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Identification of two types of neurophysins in Xenopus laevis neurointermediate pituitary homologous to mammalian MSEL- and VLDV-neurophysins
Neuropeptides (1990) 15,123-127 0 Longman Group UK Ltd 1990
Identification of two Types of Neurophysins In Xenopus Laevis Neurointermediate Pituitary Homologous to Mammalian MSEL- and VLDVNeurophysins J. CHAUVET, G. MICHEL, Y. ROUILL6, M.-T. CHAUVET and R. ACHER Laboratory of Biological Chemistry, University of Paris VI 96, Boulevard Raspail, 75006 Paris, France (Correspondence to RA, Reprint requests to JC!
Abstract-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 Xenupus 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 VLDVneurophysins according to the nature of residues in positions 2,3,6 and 7 (one-letter symbols for amino acids).
Introduction The two types of mammalian neurophysins, namely MSEL-neurophysin (vasopressin-associated neurophysin) and VLDV-neurophysin (oxytocin-associated neurophysin) (1) have previously been recognized in chicken, goose and ostrich posterior pituitaries and assumed to derive from vasotocin and mesotocin precursors, respectively (2). Furthermore a ‘big’ neurophysin Date received 10 July 1989 Date accepted 27 July 1989
has recently been isolated from the neurointermediate pituitary of the frog Rana esculentu and determination of its amino acid sequence has shown that this protein is homologous to mammalian MSEL-neurophysins extended with a C-terminal glycopeptide , copeptin (3, 4). A second neurophysin homologous to mammalian VLDVneurophysin has also been identified in this frog. Results obtained with Japanese toad Bufo juponicus through cDNA technology, allow to deduce that both types of neurophysins should be present in this amphibian (5). We have now isolated two neurophysins from 123
124
NEUROPEPTIDES
laevis neurointermediate pituitary and found that they belong to MSEL-neurophysin and VLDV-neurophysin types. From similarities between amino acid sequences and pharmacological activities of mammalian and lower vertebrate hormones, it has been suggested that vasotocin and mesotocin have been the evolutionary predecessors of mammalian vasopressin and oxytocin, respectively (6, 7). If so, two gene lineages exist in vertebrates and homologies should be found between the neurophysin domains of neurohormone precursors. Xenopus
Materials and Methods
J
Neurointermediate lobes (average wet weight: 1.2mg) from Xenopus laevis (average body weight: 1OOg) were homogenized with O.lM HCl for 4min, then stirred for 4h at 4°C. After centrifugation, biological activities were measured in an aliquot of the supernatant: uterotonic activity 0.47U and pressor activity 0.2OU per gland. Extracted neurophysins have been purified through high-pressure liquid chromatography (HPLC) on a Nucleosil Cl8 column (4.6 x 210mm, particle size 5~) using an acetonitrile linear gradient (560%) containing 0.05% trifluoroacetic acid for 55 min followed by an isocratic elution with acetonitrile 60% during 5 min. The flow rate was 1 ml/min and polypeptides were detected by absorbance at 214nm and 280nm. 0.6-ml fractions were collected and those corresponding to peaks were pooled. Homogeneity of proteins was checked by sodium dodecylsulfate polyacrylamide gel electrophoresis and molecular masses were determined by this procedure (8). Microsequencing of native neurophysins (20200pmol) was carried out by’automated Edman degradation using an Applied Biosystems model
Fig. 1 Purification of Xenopus VLDV- and MSEL-neurophysins by HPLC (Experiment III). Retention times in min are indicated (details in the text). 470 A gas-phase protein sequencer under the conditions described by Hewick et al. (9). Phenylthiohydantoins were identified and measured by high-pressure reverse-phase chromatography in an ‘on-line’ Applied Biosystems Model 120 A analyzer.
