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Neurophysins Molecular and Cellular Aspects.

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[24] A control experiment, in which n-heptanal did not give the enol silyl ethers
(6) but afforded n-C,HliOSiEt2Me under the standard reaction conditions,
led us to suggest that an aldehyde might not be an intermediate [14]. Later,
we found that this conversion into the alkoxysilane was complete before the
temperature of the reaction vessel reached at 140°C. When n-heptanal was
injected under pressure at 140 “C, the enol silyl ethers (6) were found among
the products [1 11.
1251 The silyl group migration to give the metal-carbene complex finds its topo-
logical analog in the known rearrangement of a-silyl ketones to enol silyl
ethers (22bj. Hydrogen migration to a carbene ligand similar to thar in eq.
(k) has been suggested in some reactions [26].
[26] a) K. J. Ivin, J. J. Rooney, C. D. Stewarf, M. L. H. Green, R. Mahfab, J.
Chem. SOC.Chem. Commun. 1978, 604; b) R. B. Culvert, J. R. Shapley, J.
Am. Chern. SOC.99, 5225 (1977).
[271 H. G. Ang, P. T. Lau, Organomet. Chem. Rev. A 8,235 (1972); C. S. Cundy,
R. M. Kingston, M . F. Lappert, Adv. Organomet. Chem. 11, 253 (1973).
[28] D.
L Morrison, A . P. Hagen, Inorg. Synth. 13, 65 (1972).
1291 L. H. Sommer, J. E. Lyons, H. Fujimofo, J . Am. Chem. SOC. 91, 7051
(1969).
[30] A. J. Chalk, Chem. Commun. 1970, 847.
1311 B. J. Alvlett. J. M. Camobe~l.1. Chem. SOC. A 1969. 1910: J. M. Burlitsh. J.
Am. Chem. Soc. 91,4562 (1969); J. F. Bald, Jr., A . G. MacDiarmid, J. Organomet. Chem. 22, C 2 2 (1970); H. Schaifr, A . G. MacDiarmid, Inorg. Chem.
15, 848 (1976).
E. Colomer, R. J. P. Corriu, J. Chem. Soc. Chem. Commun. 1976, 176.
E. Colomer, R. J. P. Corriu, J. C. Young, J. Chem. SOC.Chem. Commun.
1977, 73.
H. Sakurai, K. Miyoshi, Y. Nakadaira, Tetrahedron Lett. 1977, 2671.
Y. Seki, S. Murai, 1. Yamamoto, N. Sonoda, Angew. Chem. 89, 81 8 (1977);
Angew. Chem. Int. Ed. Engl. 16, 789 (1977).
1361 W. M. h g l e , G. Prefi, A. G. MacDiarmid, J. Chem. SOC.Chem. Commun.
1973, 497; B. K Nicholson. B. H. Robinson, J. Simpson, J. Organomet.
Chem. 66, C 3 (1974); B. K. Nicholson, J. Simpson, ibid. 155, 237 (1978).
(371 E. W. Coluin, J. Chem. Soc. Rev. 1977. 15.
[38] W. Reppe, H. Kroper, H. J. Pisfor, 0. Weissbarth, Justus Liebigs Ann.
Chem. 587, 87 (1953).
1391 S.Mural, 7: Kafo, N. Sonoda, Y. Seki, K Kawamoto, Angew. Chem. 91,421
(1979); Angew. Chem. Int. Ed. Engl. 18, 393 (1979).
[401 L. Marko, Proc. Chem. Soc. 1962,67; L. Marko, P. Srabo, Chem. Technol.
(Berlin) 13, 482 (1961); Chem. Abstr. 56, 7102 (1962); M. Polieuka, E. J.
Misfrik, Chem. Zvesti 26, 149 (1972); Chem. Abstr. 77, 113388 (1972). See
[12b], pp. 12 and 84, and also (48aj.
[411 J. A. Gladyz, J. C. Selouer, C. E. Sfrouse. J. Am. Chem. SOC. 100, 6766
( 1 978); see also [48 b].
142) T. Yukawa, H. Wakamatsu, Brit. Pat. 1408857 ( 1 974); S. K. Bhattacharyya,
S. K . Palit, A . D. Das, Ind. Eng. Chem. Prod. Res. Dev. 9, 92 (1970).
(431 Y. Seki, S. Murai, N. Sonoda, Angew. Chem. 90,139 (1978); Angew. Chem.
Int. Ed. Engl. 17, 119 (1978).
[a]
The possibility of “co-oligomerization” of HSiR,
and CO as an entry to persilylated sugar derivatives was suggested by Prof. A . Nakamura at the Summer Organometallic Chemistry Seminor, Yunoyama, Japan, June 1978.
(451 For reviews see: F Hudrlik, J. Organomet. Chem. Library 1, 127 (1976); S.
S. Washburne, J. Organomet. Chem. 83, 155 (1974); c) E. Cooper, Chem.
Ind. (London) 1978, 794.
I461 S. Mural, Y. Kuroki, T. Aya. N. Sonoda, S. Tsutsumi. J. Chem. SOC.Chem.
Commun. 1972, 741; S. Murai, Y. Kuroki, K . Hasegawa, S. Tsufsumi, ibid.
1972, 946. For the latest paper of this series: I. Ryu, S.Murai, Y. Hatayama,
N. Sonoda, Tetrahedron Lett. 1978. 3455.
[471 J. K. Rasmussen, Synthesis 1977, 91.
