was designed to study Sparus aurata (sea bream) biotransformation and detoxification responses to acute exposure to cadmium (Cd). Sexually immature gilthead sea bream were treated by intraperitoneal injection of Cd chloride (200 microg kg(-1)) for 6, 12, 24 and 48 h. Cd accumulation was quantified in sea bream liver by graphite furnace atomic absorption spectroscopy after the various exposure periods. The following biological responses were measured: (1) ethoxyresorufin-O-deethylase (EROD) activity as phase I biotransformation parameter, (2) liver glutathione-S-transferase (GST) activity as a phase II conjugation enzyme and metallothionein (MT) content as specific response to Cd contamination. Cd bioaccumulation in the liver resulted in an increasing uptake up to 10.3 microg g(-1) wet weight after 48 h of exposure. EROD showed a significant activation only after 6 h exposure and a return to control levels after 12 h. GST revealed significant activation starting from 12 h exposure. MT accumulation in liver showed the same behavior as GST activation.

Assessment of heavy metal contamination using real-time PCR analysis of mussel metallothionein mt10 and mt20 expression: a validation along the Tunisian coast.

In mussel Mytilus galloprovincialis tissues, metallothionein belongs to two different gene classes, mt10 and mt20, showing differential expression at both basal conditions and under heavy metal challenge. In this study, a new more highly sensitive technique, expression analysis of mt10 and mt20 mRNA levels by quantitative reverse transcription polymerase chain reaction, was used to assess the effects of heavy metal contamination in the digestive glands of mussels caged along the Tunisian coast. To validate the new assay, total metallothionein protein, amount of heavy metals (zinc, copper, cadmium), and a biomarker of oxidative stress such as malondialdehyde content, were assessed in the same tissues. At the investigated sites, the molecular assay showed variations of mt20 relative gene expression levels within one or two orders of magnitude, with maximum values at two sites severely polluted with cadmium, Mahres (100-fold) and Menzel Jemile (165-fold). Changes in mt10 expression were recorded at all sites where copper had significantly accumulated, although fold induction levels were less pronounced than those of mt20. In this paper, gene expression data are discussed in relation to the studied biomarkers, demonstrating that the molecular technique based on the differential expression of mt10 and mt20 genes represents (i) a useful and robust tool for studying and monitoring heavy metal pollution under field conditions, and (ii) an improvement in the application of metallothionein as a biomarker of response to exposure to heavy metals in marine mussels.

Effects of malathion and cadmium on acetylcholinesterase activity and metallothionein levels in the fish Seriola dumerilli.

The potential use of acetylcholinesterase (AChE) and metallothionein (MT) responses as biomarker of organophosphorous (OPs) and trace metal were assessed in fish Seriola dumerilli exposed to 0, 4, 6 mg/kg of malathion for 2, 7 and 13 days, and to 0, 50, 100, 250 mug/kg of Cd for 2 days. Brain AChE was significantly inhibited after 2 and 7 days of malathion exposure, in a dose-response manner, but no inhibition was observed after 13 days of exposure. When exposed to Cd for 2 days, S. dumerelli presented an increase in AChE activity at a concentration of 50 mug/kg, but a strong and dose-dependent AChE inhibition at 100 and 250 mug/kg. Cd treatment also caused a rapid increase in MTs concentration in liver, even at the lower concentration. Our experiments indicate that the measurement of hepatic MT concentration and brain AChE activity in S. dumerilli would be useful biomarkers of OP and Cd exposure and/or effects.

Monitoring pollution in Tunisian coasts: application of a classification scale based on biochemical markers.

Over the past decade, molecular, biochemical and cellular markers have been extensively used in pollution monitoring of aquatic environments. Biochemical markers have been selected among early molecular events occurring in the toxicological mechanisms of main contaminants. This paper assesses the marine environment quality along the Tunisian coasts using a statistical approach. Clams (Ruditapes decussatus) were collected during the four seasons of 2003 on seven different sites from the Tunisian coasts. Oxidative stress was evaluated in gills using catalase activity (Cat), neutral lipids and malonedialdehyde accumulation. Glutathione S-transferase activity is related to the conjugation of organic compounds and was evaluated in both, gills and digestive glands. Acetylcholinesterase activity was evaluated as the biomarker of exposure to organophosphorous, carbamate pesticides and heavy metals. For each biomarker, a discriminatory factor was calculated and a response index allocated. For each site, a global response index was calculated as the sum of the response index of each biomarker. Discriminant analysis shows significant differences between sites and seasons compared with control sample. Faroua (site 1) and Menzel Jemile (site 2) seem to be the less polluted with respect to the other sites for all seasons. Gargour (site 6) shows the highest Multimarker Pollution Index during the four seasons, indicating higher contamination level.