Results and Discussion Neurophysins are the main proteins extracted from neurointermediate pituitaries (Fig. 1). The retention times found in four experiments in
Table 1
Retention Times of Xenopus Laevk Neurophysins graphy (Times in Min)
VLDV-neurophysin MSEL-neurophysin
In High-Pressure
Reverse-Phase
Partition Chromato-
Experiment I (I gland)
Experiment II (2 glands)
Experiment III (3 glands)
Experiment IV (4 glands)
35.20 35.55 39.65
35.20 35.45 39.50
35.30 35.60 39.45
35.05 35.35 39.15
MAMMALIAN
MSEL- AND VLDV-NEUROPHYSINS
HPLC for the two types of neurophysins in Table 1.
125 are given
VLDV-neurophysin type: Dual peaks were usually observed for the VLDV-neurophysin-type. Although two forms of VLDV-neurophysins with M, 17000 and 20000, respectively, were detected by SDS-polyacrylamide gel electrophoresis, a single N-terminal sequence was observed (Table 2). Xenopus VLDV-neurophysin has a retention time about 35.2-35.6 min. The N-terminal sequence of Xenopus VLDV-neurophysin (Table 2) is homologous to that determined for Rana VLDV-neurophysin esculenta (unpublished results) and that deduced from cDNA for Bufo japonicus mesotocin-associated neurophysin (5)
Table 2 pmoIa)
Microsequencing
of Xenopus
Laevis
(Fig. 2). When compared with mammalian VLDV-neurophysin, a particular insertion is found in the N-terminal sequences (position 5’). This insertion is also found in the Bufo japonicus cDNA and is likely encoded in the corresponding gene. In mammalian genes, the first nine residues of each neurophysin are encoded by the same exon that codes for the associated hormone (exon A) (11-13). If it is so in lower vertebrates, an autonomous evolution of exon A could have occurred. MSEL-neurophysin
type: The MSEL-neurophysin type displays a higher retention time (about 39.439.6min) than VLDV-neurophysin. Its apparent M, is also higher, about 36000 by SDS-polyacrylamide electrophoresis. Results obtained by microsequencing are given
Neurophysins
(Amino
acid phenylthiohydantoins
VLDV-Neurophysin
1 2 3 4 5 6 7 8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Residue
Ser Val Met Asp Ile Met Asp Ile Arg LYS _ Ile Pro
RT35.45 M, 20 kD (2 glanats) pm01
Identif.b 13 13 13.5 13.5 13 11 10 7.5 11.5 8 6
in
MSEL-Neurophysin
RT 35.20 Mr17.5 kD (I gland) Cycle
are quantified
Residue
Ser Val Met Asp Ile Met Asp Ile Arg LYS _ Ile Pro GIY Pro A% Asn LYS
RT39.65 M, 36 kD (I gland) pm01
Residue
pm01
Identif.b 170 155 131 133 135 124 112 85 77
Ser Tyr Pro Asp Thr GlU Leu ArS Gln
Identif.b 17 18 21 6 16 15 9 14.5
Met Gln
12 22
GIY Pro Gly Asn LYS GIY Asn _
15 10 Identif.b 7 7 8 5
Phe GIY Pro
6 Identif.b Identif.b
89 72 _ 30 28 Identif.b 13
a Dashes correspond to half-cystines whose derivatives are not seen when native neurophysins are used. b Identif.: derivative identified but not auantified.