[481 Note added in proof: a) The reaction of HCHO with HCo(CO), is reported
to give HOCHZCHO;J. A. Rofh, M. Orchm, J. Organement. Chem. 172, C
27 (1979). b) An a-siloxyalkylmanganese complex was obtained by reaction
of C,H,CHO with (CH,),SiMn(C0)5; D. L. Johnson, J. A. Gladyre, submitted for publication.
Neurophysins: Molecular and Cellular Aspects[**]
By Roger Acher“]
Neurophysins are linear cystine-rich proteins containing 93-95 amino acid residues which
like the neurohypophysial hormones oxytocin and vasopressin are formed in the hypothalamus
and travel from there to the hypophysial posterior lobe. A species usually contains two (or
three) neurophysins which differ only slightly in chain length and/or sequence. Many observations suggest that both oxytocin and one of the neurophysins as well as vasopressin and the
other neurophysin have a common precursor whose long chain is split into neurophysin and
hormone. It can be shown on rats having considerable diabetes insipidus that a single gene
controls the biosynthesis of the vasopressin and one of the neurophysins.
1. Neurosecretion and Neurohormones: First Investigations
The history of the neurophysins cannot be dissociated
from that of neuroendocrinology. In fact, the early efforts of
biochemists to purify the “posterior lobe hormones” of the
[‘I
Prof. Dr. R. Achei
Laboratoire de Chimie Biologique
Universite de Paris VI
96 Boulevard RasDail. F-75006 Paris (France)
[**I This review IS dedicated lo the memory of Ernst Scharrer, who some fifty
years ago discovered neurosecretion
846
0 Verlag Chemie, CmbH, 6940 Weinheim, 1979
pituitary, the activities of which have been known since the
beginning of this century, were paralleled by cytologists’
studies aimed at identifying the cells synthesizing these substances and at elucidating the secretory mechanism involved.
It was Ernst Scharrer[’]who in 1928, on the basis of his observations in certain hypothalamic neurons of a teleost fish,
Phoxznus Iaeuis (Fig. l), for the first time clearly proposed the
concept of neurosecretion. At the time, this idea was indeed
revolutionary, nerve cells being considered as highly specialized for the conduction of nervous impulses and totally different from regular gland cells. For a long time thereafter,
the posterior pituitary was interpreted as an autonomic endo-
0570-0833/79/1111-0846
.$ 02.50/0
Angew. Chem. I n t . Ed. Engl. 18, 846-860 (1979)
Fig. 1 . The neurosecretory cell (a) can he considered as either a newe cell with
the characteristicsof a gland cell (b), or a gland cell having acquired the characteristics of a nerve cell. In fact, it is a neuron which has very early undergone a
second differentiation. The secretory granules have to he transported along a
particularly long pathway from the site of their synthesis (perikaryon) to the site
of their release (after [12]).
crine gland, and the synthesis of its hormones was attributed
to special glial cells, the pituicytes, present in this lobe[’].
Only after the introduction by Bargmann[31of the chrome
hematoxylin phloxin method-originally intended by Gomorif41for the demonstration of the p cells of the islets of Langerhans-was it possible to demonstrate the presence of secretory granules throughout the entire neurosecretory neuron. In 1951, Bargmann and Scharrer[’I definitely established
that the perikarya of the supraoptic and paraventricular nuclei of the hypothalamus of higher vertebrates (corresponding to the preoptic nuclei of fishes and amphibians) synthesize a substance, called neurosecretory material, which is
transported by axoplasmic flow to the posterior lobe where it
bl
a)
chiasma
nucleus paraventricularis
pars anterior
Fig. 2. Effects of severance of the pituitary stalk in the dog. Diagram of hypothalamo-hypophysial system, (a) of normal dog, and (b) of operated dog. The
neurosecretory material originating in the supraoptic and paraventricular nuclei
“migrates” along the axons and accumulates in the posterior lohe. After stalk
sectioning neurosecretory material accumulates at the proximal stump of the axons (in A) (after 1121).
is stored and eventually released (Fig. 2). Thus the concept
of a hypothalamic-neurohypophysial secretory system became established, involving a long intracellular route of
transport in the course of which the possibility of modification of the initial product cannot be excluded. The fact that
the presence of active principles demonstrated in various experiments generally coincided with that of the substance
stainable by the Gomori technique led to the conclusion that
the neurosecretory material represents either a substance associated with these hormones or the hormones themselves.
In the area of chemical characterization, the first papers by
Abel et aL[61between 1920 and 1930, suggested that the oxytocic, pressor and antidiuretic activities of the posterior pituitary belong to a single hormone. Later, two contradictory
concepts were defended by different laboratories: the ones
maintaining to have isolated a substance of high molecular
weight possessing oxytocic as well as pressor activities, the
Angew. Chem. I n t . Ed. Engl. IS, 846-860 (1979)
others having succeeded in obtaining substances with either
the one or the other activity possessing a very low molecular
weight, of the order of 1000. The investigations of Rosenfeldl’l, based on ultracentrifugation, suggested that the activities are due to one or two proteins with a molecular weight of
the order of 20000 to 30000, but that substances of low rnolecular weight appear after treatment of the extracts with hot
0.25% acetic acid. In 1942, Van Dyke et al.[*]extracted frozen
glands at low temperature and, after fractional precipitation
with sodium chloride, obtained a protein considered to be
pure and endowed with oxytocic, galactogogic, pressor and
antidiuretic activities in ratios corresponding to those in the
posterior lobe. Van DykeLs1dismissed the possibility of the
absorption of small active molecules by an inert protein on
the basis of several experiments the two principal ones of
which were as follows:
Free-boundary electrophoresis does not dissociate the activities from the protein, the unfolding of the protein as a
film 8 A thick leads to inactivation, whereas if the activities
belonged to separate peptides, they would have to be always
present, be it in the surrounding fluid or associated with the
denatured protein. The liberation of active peptides would
require cleavages which in the case of extracts are artificial,
but which could be physiological at the time of secretion.