Role of amino acid sequences flanking dibasic cleavage sites in precursor proteolytic processing. The importance of the first residue C-terminal of the cleavage site.

The amino acid sequences flanking 352 dibasic moieties contained in 83 prohormones and pro-proteins listed in a database were examined. Frequency calculations on the occurrence of given residues at positions P6 to P'4 allowed us to delineate a number of features which might be in part responsible for the in vivo discrimination between cleaved and uncleaved dibasic sites. These include the following: amino acids at these positions were characterized by a large variability in composition and properties; no major contribution of a given precursor subsite to endoprotease specificity was observed; some amino acid residues appeared to occupy preferentially certain precursor subsites (for instance, Met in P6 and P3, Asp and Ala in P'1, Pro in P6, Gly in P3 and P'2 etc.) whereas some others appeared to be excluded. Most amino acid residues occupying the P'1 position in these precursor cleavage sites were tolerated. But the beta-carbon branched side chain residues (Thr, Val, Leu, Ile) and Pro, Cys, Met and Trp were either totally excluded or poorly represented, suggesting that they might be unfavourable to cleavage. The biological relevance of these observations to the efficacy of dibasic cleavage by model propeptide convertases was in vitro tested using both pro-ocytocin convertase and Kex2 protease action on a series of pro-ocytocin related synthetic substrates reproducing the Pro7-->Leu15 sequence of the precursor in which the Ala13 residue (P'1 in the LysArg-Ala motif) was replaced by various amino acid residues. A good correlation was obtained on this model system indicating that P'1 residue of precursor dibasic processing sites is an important feature and may play the role of anchoring motif to S'1 convertase subsite. We tentatively propose that the present database, and the corresponding model, may be used for further investigation of dibasic endoproteolytic processing of propeptides and pro-proteins.

Characterization of the thermolysin-like cleavage of biologically active peptides by Xenopus laevis peptide hormone inactivating enzyme.

Peptide hormone inactivating endopeptidase (PHIE) is a metalloendopeptidase which was isolated from the skin granular gland secretions of Xenopus laevis [Carvalho, K. M., Joudiou, C., Boussetta, H., Leseney, A. M., & Cohen, P. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 84-88]. This peptidase exhibits a thermolysin-like character and hydrolyzes bonds on the amino terminus of hydrophobic amino acids, performing cleavage of Xaa-Phe, Xaa-Leu, Xaa-Ile, Xaa-Tyr, and Xaa-Trp doublets. When the enzyme recognized a doublet of hydrophobic amino acids such as Phe6-Phe7 of somatostatin-14, Phe7-Phe8 of substance P, Phe4-Leu5 of [Leu5,Arg6]enkephalin, and Tyr4-Ile5 of angiotensin II, cleavage occurred preferentially between these residues. The use of selectively modified carboxy-terminal octapeptide fragments of atrial natriuretic factor (ANF) indicated that the enzyme tolerates as substrates only peptides bearing a P'1 bulky hydrophobic amino acid residue. Although a P'1 hydrophobic residue was a necessary condition, it was found in a number of peptides that all potential cleavage sites were not recognized by the enzyme. These data suggested that this metalloendoprotease requires for its thermolysin-like activity a preferred conformation of the peptide chain. Kinetic results obtained using a series of related substrates derived from biologically active peptides of the atrial natriuretic factor, tachykinin, and enkephalin families indicated the presence of an extended binding site accommodating at least six amino acid residues, in contrast to thermolysin (EC 3.4.24.4) and neutral endopeptidase (NEP; EC 3.4.24.11), which hydrolyze shorter homologous peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of beta-turn in proteolytic processing of peptide hormone precursors at dibasic sites.

Proteolytic activation of prohormones and proproteins occurs most frequently at the level of basic amino acids arranged in doublets. Previous predictions by Rholam et al. [Rholam, M., Nicolas, P., & Cohen, P. (1986) FEBS Lett. 207. 1-6] have indicated, on the basis of 20 prohormone sequences containing 53 dibasic potential processing sites, that dibasic sites situated in, or next to, beta-turns were cleaved in vivo, whereas sites included in ordered structures like beta-sheets or alpha-helices were not. We have used peptide analogs of the proocytocin/neurophysin processing domain and a purified preparation of the putative proocytocin convertase from bovine tissues as a model to demonstrate that (1) processing at dibasic sites is associated with a prohormone sequence organized in a beta-turn structure; (2) the beta-turn is an interchangeable motif since the original sequence could be replaced by an heterologous one possessing the ability to organize as a beta-turn; and (3) this particular secondary structure participates in the catalytic reaction, most likely by favoring the interactions of the substrate with the processing endoprotease. It is concluded that, in addition to the dibasic and other amino acids around the cleavage loci, the beta-turn constitutes a key feature in the proteolytic processing reaction in participating as the favorable conformation for optimal substrate-enzyme active site recognition.