126 MSEL-Typs ox Xenopus Rana Bufo VLDV-Type ox Xenopus Rana Bufo
NEUROPEP’I’IDES Neurophysins 1 2 3 4 AMSDL SYP-T S Y P SYP-T
6 7 8 3 10 11 12 13 ELRQCLPCGPGGKGRCFGP _MQ_---N--N_------
5
_
T
5
S S
IMIM---R-I----
S-I
M _
-
_
)
-
-
15
-
-
,;___,__-_
Neurophysins 1 2 3 4 AVLDL _
_
14
5’
6 7 8 3 10 DVRTCLPCGPGGK
I
11
12
13
14
15
16
17
18
19
20
21
22
23
24
-
N
R
-
N
_
_
_
_
_
N
R
_
N
_
_
_
_
16
17
18
-K-I----RN-
-FM---K-I----RN-
Comparison of N-terminal sequences of neurophysins of ox (1) Xenopus Zaevis (this work), Rana esculenra (3) and Bufo (5). Dashes indicate residues identical to those of the upper line. An insertion (5’) is observed in amphibian VLDV-neurophysins, when alignment is made with amphibian MSEL-neurophysins or mammalian neurophysins. Fig. 2
japonicus
in Table 2. The sequence corresponding to the neurophysin with a retention time 39.65 min is homologous to that determined for Rana esculentu MSEL-neurophysin (3) and to that deduced from cDNA of Bufo japonicus for vasotocin-associated neurophysin (5) (Fig. 2). Rana MSEL-neurophysin is longer than its mammalian counterpart because the third domain of the precursor, the C-terminal copeptin, is not detached in the course of processing (3). The high molecular mass of Xenupus MSEL-neurophysin suggests that in this species copeptin is also not separated from neurophysin and there is similar evidence for several other amphibians (4). A two-step processing has been proposed for the cleavage of mammalian vasopressin precursor into three fragments, namely vasopressin, MSELneurophysin and copeptin (10). In a first step vasopressin is cut off. The intermediate twodomain neurophysin-copeptin undergoes a conformational change so that the linking sequence becomes accessible to a processing endopeptidase. This second cleavage involves a trypsin-like endopeptidase acting at the level of a single arginine residue and leads to separate neurophysin and copeptin. In lower vertebrates, vasopressin precurses is replaced by vasotocin precursor apparently built in the same way (3, 5). After the trimming of vasotocin, however, the processing of the ‘intermediate’ precursor does not occur so that a ‘big’ MSEL-neurophysin is found (3,4,10). ‘Big’ neurophysins have been found in several birds and
amphibians in contrast to mammals. Lack of the second cleavage may be due to a change in precursor sequence or in precursor conformation or to a deficiency of the processing enzyme. Absence of the second cleavage does not seem to affect the release of vasotocin from the precursor.
Acknowledgements The authors are grateful to Prof. J. Charlemagne (Laboratoire d’Immunologie Comparee, UniversitC Pierre et Marie Curie) for the gift of Xenopus laevis. They thank Mrs Danielle Thtvenet and Mrs Christine Jeanney for their skilled technical assistance. This work has been partly supported by grants from CNRS (UA 040 515 and ATP ‘Evolution’) and Minis&e de la Recherche et de I‘Enseignement Superieur (Action Incitative ‘Biologie’).
References Acher, R. (1985). Biosynthesis, processing and evolution of neurohypophysial hormone precursors. In: Kobayashi, H., Bern, H.A. and Urano, A. (eds) Neurosecretion and the Biology of Neuropeptides. Proceedings of the Ninth International Symposium on Neurosecretion. Japan. Sci. SOC.Press. Tokyo-Springer Verlag, Berlin, 11-25. Chauvet, M. T., L&y, B., Michel, G. Chauvet, J. and Acher, R. (1986). Precursors of mesotocin and vasotocin in birds: Identification of VLDV- and MSEL-neurophysins in chicken, goose and ostrich. Bioscience Rep. 6: 381-385. Michel, G., Chauvet, J., Chauvet, M. T. and Acher, R. (1987). One-step processing of the amphibian vasotocin precursor: structure of a frog (Ram esculenta) ‘big’ neurophysin Biochem. Biophys. Res. Common. 149: 538-544.
MAMMALIAN
MSEL- AND VLDV-NEUROPHYSINS
4. Chauvet, J., Michel, G., Chauvet, M. T. and Acher, R.
5.
6. 7.
8.