Nevertheless, Kamm[’’, followed by Du Vigneaudllol,Fromageot[“] and their collaborators, in 1953, isolated from acetone-treated glands (“acetone powder”) by extraction with
hot 0.25% acetic acid two fractions, one with oxytocic and
galactogogic, the other with pressor and antidiuretic activities. This then presented the problem of determining the natural form of the hormones, protein or peptides, and of clarifying the relationships between the different active principles.
Moreover, the intracellular localization of the hormones
and the endocrine significance of the substance stainable
with the Gornori technique remained to be established. The
Gomori reaction with chrome hematoxylin is not specific for
a given chemical substance, but rather constitutes a staining
process; not only can certain other neurosecretory systems,
i. e. those of invertebrates[‘’], be demonstrated by this method, but also different endocrine cell types, e. g. the p cells of
the islets of Langerhans. Thus the chemical nature of the
stainable substance cannot be determined in this way. Despite the fact that Hild and Zetler[131suggested that one is
dealing with a phosphatide soluble in organic solvents and
not with hormonal proteins or peptides, the histochemical
studies of Sloper et al. [I4] showed that the “Gomori-positive”
granules stain equally well with performic acid/alcian blue,
which are specific for cystine and cysteine, and that consequently the material has to be either a protein or a peptide
rich in these amino acids. This applies specifically to the neurohypophysial peptide hormones or to the proteins with
which they are associated, i. e. the neurophysins. It was,
therefore, particularly interesting to find that the neurophysins, be they alone or in association with the hormones, stain
with the Gomori method[l51.
Since the first review on the neurophysins by Acher and
Fromageot[”] several monographs on this subject have been
published (see e. g. [I6* ”I). Furthermore, the proceedings of a
symposium on “Neurophysins: carriers of peptide hormones” has appeared as a separate volume[181.
841
2. The Reversible
Hormone Complex
Neurophysin-Neurohypophysial
ponents was obtained likewise, either by counter-current distribution (Fig. 3) or by treatment with 5% trichloracetic acid,
Without denying the existence of small peptides endowed
with hormonal activity, the followers of the unitary theory
considered them as degradation products formed in the
course of purification of the active protein. In fact, the posterior lobe extracts obtained at cold temperature appeared to
be devoid of these small molecules. Besides, the possibility of
non-covalent binding between a protein and peptides, at the
time visualized as purely fortuitous, seemed hardly plausible,
since the different protein preparations possessed specific reproducible activities and in ratios corresponding to the glandular content.
Since the active protein could play the role of a common
precursor for oxytocin and vasopressin, it seemed to us important to elucidate the mechanism of formation of the peptides from a macromolecule1"~20~.
The material obtained, under the conditions of Van Dyke, from fresh or acetone dried
glands had all the characteristics described, namely: oxytocic
and pressor activities of 18 U/mg, a sedimentation constant
of 2.6-2.8 S, and a diffusion constant of 8.5 x lo-' cmz s-',
from which it could be concluded that the molecular weight
was close to 30000. Based on this value, the estimated activities appeared in the neighborhood of 500 U per pmol, i.e.
about equal to those of 1 pmol of oxytocin and 1 pmol of vasopressin. It was tempting to assume a stoichiometric relationship between the putative precursor and the active peptides. What then became a primary task was the rigorous verification of the homogeneity of the active protein. In the
electrophoresis experiments of Van Dykela]the protein activities were not separated; this was accomplished by Irving and
Du Vigneaud"] who used the juice pressed from freshly
slaughtered glands that had been immediately frozen and
then ground up in the cold in the laboratory. This suggests
that, under conditions of apparently weak proteolysis, the
pressor principle migrates more quickly towards the cathode
than the oxytocic principle.
We first carried out the free-boundary electrophoresis of
the Van Dyke protein at low temperature (so as to minimize
hydrolysis), and quantitatively determined the activities and
the nitrogen content in the three parts of the U-shaped cell.
Under conditions where the yields of the active principles
and of nitrogen were of the order of 90-95% at the end of
the experiment, the pressor activity per mg nitrogen was 5
times higher on the cathode side than on the anode side. The
electrophoretic dissociation thus accomplished was then further exploited by subjecting the material to electrodialysis
which separated proteins from small peptides. On using an
electrodialyzer with three compartments separated by cellophane membranes and a current of only 12-15 mA (so as to
avoid heating), it was possible to collect, from the active protein placed in the central compartment, practically all of the
oxytocic and pressor activities in the cathode compartment.
90% of the nitrogen was recovered from the original, i. e. central compartment, the protein having remained
The only constituents identified in the cathode compartment
were oxytocin and vasopressin, thus indicating that the entire
activities of the protein complex were due to these peptides
associated with the protein by non-covalent bonds. The
quantitative dissociation of the active protein into three com848
Fraction
-
Fig. 3. Separation of the components of the neurophysin-neurohypophysial hormone complex by counter-current distribution in the system sec-butanol/0.5%
oxytocin activity, -- nitrotrichloroacetic acid. '... vasopressin activity,
gen.
~~~~
which precipitates the protein but leaves the peptides in the
supernatant fluid (Fig. 4).This latter method opened the way
to the simultaneous purification of the two hormones: the
+
0
0
I
r
.
&.--i-
I
i*/
\
.....m....c> ._..
16
20
-
8
12
I[cml
Fig. 4. Identification by paper electrophoresis of oxytocin and arginine vasopressin in the cathodic material obtained after electrodialysis of the supernate resulting from the dissociation of the "active" complex by 5% trichloroacetic acid.
