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Biomedical subjects

R Acher

Publications and source records attributed to R Acher.

At least 37 records · Page 2Linked to original sources
PubMed1993

Vasotocin and hydrin 2 (vasotocinyl-Gly) in the African toad Bufo regularis: study under various environmental conditions.

1. The neurohypophysial osmoregulatory hormones of the African toad Bufo regularis, a species adapted to estivate under dry and hot conditions, have been investigated. Vasotocin and hydrin 2 (vasotocinyl-Gly) have been identified by their retention times in high-pressure reverse-phase liquid chromatography and coelution with synthetic peptides, their pharmacological properties (vasotocin) and microsequencing. 2. Vasotocin-associated neurophysin (MSEL-neurophysin type) has been characterized by its N-terminal amino acid sequence. 3. In toads subjected to dehydration by evaporation (20% weight loss) or to osmotic stress by immersion in 2% NaCl for 3 hr (6% weight loss), the molar ratio hydrin 2/vasotocin (about 2:1) remained similar to the one observed in control animals. 4. In toads exposed to saline solution, there was a large decrease (roughly 30%) in the amounts of both hormones in the neuro-hypophysis. Environmental conditions for distinct secretions of vasotocin and hydrin 2 remain to be found.

Amino Acid Sequence↗
PubMed1992

Evidence for distinct dibasic processing endopeptidases with Lys-Arg and Arg-Arg specificities in neurohypophysial secretory granules.

Two Ca(2+)-dependent endopeptidases endowed with specificities for paired basic residues have been disclosed in rat and ox neurohypophysial secretory granules. Specificities investigated by using synthetic fluorogenic substrates showed the presence of a Lys-Arg endopeptidase with optimum pH close to the granule pH (5.5) and of an Arg-Arg endopeptidase more active at pH 7.0. Granule extracts have virtually no activity towards Lys-Lys-containing substrate or monobasic substrates. Pro-Gly-Lys-Arg-chloromethylketone appears a very efficient inhibitor for the Lys-Arg enzyme. Soluble and membrane-bound forms of both endopeptidases have been detected. pH-dependence of membrane binding and partitioning into Triton X-114 suggest that the membrane-bound form of Lys-Arg endopeptidase is associated through an amphiphilic alpha-helix. It is proposed that the enzyme Lys-Arg cleaves prooxytocin and provasopressin at their signal sequence Gly-Lys-Arg when these precursors arrive in the neurosecretory granules. The processing proceeds in the granules through carboxypeptidase E and alpha-amidating enzyme complex for giving mature pharmacologically active nonapeptide hormones.

Animals↗
PubMed1992

Processing endopeptidase deficiency in neurohypophysial secretory granules of the diabetes insipidus (Brattleboro) rat.

The homozygote Brattleboro rat exhibits a hereditary diabetes insipidus due to a deficiency of vasopressin, the antidiuretic hormone. It has previously been shown that in this animal a single nucleotide deletion in the provasopressin gene leads to a mutant precursor with a C-terminal amino acid sequence different from that of the wild-type. However the N-terminal region including the hormone moiety, the processing signal as well as the first two-thirds of the neurophysin is entirely preserved and absence of maturation has to be explained by an additional cause. We show here that the neurohypophysis of the homozygote Brattleboro rat, in contrast to the adenohypophysis, displays a significant decrease in the Lys-Arg processing endopeptidase activity when compared to the heterozygote or the wild-type Wistar. It is suggested that hypothalamic vasopressinergic neurons of the homozygote Brattleboro rat display a deficiency in the processing enzyme in contrast to the oxytocinergic neurons in which processing of prooxytocin is normal.

Amino Acid Sequence↗
PubMed1992

A neurosecretory granule Lys-Arg Ca(2+)-dependent endopeptidase putatively involved in prooxytocin and provasopressin processing.

A Ca(2+)-dependent endopeptidase cleaving at the carboxyl side of the paired Lys-Arg residues has been found in the neurosecretory granules of the rat neurointermediate pituitary. The specificity pattern on synthetic fluorogenic substrates, the inhibitor profile, the pH optimum of 5.0 and the Ca(2+)-dependence are compatible with an involvement of this enzyme in the prooxytocin and the provasopressin processing within the granules. The enzymatic features of the neurohypophysial granule endopeptidase resemble those of the insulinoma granule type II endopeptidase and suggest that the same Ca(2+)-dependent protease or closely related enzymes could be involved in processing Lys-Arg-containing prohormones in neuroendocrine cells.