(1988). An amphibian two-domain ‘big’ neurophysin: conformational homology with the mammalian MSELneurophysin/copeptin intermediate precursor shown by trypsin-Sepharose proteolysis. FEBS Lett. 230: 77-80. Nojiri, H., Ishida, I., Miyashita, E., Sato, M., Urano, A. and Deguchi, T. (1987). Cloning and sequence analysis of cDNAs for neurohypophysial hormones vasotocin and mesotocin for the hypothalamus of toad, Bufo japonicus. Proc. Natl. Acad. Sci. USA 84: 3043-3046. Acher, R. (1980). Molecular evolution of biologically active polypeptides. Proc. R. Sot. London B 210: 21-43. Acher, R., Chauvet, J., Chauvet, M. T. and Hurpet, D. (1985). Evolution of neurohypophysial hormones and their precursors. In: Current Trends in Comparative Endocrinology. B. Lofts and W.N. Holmes (eds) pp. 1147-1152. Hong Kong, Hong Kong University Press. Weber, K. and Osborn, M. (1969). The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 244: 4406-4412.
127 9. Hewick, R. M., Hunkapiller, M. W., Hood, L. E. and Dreyer, W. J. (1981). A gas-liquid-solid phase peptide and protein sequenator. J. Biol. Chem. 256: 7990-7997. 10. Acher, R., Chauvet, M. T., Chauvet, J., Levy, B.. Michel, G. and Rouillt, R. (1988). The Processing of neurohypophysial hormone-neurophysin precusors: A conformation-directed stepwise maturation. In: Proceedings of the Symposium ‘Recent Progress in Posterior Pituitary Hormones’ S. Yoshida and L. Share (eds) Elsevier, pp. 419-426. 11. Ivell, R. and Richter, D. (1984). Structure and comparison of the oxytocin and vasopressin genes from rat. Proc. Nat]. Acad. Sci. USA 81: 2006-2010. 12. Ruppert. S., Scherer, G. and Shiitz, G. (1984). Recent gene conversion involving bovine vasopressin and oxytocin precursor genes suggested by nucleotide sequence. Nature 308: 554-557. 13. Sausville, E., Carney, D. and Battey, J. (1985). The human vasopressin gene is linked to the oxytocin gene and is selectively expressed in a cultured lung cancer. J. Biol. Chem. 260: 10236-10241.
Identification of two Types of Neurophysins In Xenopus Laevis Neurointermediate Pituitary Homologous to Mammalian MSEL- and VLDVNeurophysins J. CHAUVET, G. MICHEL, Y. ROUILL6, M.-T. CHAUVET and R. ACHER Laboratory of Biological Chemistry, University of Paris VI 96, Boulevard Raspail, 75006 Paris, France (Correspondence to RA, Reprint requests to JC!
Abstract-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 Xenupus 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 VLDVneurophysins according to the nature of residues in positions 2,3,6 and 7 (one-letter symbols for amino acids).
Introduction The two types of mammalian neurophysins, namely MSEL-neurophysin (vasopressin-associated neurophysin) and VLDV-neurophysin (oxytocin-associated neurophysin) (1) have previously been recognized in chicken, goose and ostrich posterior pituitaries and assumed to derive from vasotocin and mesotocin precursors, respectively (2). Furthermore a ‘big’ neurophysin Date received 10 July 1989 Date accepted 27 July 1989
has recently been isolated from the neurointermediate pituitary of the frog Rana esculentu and determination of its amino acid sequence has shown that this protein is homologous to mammalian MSEL-neurophysins extended with a C-terminal glycopeptide , copeptin (3, 4). A second neurophysin homologous to mammalian VLDVneurophysin has also been identified in this frog. Results obtained with Japanese toad Bufo juponicus through cDNA technology, allow to deduce that both types of neurophysins should be present in this amphibian (5). We have now isolated two neurophysins from 123
124
NEUROPEPTIDES
laevis neurointermediate pituitary and found that they belong to MSEL-neurophysin and VLDV-neurophysin types. From similarities between amino acid sequences and pharmacological activities of mammalian and lower vertebrate hormones, it has been suggested that vasotocin and mesotocin have been the evolutionary predecessors of mammalian vasopressin and oxytocin, respectively (6, 7). If so, two gene lineages exist in vertebrates and homologies should be found between the neurophysin domains of neurohormone precursors. Xenopus
Materials and Methods
J