Above: electrophoresis strips developed with Bromophenol blue, numbered acvasopressin
cording to fraction. Below: activity test; .... oxytocin activity,
activity. I = migration towards cathode (after [20]).
~~~~
protein-peptide complex was first separated from the extract
by fractionated precipitation; after dissociation of the complex with trichloracetic acid, oxytocin and vasopressin were
isolated by chromatography on an ion-exchange column[22'
(Fig. 5).
We have proposed the term neurophysin for the protein
which seems to be specifically associated with the horAngew. Chem. Inr. Ed. Engl. 18, 846-860 (1979)
electrophoresis at pH 8.1, Hope[’61observed that the neurophysin isolated under our conditions contained several constituents. He attributed a part of these to proteolytic degradation occurring during the extraction at pH 4.0, and recommended acid extraction by means of 0.1 N hydrochloric acid
in order to denature the proteolytic enzymes irreversibly.
However, under these conditions, some contaminating proteins of higher molecular weight are extracted together with
the complex and must be eliminated by a supplementary step
of molecular sieving. HopP“ recognized the presence of two
major components and one minor component in the neurophysins of beef and pig and proposed for them the names
Neurophysin I, 11, etc., in the order of their decreasing mobility toward the anode. This nomenclature, thus far accepted, can however suggest erroneous homologies between one
species and another, since the number and mobility of the
components vary in each species and since it is difficult to
distinguish the authentic molecular species from degradation
products. Thus one observed several neurophysins in the
sheep although only one major protein can be detected[26.26a,27,27a].
Polymorphism has been noted in several
mammals, including man[”] and rat[29];however, it is important to note that in the guinea pig, Suchs et al.13’l demonstrated only a single neurophysin.
In the meantime at least two sequentially different neurophysins have also been found in guinea
The purification of neurophysins is primarily accomplished by following two kinds of procedures which differ essentially in the first steps. In general, the initial material is an
acetone powder of posterior lobes, but it can consist of fresh
or frozen glands. An essential factor seems to be the speed
with which the glands are removed after death. In the first
p r o ~ e s s [ ~the
’ ~ ~powder
~ ~ , is extracted at 4 “C with 0.01 N sulfuric acid, the pH being about 4. Under these conditions, the
major part of the extracted material consists of the neurophysin-neurohypophysial hormone complex, which is then
purified by fractionated precipitation with sodium chloride
at pH 3.9 (Table 1).
After removal of the salts, the complex is dissociated into
the proteins and peptide hormones by gel filtration on Sephadex G-25 with 0 . 2 ~
acetic acid. The proteins consist es-
i3~
I
-lOlM,pH5.0
-0.5M,pH7.7)
Fraction
-
++i+OZSM,pH7.7)-
Fig. 5. Simultaneous isolation of oxytocin and arginine vasopressin by gradient
chromatography, on Amberlite CG-50, of the supernatant material resulting
from the dissociation of bovine “active” complex by 5% trichloroacetic acid.
Right-hand ordinate: oxytocin (OX-) and vasopressin (VP-) activity [units] (after
[221).
m ~ n e [ ‘ ~This
I . specificity was confirmed by the reconstitution
of the complex from its components, neurophysin, oxytocin
and vasopressin, at the same ratio as that observed in the initial
Fu~hermore,“hybrid” complexes were obtained with the neurophysin from one species and the hormones from another; mammalian neurophysins were thus
used as selective adsorbents in the purification of active principles of lower vertebrates[241.
Neurophysin-hormone complexes were prepared from the
glands of various mammalian species and it seems that this
type of association is general among vertebrates. In the case
of the ~hiting-pout[~’],
the precipitation of the complex required a higher sodium chloride concentration than in mammals. This might suggest a different constitution of the neurophysins of lower vertebrates or a degradation. In general,
the yield in these species was increased by the addition of bovine neurophysin to the extracts.
3. Characterization of Neurophysins
3.1. Purification of Neurophysins
In spite of the fact that a certain heterogeneity of the neurophysin became apparent in the course of the counter-cur-
.~
Posterior pituitary powder
Activities
IU/mgl
ox
VP
VP/OX
ox
ox
1.O
Calf (new born)
Sheep
Pig
Horse
Whale
Rat
Rabbit
0.4
0.8
Species
1.4
0.9
0.5
1.2
0.9
Complex
1.1
18-20
.o
2.5
0.8
1.4
0.8
2.5
1.4
1.7
1
1.o
.o
9
18
0.9
5.0
18
20
5
1.1
1
18-20
21
18
18
Weight
[g/lW 91
Yield in
activities
[%I
VP/OX
10
1.O
2.3
1.O
1 .O
0.5
20
4.0
1.5
3.0
4.2
1.5
0.8
6.0
27
61
92
19
18
47
30
61
90
19
10
57
1.2
1.9
rent distribution (Fig. 3) and paper electrophoresis experiments at alkaline pH, the protein separated from the complex seemed to correspond to a single molecular species in
the course of ultracentrifugation. However, during starch-gel
Angew. Chem. inr. Ed. Engl. 18, 846-860 (1979)
Activities
[U/mgl
VP
sentially of neurophysins, which are then separated by chromatography on a basic ion-exchanger, diethylaminoethyl-sephadex A-50,using a gradient ionic strength (0.2-1 M pyridine acetate buffers) (Fig. 6). Under these conditions two well
849
separated fractions are obtained Fraction A in the case of
beef, sheep, and pig contains a neurophysin which, accord3001
,
a)
t
b’
t
c’
U
‘0
500 1000 1500 0
500 1000 1500 0
500 1000 1500
+l-Z--I
&--24
i-1-2-9I
mlrnl
rnl-
-
Fig. 6. Isolation of a) bovine, b) equine and c) ovine MSEL-neurophysins by gradient chromatography on DEAE-Sephadex A-50. Ordinate: absorption at 700
nm (Folin). A, B see text. 1: 0 . 2 ~ + 0 . 4 ~2:: 0 . 4 ~ + 1 . 0 ~
pH=5.9
.