Amino Acid Sequence↗
PubMed1992

Partial conversion of vasopressinyl-Gly-Lys-Arg into pharmacologically active vasopressin through secretory granule carboxypeptidase E and alpha-amidating processing enzymes.

Vasopressinyl-Gly-Lys-Arg, the first intermediate derived from vasopressin protein precursor, has been converted into mature vasopressin by an "in vitro" two-step reaction through neurohypophysial secretory granule enzymes. Whereas the conversion into vasopressinyl-Gly is virtually complete at pH 5.5 as judged by HPLC, the conversion of vasopressinyl-Gly into vasopressin is weak at pHs 6.0 or 8.0 as judged by HPLC and measure of generated pressor activity. It is suggested that the high conversion yield usually seen in mammalian neurohypophysis, where no intermediate is detected, might be due to additional "in vivo" factors such as particular membrane-association or binding of the intermediate onto a neurophysin carrier.

Amino Acid Sequence↗
PubMed1991

The hormone-binding site of neurophysins: binding of vasopressin to the N-terminal sub-domain dissected from human MSEL-neurophysin through endopeptidase Lys-C.

Human MSEL-neurophysin has been dissected into two halves by endopeptidase Lys-C, taking advantage of a peculiar Lys59-Ala60 bond. Two sub-domains, N-terminal (1-59) and C-terminal (60-93), have been separated. These sub-domains have been purified by reverse-phase high pressure liquid chromatography and identified by their N-terminal sequences. The N-terminal fragment comprises two chains 1-18 and 19-59, because of the presence of a second lysine residue in position 18, whereas the C-terminal fragment (60-93) is a single chain. Hormone-binding experiments have been carried out using vasopressin or vasopressinyl-Gly-Lys-Arg and testing the ability of the hormone-neurophysin complex to precipitate at pH 3.9 with 10% NaCl. The N-terminal sub-domain precipitates in presence of vasopressin in the same way as native neurophysin whereas the C-terminal sub-domain does not. It can be concluded that the hormone-binding site is located in the 1-59 region of neurophysin.

Amino Acid Sequence↗
PubMed1991

Study of frog (Rana esculenta) proopiomelanocortin processing in the intermediate pituitary. Identification of alpha-melanotropin, beta-melanotropin, Lys-gamma-melanotropin, and corticotropin-like intermediate lobe peptide.

The proteolytic processing of frog (Rana esculenta) proopiomelanocortin in melanotropic cells of the intermediate pituitary gland has been examined through purification of the mature fragments by reverse-phase high-pressure liquid chromatography and microsequencing of isolated peptides. alpha-Melanotropin, beta-melanotropin, Lys-gamma-melanotropin, corticotropin-like intermediate lobe peptide, and hinge peptide have been isolated and chemically characterized. The results show a high preservation in the processing sites of frog proopiomelanotropin when compared to bovine counterparts. They reveal also a great conservation of the processing enzyme equipment of melanotropic cells in tetrapods species. Identification of Lys-gamma-melanotropin suggests the occurrence of an endopeptidase able to cleave between two basic residues. On the other hand alpha-melanotropin does not appear to be N-acetylated, as previously found in the clawed-toad Xenopus laevis, and this feature might distinguish amphibian from mammalian proopiomelanocortin processing.

Adrenocorticotropic Hormone↗
PubMed1991

[Adaptative differential maturation of neurohypophyseal provasotocin in amphibians: presence of hydrin 2(vasotocinyl-glycine) in anura but not in urodela].

Occurrence of hydrin 2 (vasotocinyl-glycine), an intermediate in the provasotocin processing, has been shown in the neurointermediate lobe of the pituitary of semi-aquatic or terrestrial amphibian species belonging to the order Anura, namely Rana esculenta, R. temporaria, R. pipiens (Ranidae), and Bufo bufo, B. marinus, B. ictericus (Bufonidae). In contrast hydrin 2 appears absent in species belonging to the order Urodela, namely the axolotl Ambystoma mexicanum (Ambystomidae) and the pleurodele newt, Pleurodela waltlii (Salamandridae). Whereas water transport through the skin and the urinary bladder is stimulated by vasotocin and hydrin 2 in anuran species, this stimulation is known not to occur in urodele species.

Amphibians↗
PubMed1990

Evolutionary specificity of hydrins, new hydroosmotic neuropeptides: occurrence of hydrin 2 (vasotocinyl-Gly) in the toad Bufo marinus but not in the viper Vipera aspis.