Neurointermediate lobes (average wet weight: 1.2mg) from Xenopus laevis (average body weight: 1OOg) were homogenized with O.lM HCl for 4min, then stirred for 4h at 4°C. After centrifugation, biological activities were measured in an aliquot of the supernatant: uterotonic activity 0.47U and pressor activity 0.2OU per gland. Extracted neurophysins have been purified through high-pressure liquid chromatography (HPLC) on a Nucleosil Cl8 column (4.6 x 210mm, particle size 5~) using an acetonitrile linear gradient (560%) containing 0.05% trifluoroacetic acid for 55 min followed by an isocratic elution with acetonitrile 60% during 5 min. The flow rate was 1 ml/min and polypeptides were detected by absorbance at 214nm and 280nm. 0.6-ml fractions were collected and those corresponding to peaks were pooled. Homogeneity of proteins was checked by sodium dodecylsulfate polyacrylamide gel electrophoresis and molecular masses were determined by this procedure (8). Microsequencing of native neurophysins (20200pmol) was carried out by’automated Edman degradation using an Applied Biosystems model
Fig. 1 Purification of Xenopus VLDV- and MSEL-neurophysins by HPLC (Experiment III). Retention times in min are indicated (details in the text). 470 A gas-phase protein sequencer under the conditions described by Hewick et al. (9). Phenylthiohydantoins were identified and measured by high-pressure reverse-phase chromatography in an ‘on-line’ Applied Biosystems Model 120 A analyzer.
Results and Discussion Neurophysins are the main proteins extracted from neurointermediate pituitaries (Fig. 1). The retention times found in four experiments in
Table 1
Retention Times of Xenopus Laevk Neurophysins graphy (Times in Min)
VLDV-neurophysin MSEL-neurophysin
In High-Pressure
Reverse-Phase
Partition Chromato-
Experiment I (I gland)
Experiment II (2 glands)
Experiment III (3 glands)
Experiment IV (4 glands)
35.20 35.55 39.65
35.20 35.45 39.50
35.30 35.60 39.45
35.05 35.35 39.15
MAMMALIAN
MSEL- AND VLDV-NEUROPHYSINS
HPLC for the two types of neurophysins in Table 1.
125 are given
VLDV-neurophysin type: Dual peaks were usually observed for the VLDV-neurophysin-type. Although two forms of VLDV-neurophysins with M, 17000 and 20000, respectively, were detected by SDS-polyacrylamide gel electrophoresis, a single N-terminal sequence was observed (Table 2). Xenopus VLDV-neurophysin has a retention time about 35.2-35.6 min. The N-terminal sequence of Xenopus VLDV-neurophysin (Table 2) is homologous to that determined for Rana VLDV-neurophysin esculenta (unpublished results) and that deduced from cDNA for Bufo japonicus mesotocin-associated neurophysin (5)
Table 2 pmoIa)
Microsequencing
of Xenopus
Laevis
(Fig. 2). When compared with mammalian VLDV-neurophysin, a particular insertion is found in the N-terminal sequences (position 5’). This insertion is also found in the Bufo japonicus cDNA and is likely encoded in the corresponding gene. In mammalian genes, the first nine residues of each neurophysin are encoded by the same exon that codes for the associated hormone (exon A) (11-13). If it is so in lower vertebrates, an autonomous evolution of exon A could have occurred. MSEL-neurophysin
type: The MSEL-neurophysin type displays a higher retention time (about 39.439.6min) than VLDV-neurophysin. Its apparent M, is also higher, about 36000 by SDS-polyacrylamide electrophoresis. Results obtained by microsequencing are given
Neurophysins
(Amino
acid phenylthiohydantoins
VLDV-Neurophysin
1 2 3 4 5 6 7 8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Residue
Ser Val Met Asp Ile Met Asp Ile Arg LYS _ Ile Pro
RT35.45 M, 20 kD (2 glanats) pm01
Identif.b 13 13 13.5 13.5 13 11 10 7.5 11.5 8 6
in
MSEL-Neurophysin
RT 35.20 Mr17.5 kD (I gland) Cycle
are quantified
Residue
Ser Val Met Asp Ile Met Asp Ile Arg LYS _ Ile Pro GIY Pro A% Asn LYS
RT39.65 M, 36 kD (I gland) pm01
Residue
pm01
Identif.b 170 155 131 133 135 124 112 85 77
Ser Tyr Pro Asp Thr GlU Leu ArS Gln
Identif.b 17 18 21 6 16 15 9 14.5
Met Gln
12 22
GIY Pro Gly Asn LYS GIY Asn _
15 10 Identif.b 7 7 8 5
Phe GIY Pro
6 Identif.b Identif.b
89 72 _ 30 28 Identif.b 13
a Dashes correspond to half-cystines whose derivatives are not seen when native neurophysins are used. b Identif.: derivative identified but not auantified.