(after 1271).
ing to the nature of the amino acids in positions 2 , 3 , 6 and 7,
we call MSEL-neurophysin (cf. Table 2). This material, depending on the conditions of preparation of the acetone
powder, may contain derivatives truncated by several residues at the N- and C-terminus. Fraction B usually contains a
second type of neurophysin (VLDV-neurophysin), as well as
degradation products that have not yet been clearly identified.
In the second procedure used by Hope et aZ.[16],
BresZow[”]
and WatkinslZ61,the extraction of the acetone powder is carried out with 0.1 N HC1 at a pH of about 2.0. Neutralization
to pH 7.0 leads to formation of a precipitate which can be
separated by centrifugation. Readjustment of the pH to 3.9
and precipitation with sodium chloride furnishes a material
containing the complex together with contaminating proteins
of higher molecular weight. The peptide fraction of the extract is separated off by gel filtration on Sephadex G-25 in
0.1 M formic acid; the protein fraction is then passed through
a gel of Sephadex G-75 once again so as to remove the proteins of higher molecular weight. The “neurophysin” fraction is then chromatographed on the basic ion-exchanger
diethylaminoethyl-Sephadex A-50 at a constant pH of 8.1
(Tris-HC1) with increasing sodium chloride gradient. Two or
three fractions are thus separated, from which at least three
components possessing different electrophoretic mobilities
can be isolated by repeated chromatography. Nevertheless,
so far only two distinct neurophysins have been identified
beyond doubt by means of sequential analysis of proteins
from beef, pig and sheep glands. Other purification procedures, including selective extraction, isoelectric focusing or
affinity chromatography on “immobilized” hormones, have
been tried occasionally, but have thus far not been used extensively.
“crude” or purified n e u r o p h y s i n ~ [ ~ ~In. ~ contrast,
” ~ ~ ~ . the
molecular weight determinations by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate
give lower values of the order of 12500 for the neurophysins[321.Finally, sequential analysis of the peptides furnished
by trypsin hydrolysis of the oxidized neurophysins has revealed that the molecular weight of the monomer lies around
10000. A “dimerization” might therefore easily occur at pH
4 to 8, yielding associations stable in the course of molecular
sieving or of ultracentrifugation.
Another interpretation might be that the molecular
weights evaluated by ultracentrifugation or by gel filtration
are too high owing to the unusual conformation of the molecule[44“l.
Table 2. Amino acid sequence of the MSEL neurophysins of sheep [34, 34a], ox
134, 34a1, pig 137, 37a, b], horse [37c] and whale [37d].
Sheep
1
5
I0
Ala- Met-Ser - Asp- Leu- Glu- Leu- Arg- Gln-Cys- Leu- Pro -
ox
Pig
Horse
Whale
IS
20 21
25
Cys- Gly - Pro - Gly -Gly -Lys- Gly - Arg- Cys- Phe- Gly - Pro - Ser - Ile - Cys-
30
35 36
40 41
Cys- Gly- Asp-Glu- Leu- Gly- Cys- Phe- Val - Gly- Thr- Ala - Glu- Ala - Leu-
- Met -
45
48
50
55
Arg- Cys- Gln- Glu- Glu- Ile - Tyr - Leu- Pro - Ser - Pro - Cys- Gln- Ser - Gly
- Asn .
-Am.
- Asn -Am.
65
70
60 61
Gln-Lys- Pro - Cys- Gly - Ser - Gly -Gly - Arg- Cys- Ala- Ala - Ala - Gly- Ile -
7s
80 81
85
Cys- Cys- Asn- Asp- Glu- Ser - Cys- Val - Thr- Glu- Pro - Glu-Cys- Arg- Glu-
3.2. Amino Acid Sequence of the Neurophysins
The molecular weight of the neurophysins is still under
discussion. By ultracentrifugation at pH 4.8,Hope et al. obtained values of 19000 and 21 000, respectively, for bovine
neurophysins I and IP”. On assuming a molecular weight of
25 000-30000 for the complex one could roughly calculate
that one molecule of neurophysin is associated with one molecule of oxytocin and one molecule of arginine vasopressin.
If the molecular weight is measured by molecular sieving on
Sephadex G-75 at pH 7.0, values of the order of 1500@20000 are obtained, both for the complex as well as the
850
89 90 91 92
95
Gly -1le - Gly - Phe- Pro - Arg- Arg- Val
Ile
- -Val-
- -Ala- Ser ---Leu-
__ -Ala
- -Ala---Leu-
-Ala
- -Ah-Ser-
-Ala
Angew. Chem. Inl. Ed. Engl. 18,846-860 (1979)
The neurophysins isolated thus far are acid proteins having isoelectric points between 4 and 5. They are distinguished from other proteins by a very high content of cystine
(seven disulfide bridges for a chain of 93-95 residues) and
proline (seven to eight residues) and by a very low content of
aromatic amino acids: they do not contain tryptophan, and
have only one tyrosine residue, and two to four phenylalanine residues per molecule. This explains the atypical absorption spectrum in the ultraviolet with a 260/280 nm absorbance ratio greater than unity.
the three neurophysins demonAccording to Hope et
strable in beef and pig glands all possess an N-terminal alanine. A comparable result was then obtained for the three
components of
On the other hand, in the case of the
beef neurophysins, amino acid analyses have shown that
neurophysin I contains histidine but no methionine, whereas
neurophysins I1 and C contain methionine but no histidine1Ib1.In the pig, none of the three neurophysins contains
histidine and only two of them contain methionine. The
presence or absence of methionine often serves for the distinction of the different neurophysins in a given species.