A peptide-hormone-inactivating endopeptidase in Xenopus laevis skin secretion.

An endopeptidase was isolated from Xenopus laevis skin secretions. This enzyme, which has an apparent molecular mass of 100 kDa, performs a selective cleavage at the Xaa-Phe, Xaa-Leu, or Xaa-Ile bond (Xaa = Ser, Phe, Tyr, His, or Gly) of a number of peptide hormones, including atrial natriuretic factor, substance P, angiotensin II, bradykinin, somatostatin, neuromedins B and C, and litorin. The peptidase exhibited optimal activity at pH 7.5 and a Km in the micromolar range. No cleavage was produced in vasopressin, ocytocin, minigastrin I, and [Leu5]enkephalin, which include in their sequence an Xaa-Phe, Xaa-Leu, or Xaa-Ile motif. The endopeptidase activity was inhibited by divalent cation chelators and by phosphoramidon only at high concentrations (IC50 = 50 microM), whereas it was insensitive to classical inhibitors of chymotrypsin, angiotensin convertase, and serine and cysteine peptidases, as well as carboxypeptidases. It is hypothesized that this enzyme, which is distinct from neutral endopeptidase (EC 3.4.24.11), constitutes the prototype of a family of related metalloendopeptidases that inactivate peptide substrates by cleavage at the Xaa-Phe, Xaa-Leu, or Xaa-Ile bond.

Isolation and functional properties of an arginine-selective endoprotease from rat intestinal mucosa. A putative prosomatostatin convertase.

The endoproteolytic activity previously detected in rat intestinal mucosal extracts (Beinfeld M., Bourdais, J., Kuks, P., Morel, A., and Cohen, P. (1989) J. Biol. Chem. 264, 4460-4465), was purified to homogeneity as a 65-kDa molecular species. This putative proprotein-processing enzyme cleaves the peptide bond on the carboxyl side of a single arginine residue in hepta-[Leu62-Gln-Arg-Ser-Ala-Asn-Ser68] or trideca-[Asp56-Glu-Met-Arg-Leu-Glu-Leu-Gln-Arg-Ser-Ala-Asn-+ ++Ser68] peptides, reproducing the prosomatostatin sequence around Arg64, the locus for endoproteolytic release of either somatostatin-28 or its NH2-terminal fragment, somatostatin-28-(1-12), from their common precursor. This enzyme exhibits a strict selectivity for arginyl residues, as demonstrated with related substrates, and did not cleave at lysyl residues. Moreover, only arginyl residues belonging to peptides of the prosomatostatin family were cleaved, since no hydrolysis of peptides from other prohormones was detected. In addition, the arginine residue situated at position -5 on the NH2-terminal side of Arg64 not only did not function as a cleavage locus, but had no effect on the overall cleavage kinetics of the prosomatostatin-(56-68) peptide substrate. This enzyme also cleaved, but with much less efficiency, the peptide bond on the carboxyl side of an arginine in peptides containing either an Arg-Lys or a Lys-Arg doublet corresponding to prohormone cleavage sites. This enzyme was insensitive to divalent cation chelators, was completely inhibited by aprotinin and leupeptin, and was somewhat inhibited by other serine-protease inhibitors. It is concluded that this endoprotease is a serine protease and could be involved in prohormone or proprotein post-translational processing at single arginine cleavage sites.

Processing endoprotease recognizes a structural feature at the cleavage site of peptide prohormones. The pro-ocytocin/neurophysin model.

Pro-ocytocin/neurophysin convertase is a divalent cation-dependent endoprotease isolated from both bovine corpus luteum and neurohypophyseal secretory granules. The putative pro-ocytocin/neurophysin converting enzyme cleaves the Arg12-Ala13 bonds of both pro-ocytocin/neurophysin (1----20) and pro-ocytocin/neurophysin obtained by hemisynthesis. The minimal efficient substrate structure allowing recognition by this processing endoprotease was defined by measuring its cleavage efficiency and the inhibitory properties of a set of 34 selectively modified derivatives of the (1----20) NH2-terminal domain of the ocytocin/neurophysin precursor. The data demonstrate that: (i) the basic Lys11-Arg12 doublet, although necessary, is not sufficient; (ii) a minimal substrate length of nine amino acids (residues 7-15 or 8-16) is essential; (iii) those amino acids around the Lys-Arg doublet which contribute to the formation of a possible beta-turn-alpha-helix secondary structure are critical; (iv) substrate recognition by the enzyme may involve several subsites in which structural determinants, situated on both sides of the basic doublet, participate; (v) the NH2-terminal sequence of neurophysin plays a critical role in the correct reading of the cleavage sequence by the processing endoprotease. It is proposed, first, that this type of structural feature may constitute the basis of a general coding system for endoproteases involved in the processing of polypeptide hormone precursors; second, that in addition to its role in the intragranular packaging of the nonapeptide hormone, neurophysin plays a key role in the correct processing of its common precursor with ocytocin.