126 MSEL-Typs ox Xenopus Rana Bufo VLDV-Type ox Xenopus Rana Bufo
NEUROPEP’I’IDES Neurophysins 1 2 3 4 AMSDL SYP-T S Y P SYP-T
6 7 8 3 10 11 12 13 ELRQCLPCGPGGKGRCFGP _MQ_---N--N_------
5
_
T
5
S S
IMIM---R-I----
S-I
M _
-
_
)
-
-
15
-
-
,;___,__-_
Neurophysins 1 2 3 4 AVLDL _
_
14
5’
6 7 8 3 10 DVRTCLPCGPGGK
I
11
12
13
14
15
16
17
18
19
20
21
22
23
24
-
N
R
-
N
_
_
_
_
_
N
R
_
N
_
_
_
_
16
17
18
-K-I----RN-
-FM---K-I----RN-
Comparison of N-terminal sequences of neurophysins of ox (1) Xenopus Zaevis (this work), Rana esculenra (3) and Bufo (5). Dashes indicate residues identical to those of the upper line. An insertion (5’) is observed in amphibian VLDV-neurophysins, when alignment is made with amphibian MSEL-neurophysins or mammalian neurophysins. Fig. 2
japonicus
in Table 2. The sequence corresponding to the neurophysin with a retention time 39.65 min is homologous to that determined for Rana esculentu MSEL-neurophysin (3) and to that deduced from cDNA of Bufo japonicus for vasotocin-associated neurophysin (5) (Fig. 2). Rana MSEL-neurophysin is longer than its mammalian counterpart because the third domain of the precursor, the C-terminal copeptin, is not detached in the course of processing (3). The high molecular mass of Xenupus MSEL-neurophysin suggests that in this species copeptin is also not separated from neurophysin and there is similar evidence for several other amphibians (4). A two-step processing has been proposed for the cleavage of mammalian vasopressin precursor into three fragments, namely vasopressin, MSELneurophysin and copeptin (10). In a first step vasopressin is cut off. The intermediate twodomain neurophysin-copeptin undergoes a conformational change so that the linking sequence becomes accessible to a processing endopeptidase. This second cleavage involves a trypsin-like endopeptidase acting at the level of a single arginine residue and leads to separate neurophysin and copeptin. In lower vertebrates, vasopressin precurses is replaced by vasotocin precursor apparently built in the same way (3, 5). After the trimming of vasotocin, however, the processing of the ‘intermediate’ precursor does not occur so that a ‘big’ MSEL-neurophysin is found (3,4,10). ‘Big’ neurophysins have been found in several birds and
amphibians in contrast to mammals. Lack of the second cleavage may be due to a change in precursor sequence or in precursor conformation or to a deficiency of the processing enzyme. Absence of the second cleavage does not seem to affect the release of vasotocin from the precursor.