The actual data concerning complete amino acid sequences of neurophysins are so far limited to those of beef,
sheep, pig, horse and whale. N-terminal sequences are
known for the neurophysins of rat, dog, cod and man. It
seems that every species possesses at least two neurophysins
which differ essentially from each other by the N- and C-terminal sequences. Consequently, it has been proposed to distinguish between two families, the MSEL-neurophysins and
the VLDV-neurophysins, according to the residues in positions 2,3,6 and 7[331.
All neurophysins are single-chained polypeptides of 93-95 amino acids.
The MSEL-neurophysins are actually the best known. The
complete amino acid sequences that have so far been determined are collected in Table 2. The amino acid sequence
proposed by Walter et al. [35,361 for beef MSEL-neurophysin
would appear by way of analogy[381
to be less likely than the
sequence in Table 2. The amino acid sequence suggested by
chain of 95 residues. A large N-terminal part (residues 188) is nearly invariant since substitutions occur only once in
position 36 (valine in all, except methionine in whale) and
position 48 (asparagine in all, except isoleucine in sheep). In
contrast, for the C-terminal part of the chain, variations can
be observed in positions 89,90,91,92 and 95. In the ox, a microheterogeneity is found in position 89, since two forms of
MSEL-neurophysins are found, one with isoleucine, the other with valine.
Except for position 90 in which glycine or serine is found,
the “mutable” positions of the C-terminal sequence are occupied by hydrophobic residues. ‘Curiously,positions 89 and 95
seem related since both are occupied either by alanine (pig,
horse, whale) or by isoleucine or valine (ox and sheep).
The five species examined belong to three orders of eutherian mammals: Artiodactyla (ox, sheep, pig), Perissodactyla (horse) and Cetacea (whale). These orders diverged
about 70-80 million years ago. If the sheep is taken as reference, the percentage of substitutions between two different
orders is 4-5%. The “drift” of MSEL-neurophysin in the
course of evolution is small when compared with those of hemoglobin chains (12-20%). On the other hand, the close resemblance between ox and sheep MSEL-neurophysins is
reminiscent of the similarity found for several pituitary polypeptide hormones (Table 3). A “neutral” drift is hard to
imagine because both species (which belong to the suborder
Ruminantia) diverged perhaps 30 million years ago and the
number of substitutions is not proportional to time.
All the MSEL-neurophysins contain 14 cysteine residues
per molecule (monomer) which form seven disulfide bonds;
a certain mode of bonding has been suggested in the case of
the ox protein[391,but since the sequence from which it was
deduced is questionable, the definite location of the disulfide
bridges still remains unclear.
The VLDV-neurophysins seem less abundant than the
MSEL-neurophysins and the proportion of them appears to
vary from species to species, so that their characterization is
less advanced. The complete amino acid sequence of pig
Table 3. Amino acid sequence of the neurohypophysial hormones
1
2
3
4
5
6
7
8
4
5
6
7
8
-
9
Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-GlyNH2
Oxytocin
9
C:s-T2yr-P~e-GIn-Asn-Cys-Pro-Arg-GlyNHZ Arginine-vasopressin
Oxytocin type
4
Vasopressin type
3
8
8
Mammals (except pig)
Gln
Leu
Oxytocin
Phe
Arg
Pig
Birds, reptiles,
Amphibians, lung
fishes
Bony fishes
(poleopterygio and
Gln
Leu
Oxytocin
Phe
LY s
Arginine-vasopressin
Ly sine-vasopressin
Gln
Ile
Mesotocin
Ile
A%
Vasotocin
Ser
Ile
Isotocin
Ile
Arg
Vasotocin
Ser
Gln
Asn
Gln
Val
Leu
Glumitocin
Valitocin
Aspargtocin
Ile
Ile
Ile
Arg
Arg
Arg
Vasotocin (?)
Vasotocin (?)
Vasotocin (?)
neopterygia)
I
I
Cartilaginous fishes (rays)
Cartilaginous fishes (sharks)
Chauvet et al.[341
for beef MSEL-neurophysin has been confirmed by Wuu and C r ~ r n r n ‘ ~ ~ ~ ~ .
The MSEL-neurophysins characterized up to now (ox,
sheep, pig, horse and whale) consist of a single polypeptide
Angew. Chem Int. Ed. Engl. 18, 846-860 (1979)
VLDV-neurophysin (neurophysin 11) has been indicatedl4’].
The N-terminal sequences of VLDV-neurophysins from ox
(neurophysin
man (neurophysin I)[421,rat (neurophysin
II)[42a1
and dog (neurophysin
are known. VLDV-neu851
rophysins contain 93 residues per molecule and 14 cysteine
residues, i. e. they resemble the MSEL-neurophysins.
The complete amino acid sequences of
and
OX[^^^,^^^^ VLDV-neurophysins have recently been published
(Table 4)[40a1.
The sequence for bovine VLDV-neurophysin
in Table 4 differs from that published by Schlesinger et ul.[41a1
Table 4. Amino acid sequence of porcine and bovine VLDV-neurophysin. The
arrangement of the tryptic peptides (pig: TI-T7; ox: Tt-T6) has been determined with overlapping peptides which were obtained with the protease from
Staphylococcus (S1 -S5) and by sequencing (N-terminal sequence) (after
Wal).