Synthetic peptide substrates as models to study a pro-ocytocin/neurophysin converting enzyme.

The selectivity and mechanism of processing at paired basic amino acids in hormone precursors was studied on several analogues of the (1-20)-aminoterminal domain of the ocytocin/neurophysin precursor in a cleavage assay by an endoprotease partially purified from bovine pituitary secretory granules. Peptide analogues with amino acid substitutions in, and around, the basic doublet were synthesized and used as substrates. The data obtained demonstrate the strict requirement of the processing enzyme for basic amino acids in tandem within a possibly preferred conformation which may be highly conserved in the aminoterminal domain of this hormone precursor.

Somatostatin-28 and pro-ocytocin/neurophysin convertases: basic pair selective endoproteases involved in pro-hormone processing in the rat brain cortex and bovine corpus luteum.

Two neuropeptide precursor processing enzyme systems were characterized in the rat brain cortex and bovine neurohypophysis and corpus luteum. The first one combines the action of a 90 kDa endoprotease which cleaves somatostatin-28 before the Arg-Lys doublet and that of an aminopeptidase B-like enzyme. The second system associates the action of a 58 kDa endoprotease cleaving pro-ocytocin/neurophysin (1-20) after the Lys-Arg dibasic moiety and a carboxypeptidase B-like activity. Both systems appear to be located in membrane-limited secretory vesicles of the producing organs, and to exhibit the properties of metallo-enzymes sensitive to divalent cation chelators. In contrast, they do not show the characteristics of serine-proteases and of trypsin-like enzymes. Studies with substrate analogs selectively modified at the basic doublet indicated that the integrity of both basic amino acids is essential but that conformational parameters, probably governed by the amino acid sequences flanking the basic doublet, play an important role. These data will be discussed in relation to a hypothesis on the predicted preferred secondary structure of these restriction loci.

Partial purification and functional properties of an endoprotease from bovine neurosecretory granules cleaving proocytocin/neurophysin peptides at the basic amino acid doublet.

An enriched preparation of neurosecretory granules from bovine pituitary neural lobes was used as a source of processing enzymes possibly involved in the cleavage of the proocytocin/neurophysin precursor. A synthetic eicosapeptide reproducing the entire (1-20) sequence of the NH2-terminal domain of the bovine ocytocin/neurophysin precursor was used as a substrate to monitor an endoprotease activity cleaving at the Lys11-Arg12 doublet. The 58-kDa endoprotease detected in the lysate of neurohypophyseal granules produced a single cleavage, after the doublet, at the Arg12-Ala13 peptide bond. This endoprotease with pHi 6.9 and 7.2 exhibits maximal activity at pH around neutrality (7.0) and was strongly inhibited by divalent cation chelating agents [ethylenediaminetetraacetic acid and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',-N'-tetraacetic acid] and to some extent by p-(chloromercuri)benzoate and p-(chloromercuri)benzenesulfonic acid, while phenylmethanesulfonyl fluoride and pepstatin were not active. This endoprotease action was sensitive to any modification of the substrate at either basic amino acid of the doublet since replacement of either L-Lys11 or L-Arg12 by D-Lys or D-Arg and by L-Nle abolished the cleavage reaction. In contrast, reversal of the polarity of the doublet in [Arg11,Lys12]proocytocin/neurophysin(1-20) had no effect on the mode of endoproteolytic cleavage as well as modifications of Gly10 (replaced by Ala10). It is concluded that the selectivity of this endoprotease, which may be involved in the primary event occurring in proocytocin/neurophysin processing, is strictly dependent upon the integrity of the basic doublet but that other parameters determined by the amino acid sequence around this doublet may play an important role.

Solid phase synthesis of somatostatin-28 II. A new biologically active octacosapeptide from anglerfish pancreatic islets.

Somatostatin-28 II, an octacosapeptide recently isolated from anglerfish pancreatic islets, was synthetized by the solid phase method along with its somatostatin-14 II and somatostatin-28 II-(1-12) corresponding domains. Homogeneity of the synthetic peptides was demonstrated by analytical RP-HPLC, thin layer chromatography and electrophoresis. The peptides were further characterized by amino acids analysis, fast atomic bombarding mass spectrometry and/or 252Cf plasma desorption mass spectrometry. Synthetic somatostatin-28 II and somatostatin-14 II displace equally well the potent agonist (Tyr0,D-Trp8)-somatostatin-14 from its specific binding sites on anterior pituitary cells membranes. Both peptides activate adenylate cyclase from dispersed rat anterior pituitary cells.

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.

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.