Hydrin 2 (vasotocinyl-Gly), a hydroosmotic peptide resulting from differential processing of provasotocin and recently identified in frog neurohypophysis, has been looked for in the pituitary gland of an exotic toad (Bufo marinus) and of a reptile (Vipera aspis). Hydrin 2 has been found in the amphibian but not in the reptile. This result confirms the evolutionary specificity of hydrin 2 that has been identified only in frogs and toads but not in birds and reptiles. Occurrence of hydrin 2 is explained by its regulatory function on the water permeability of the skin of anurans.

Animals↗
PubMed1990

Identification of two types of neurophysins in Xenopus laevis neurointermediate pituitary homologous to mammalian MSEL- and VLDV-neurophysins.

UNLABELLED: Xenopus laevis neurophysins have been purified from neurointermediate pituitaries through high-pressure reverse-phase liquid chromatography and their N-terminal amino acid sequences have been determined by microsequencing. Two types of neurophysins, corresponding to mammalian MSEL- and VLDV-neurophysins, have been distinguished. A strong homology exists between neurophysins of Xenopus (Pipidae), frog (Ranidae) and toad (Bufonidae). Xenopus MSEL-neurophysin, as frog MSEL-neurophysin, has a high molecular mass suggesting that the C-terminal domain of the vasotocin precursor is not processed in contrast to the two-step processing observed for mammalian vasopressin precursor. ABBREVIATIONS: Mammalian neurophysins are termed MSEL- and VLDV-neurophysins according to the nature of residues in positions 2, 3, 6 and 7 (one-letter symbols for amino acids).

Amino Acid Sequence↗
PubMed1990

Non-mammalian "big" neurophysins--complete amino acid sequence of a two-domain MSEL-neurophysin from goose.

Vasotocin-associated neurophysin (MSEL-neurophysin) has been purified from goose neurohypophysis through molecular sieving and high-pressure reverse-phase liquid chromatography (HPLC). The protein has a molecular mass (measured by SDS-polyacrylamide gel electrophoresis) of 17 kDa in contrast to 10 kDa found for the mammalian MSEL-neurophysins. Complete amino acid sequence (131 residues) has been determined mainly through tryptic or staphylococcal proteinase peptides derived from carboxyamidomethylated neurophysin, isolated by HPLC and microsequenced. N- and C-terminal sequences have been established by Edman degradation or action of carboxypeptidase Y, respectively, applied on the native protein. Goose MSEL-neurophysin is homologous to the two-domain "big" MSEL-neurophysin previously identified in the frog. It appears that in non-mammalian tetrapods, namely birds and amphibians, the proteolytic processing of the pro-vasotocin involves only one cleavage, releasing the hormone moiety and a "big" neurophysin with two domains homologous to mammalian MSEL-neurophysin and copeptin, respectively. Comparison of the avian protein with its mammalian and amphibian counterparts reveals that the first half of the polypeptide chain is evolutionarily much less variable than the second and that the goose protein resembles the frog protein much more than the mammalian one.

Amino Acid Sequence↗
PubMed1990

Complete amino acid sequence of goose VLDV-neurophysin. Traces of a putative gene conversion between promesotocin and provasotocin genes.

Goose VLDV-neurophysin (mesotocin-associated neurophysin) has been purified from posterior pituitary glands through molecular sieving on Sephadex G-75 and high-pressure reverse-phase liquid chromatography on Nucleosil C-18 columns. Despite apparent molecular mass of unreduced VLDV-neurophysin measured by polyacrylamide gel electrophoresis with sodium dodecylsulfate appeared near 17 kDa, this value fell to 11 kDa after reduction with mercaptoethanol, suggesting the existence of a homodimer. Complete amino acid sequence (93 residues) of goose VLDV-neurophysin has been determined. N- and C-terminal sequences of the protein have been established by Edman degradation (microsequencing) and use of carboxypeptidase Y, respectively. Peptides derived from oxidized or carboxamidomethylated neurophysin by trypsin or staphylococcal proteinase hydrolyses have been isolated by high-pressure liquid chromatography and microsequenced, allowing determination of the complete sequence. Comparison within the vertebrate VLDV-neurophysin lineage, namely goose VLDV-neurophysin to mammalian VLDV-neurophysins and to deduced toad VLDV-neurophysin, reveals a residue insertion between positions 66 and 67 in the nonmammalian VLDV-neurophysins. When goose MSEL-neurophysin (vasotocin-associated neurophysin) and goose VLDV-neurophysin are compared to their bovine counterparts, identical substitutions are found in positions 17 (Asn in both goose neurophysins instead of Gly in both ox neurophysins), 18 (Arg instead of Lys), 35 (Tyr instead of Phe), and 41 (Thr instead of Ala). Identity of the sequences 10-74 in both ox neurophysins has been explained by partial gene conversion between oxytocin and vasopressin genes, and identical substitutions in both goose neurophysins might reveal a similar gene conversion between mesotocin and vasopressin genes in birds.