TI
19
Ala - Val - Leu- Asp- Leu- Asp-Val - Arg- Lys - Cys- Leu- Pro T1
LThr-
Table 5. Comparison of MSEL and VLDV neurophysins. Upper series: Sequence of the MSEL neurophysins of sheep, ox, pig, horse and whale. Lower series: Sequence of the VLDV neurophysins of pig and ox. Interchanged amino
acids within the two groups are given (after [4Oa]).
MSEL
1
2
3
5
6
7
9
10
Ala - Met-Ser - Asp- Leu- Glu- Leu - Arg- Gln- Cys- Leu- Pro - Cys-
VLDV
--
Val - Leu--
- ~ s p - v a -i --
Lys Thr
15
20 21
25
Gly - Pro - Gly -Gly - Lys - Gly - Arg- Cys- Phe- Gly- Pro - Ser - Ile
30
35
40
36
Gly- Asp- Glu- Leu -Gly -Cys- Phe-:(-Gly-Thr-
-
Cys- Cys-
41
Ala - Glu- Ala - Leu- Arg-
1
Pig
ox
45
50
48
55
Cys- Gln -Glu - G l u - i T - Tyr - Leu - Pro - Ser - Pro - Cys- Gln -Ser - Gly - Gin -
T2
1-3
20 121
Cys- Gly - Pro - Gly -Gly - Lys- Gly - Arg- Cys- Phe- Gly - Pro - Ser - Ile - CysT2
T3
1
60 61
64 65
70
Lys - Pro - Cys- Gly -Ser - Gly -Gly - Arg- Cys- Ala - Ala - Ala - Gly - Ile
Glu-
-
Cys-
Gly
s2
SI
1
40
h1
Cys- Gly - Asp- Glu- Leu- Gly -Cys- Phe- Val - Gly -Thr- Ala - Glu- Ala - Leust
s2
1
T4
53
I
1
T?
s,3
75
xo 81 82
85
Cys- Asn- Asp-Glu-Ser - Cys- Val - Thr- Glu- Pro - Glu-Cys- Arg- Glu-Gly Asn
Arg Phe
--Ser
-Pro- Asp-Gly- -His - G l u - A ~ p- -Ala - -Asp-Pro - Glu-
1
Arg- Cya- Gln- Glu- Glu- Asn- Tyr - Leu- Pro - Ser - Pro - Cys- Gln-Ser - Gly -
89 90 91 92 93
95
lle Gly Phe Pro
Val
Val- -Arg-ArgAla Ser Leu Leu
Ala
A1a-Z;:-
T5
60 61
64
1
Gln-Lys- Pro- Cys- Gly-Ser - Glu-Gly- Arg- Cys- Ala- Ala - Ala- Gly-Ile
T5
Phe-Ser - Gln
tion of a primitive gene coding for 55-60
sion of the daughter genesc4'].
residues and fu-
T6
75
80 1x1
Cys-Cys- Asn-Pro - Asp-Gly-Cys- Arg- Phe - Asp- Pro - Ala - Cys- Asp- Pro
- Ser -
~
-His - Glu-
T7, 55
93
Glu-Ala - Thr- Phe-Ser - Gin
s4
T6, S5
A - A l a s4
I
90
by the C-terminal residue: glutamine instead of leucine. Table 5 compares the five MSEL-neurophysins and the two
VLDV-neurophysins known to date. The homology between
the MSEL- and VLDV-neurophysins of the same species is
about 80%. The homology within the MSEL-family (five
species) ranges from 95 to 99%. The two types of neurophysins have now been recognized also in man[40b';so the duality
exists in all the mammalian species investigated.
The two families of neurophysins have in common a long
central sequence of about 65 residues (between positions 10
to 75) and seem to differ mainly in their N-terminal and Cterminal sequences. This fact strongly suggests that the origin
of the polymorphism is duplication. On the other hand an internal homology between two parts of the same chain has led
to the hypothesis that the chain is formed by partial duplica852
3.3. Conformation of the Neurophysins
The three-dimensional structure of the neurophysins has
not yet been determined. The CD spectrum of beef MSELand VLDV-neurophysins are very similar and suggest a
small percentage of a-helix (about 5%) but a very high percentage of @-structure(about 40%)[17.431.
The marked accessibility of the disulfide bonds to reducing agents in the absence
of ~ r e a [ ~suggests
. ~ ~ ~a conformation
l
of low compactness. In
the absence of urea, routinely used to unfold proteins, the
seven disulfide bonds of the molecule are entirely reduced by
dithiothreitol, which could be explained if the protein had a
naturally extended structure. Its cleavability by enzymes
leads to the same conclusion: the protein with its seven disultide bonds intact is split by trypsin or chymotrypsin in practically the same way as in the unfolded state after reduction
and alkylationI4"! This extended (or easily extendable) conformation can be explained by the great number of proline
residues, which for proteins of animal origin is most uncommon.