Acknowledgements The authors are grateful to Prof. J. Charlemagne (Laboratoire d’Immunologie Comparee, UniversitC Pierre et Marie Curie) for the gift of Xenopus laevis. They thank Mrs Danielle Thtvenet and Mrs Christine Jeanney for their skilled technical assistance. This work has been partly supported by grants from CNRS (UA 040 515 and ATP ‘Evolution’) and Minis&e de la Recherche et de I‘Enseignement Superieur (Action Incitative ‘Biologie’).
References Acher, R. (1985). Biosynthesis, processing and evolution of neurohypophysial hormone precursors. In: Kobayashi, H., Bern, H.A. and Urano, A. (eds) Neurosecretion and the Biology of Neuropeptides. Proceedings of the Ninth International Symposium on Neurosecretion. Japan. Sci. SOC.Press. Tokyo-Springer Verlag, Berlin, 11-25. Chauvet, M. T., L&y, B., Michel, G. Chauvet, J. and Acher, R. (1986). Precursors of mesotocin and vasotocin in birds: Identification of VLDV- and MSEL-neurophysins in chicken, goose and ostrich. Bioscience Rep. 6: 381-385. Michel, G., Chauvet, J., Chauvet, M. T. and Acher, R. (1987). One-step processing of the amphibian vasotocin precursor: structure of a frog (Ram esculenta) ‘big’ neurophysin Biochem. Biophys. Res. Common. 149: 538-544.
MAMMALIAN
MSEL- AND VLDV-NEUROPHYSINS
4. Chauvet, J., Michel, G., Chauvet, M. T. and Acher, R.
5.
6. 7.
8.
(1988). An amphibian two-domain ‘big’ neurophysin: conformational homology with the mammalian MSELneurophysin/copeptin intermediate precursor shown by trypsin-Sepharose proteolysis. FEBS Lett. 230: 77-80. Nojiri, H., Ishida, I., Miyashita, E., Sato, M., Urano, A. and Deguchi, T. (1987). Cloning and sequence analysis of cDNAs for neurohypophysial hormones vasotocin and mesotocin for the hypothalamus of toad, Bufo japonicus. Proc. Natl. Acad. Sci. USA 84: 3043-3046. Acher, R. (1980). Molecular evolution of biologically active polypeptides. Proc. R. Sot. London B 210: 21-43. Acher, R., Chauvet, J., Chauvet, M. T. and Hurpet, D. (1985). Evolution of neurohypophysial hormones and their precursors. In: Current Trends in Comparative Endocrinology. B. Lofts and W.N. Holmes (eds) pp. 1147-1152. Hong Kong, Hong Kong University Press. Weber, K. and Osborn, M. (1969). The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 244: 4406-4412.
127 9. Hewick, R. M., Hunkapiller, M. W., Hood, L. E. and Dreyer, W. J. (1981). A gas-liquid-solid phase peptide and protein sequenator. J. Biol. Chem. 256: 7990-7997. 10. Acher, R., Chauvet, M. T., Chauvet, J., Levy, B.. Michel, G. and Rouillt, R. (1988). The Processing of neurohypophysial hormone-neurophysin precusors: A conformation-directed stepwise maturation. In: Proceedings of the Symposium ‘Recent Progress in Posterior Pituitary Hormones’ S. Yoshida and L. Share (eds) Elsevier, pp. 419-426. 11. Ivell, R. and Richter, D. (1984). Structure and comparison of the oxytocin and vasopressin genes from rat. Proc. Nat]. Acad. Sci. USA 81: 2006-2010. 12. Ruppert. S., Scherer, G. and Shiitz, G. (1984). Recent gene conversion involving bovine vasopressin and oxytocin precursor genes suggested by nucleotide sequence. Nature 308: 554-557. 13. Sausville, E., Carney, D. and Battey, J. (1985). The human vasopressin gene is linked to the oxytocin gene and is selectively expressed in a cultured lung cancer. J. Biol. Chem. 260: 10236-10241.