Alkylation↗
PubMed1990

Occurrence of hydrin 2 (vasotocinyl-Gly), a new hydroosmotic neurohypophyseal peptide, in secretory granules isolated from the frog (Rana esculenta) neurointermediate pituitary.

Neurohypophyseal secretory granules have been purified from the frog (Rana esculenta) neurointermediate pituitary gland by sucrose gradient centrifugation, and their polypeptide content has been analyzed by reverse-phase high-pressure liquid chromatography. Aside from vasotocin, mesotocin, and their associated neurophysins, hydrin 2 (vasotocinyl-Gly), previously identified in hydrochloric acid extracts, has been recognized. This finding supports the previous suggestion that hydrin 2, a peptide active on the water permeability of frog bladder and frog skin, is a secreted hormone involved in osmoregulation specific to amphibians. Hydrin 2 has not been found in neurosecretory granules of birds such as the goose.

Animals↗
PubMed1989

Particular processing of pro-opiomelanocortin in Xenopus laevis intermediate pituitary. Sequencing of alpha- and beta-melanocyte-stimulating hormones.

alpha- and beta-melanocyte-stimulating hormones (alpha-MSH and beta-MSH) have been isolated from Xenopus laevis neurointermediate pituitary and microsequenced. Intracellular alpha-MSH is not N-acetylated after proteolytic processing of pro-opiomelanocortin in contrast to mammalian alpha-MSHs. There is a high preservation of the melanotropic amino acid sequence common to all MSHs although in Xenopus beta-MSH a histidine residue replaces the glutamic acid residue found in position 8 of mammalian beta-MSHs.

Acetylation↗
PubMed1989

Isolation of neurosecretory granules containing vasotocin, mesotocin, MSEL- and VLDV-neurophysins from goose neurohypophysis.

Neurosecretory granules have been isolated from goose posterior pituitaries and their contents have been analyzed by reverse-phase high-pressure liquid chromatography and polyacrylamide gel electrophoresis. Vasotocin and mesotocin have been identified by their biological activities and their retention times compared with those of synthetic peptides. MSEL- and VLDV-neurophysins have been characterized by their N-terminal sequences, their electrophoretic migrations and their retention times, compared with those of purified goose neurophysins. In contrast to the two-step processing of mammalian provasopressin, processing of the vasotocin - MSEL-neurophysin precursor appears to involve only one cleavage giving the hormone and a "big" MSEL-neurophysin homologous to mammalian MSEL-neurophysin extended by copeptin.

Animals↗
PubMed1989

Hydrins, hydroosmotic neurohypophysial peptides: osmoregulatory adaptation in amphibians through vasotocin precursor processing.

From neurointermediate pituitary glands of Xenopus laevis and Rana esculenta, previously unreported peptides termed hydrins, active on water permeability of frog urinary bladder and frog skin (Brunn or "water-balance" effect), have been isolated and sequenced. These peptides seem to be derived from the pro-vasotocin-neurophysin precursor. Hydrin 1, found in Xenopus, has been identified as vasotocin C-terminally extended with the Gly-Lys-Arg sequence; hydrin 2, found in Rana, has been identified as vasotocin C-terminally extended with glycine. Hydrin 2 has been detected in several Ranidae (R. esculenta, Rana temporaria, Rana pipiens) and Bufonidae (Bufo bufo, Bufo ictericus) and appears to have a large distribution in terrestrial or semiaquatic anurans. Hydrins, in contrast to vasotocin, are not active on rat uterus or rat blood pressure. They are absent from other vasotocin-bearers such as birds and could be involved specifically in water-electrolyte regulation of amphibians.

Acclimatization↗
PubMed1988

Neurohypophysial hormones of the 1-month-old bovine fetus: absence of vasotocin during mammal development.

The neurohypophysial hormones of the 1-month-old bovine fetus have been identified by their positions in ion-exchange chromatography and their retention times in high-pressure reverse-phase partition chromatography. Arginine vasopressin and oxytocin have been recognized. The molar ratio vasopressin/oxytocin in neurohypophysis is about 6 in the 1-month-old fetus compared with 4 in the 3-month-old fetus, 2.7 in the 7-month-old fetus and 1 in the adult. Vasotocin is virtually absent even in the early fetus (less than 0.1% of arginine vasopressin). The occurrence of a vasotocin gene expressed in the fetus but silent in the adult appears unlikely.

Animals↗