Molecular weight determinations in the absence of dissociating agents suggest that at pH 3 to 8 two subunits are associated to form a dimer, and even more aggregated forms
could exist. It seems probable that the binding of the horAngew. Chem. In[. Ed. Engl. 18, 846-860 (1979)
mones takes place on the dimer, but the latter’s conformation
is still unknown.
r,
b)
4. Interactions between Neurophysins and Neuro-
hypophysial Hormones
The relatively small size of the neurophysins and especially of the neurohypophysial hormones, which belong to the
smallest biologically active peptides, makes the complex a
particularly simple model for the study of protein-protein interactions. Besides, the association is not followed by a chemical modification, as for example in proteolysis, which would
complicate the observations. A very large spectrum of synthetic analogues of the hormones (over 300) has been prepared, primarily due to the efforts of du Vigneaud, Boissonnus and their co-workers, and it has been possible to determine the residues that intervene in the association. Though
complexes of beef neurophysin with arginine vasopressin
have been obtained in crystalline form by Hope et al.1“’, the
crystals have thus far not lent themselves for a radiocrystallographic X-ray study, so the three-dimensional structure of
the complex has not yet been established. Indirect information is obtainable, however, from equilibrium dialysis studies
as well as from variations in ultraviolet absorbance, circular
dichroism, and nuclear magnetic resonance following the
binding of the hormones to the neurophysins.
Fig. 8. Near-ultraviolet CD spectra of bovine neurophysin 11 (MSEL-neuropbysin) in the presence of a) 1 x I O-’ M S-methyl-Cys-Phe-lleNH2 and b) Met-TyrPheNH1. - observed, .... theoretical. “Theoretical” curves represent calculated
spectra for a solution of bovine neurophysin I1 that is I x I O - ’ M in the appropriate peptide with neglection of interactions (after 1431).
4.1. Stoichiometry of the Association
According to the experiments of B r e ~ l o w [ ”the
~ ~ changes
~]
in the CD spectrum reach their maximum when one mole of
oxytocin or one mole of lysine vasopressin is combined with
one mole of monomeric neurophysin (molecular weight of
10000) (Fig. 7-9). By means of an equilibrium dialysis
study at pH 7.4 using labeled oxytocin, lysine vasopressin
and beef neurophysin I1 (MSEL-neurophysin), Breslow and
Walter[46]found that the binding of the two hormones is
b)
LVP/NP
-
OX/NP
.
Angew. Chem I n t Ed. Engl 18. 846-860 (1979)
-
competitive, with association constants of 1.2 x lo4 M for
oxytocin and 8 x l o 3 M I for lysine vasopressin. Moreover,
the authors point out that the affinities for neurophysin I
(VLDV-neurophysin) are similar, so that it is not possible to
demonstrate the specificity of association of one neurophysin
with one given hormone.
On the other hand, the competition and the very similar
association constants suggest that the binding sites of the
neurophysin for the hormones largely overlap and are perhaps even identical; one direct deduction from this is that the
binding sites of the hormones comprise especially the residues which they have in common (seven residues out of
nine). The interpretation by Breslow and Walter does not
take into account the fact that, at the pH at which the binding takes place, neurophysin is likely to be present in the
form of a dimer, and assumes implicitly that each monomer
fixes its ligand in an independent manner. However, the existence of cooperative effects in the binding process has recently been e v ~ k e d [ ~ ’ ,If~ *one
~ . assumes a stoichiometric relationship of 1 : 1 for neurophysin and hormone, one is led to
reckon with a separate binding site in each monomer, and to
identify the residues that constitute it. The relative instability
~
-
Fig. 7. Alteration of C D spectrum on titration of native or nitrated bovine neurophysin I1 (MSEL-neurophysin, NP) with a) lysine-vasopressin (LVP) and (b)
oxytocin (OX). Results are expressed as the percentage of the total change in ellipticity generated by different hormone/protein ratios, where the total change is
defined as the ellipticity of the saturated protein minus the sum of its isolated
components. - One high affinity site, .... one site with K = 2 x lo’, ---- two
equal high affinity sites. 280 nm, native bovine NP-11; o 245 nm, native bovine
NP-11; 350 nm, nitrated bovine NP-I1 (after (451).
.
h [nml
Fig. 9. a) Difference C D spectrum obtained by subtracting the C D spectra of the
individual components of the mixture from the spectrum of the protein-peptide
complex. Spectra obtained in the presence of oxytocin (-) or of lysine-vasopressin (---) represent 100% saturation of protein with hormone. Spectra obtained
in the presence of S-methyl-Cys-Tyr-PheNH2 1....) represent 90% saturation of
the protein. b) Direct circular dicbroism spectra of neurophysin 11 alone (and in the presence of 2 equivalents of oxytocin (-) or lysine-vasopressin (---)
(after [45]).
853
of the complexes, shown by the weak association constants,
makes it difficult to identify the shielded residues as was possible in the case of the associations of trypsin or chymotrypsin with the pancreatic Kunitz i n h i b i t ~ r [ ~ ~ . ~ ~ l .
4.2. Binding Site of the Hormones
Beef neurophysins bind not only the mammalian neurohypophysial hormones (oxytocin, arginine vasopressin, lysine
vasopressin) but also the six active peptides found in the other classes of vertebrates (see Table 3). All these hormones are
nonapeptides, in which only the amino acids in positions 3,4
and 8
The investigations carried out to date have
mainly demonstrated the role of the first, second and third
amino acid residues of the hormones in the binding to the
neurophysins.
Amino Acid Residue No. 1: Acher, Chauvet and Olivry[201
had observed that dialysis at pH 2.75 against 0 . 1 acetic
~
acid leads to dissociation of the complex, permitting the passage of the hormones from the dialysis sac, whereas the same
procedure against water elicits practically no dissociation.
This observation suggested the existence of ionic binding between hormones and neurophysins. Since active peptides
possess all their carboxy groups in amide form, the only possible group with a charge at the pH of fixation is the a-amino
group, which in all cases belongs to a cysteine residue.
The discovery by du Vigneaud et aZ.[s‘lthat this group is
not only unnecessary for hormonal activity, but that its suppression even leads to an increase in activity, has naturally
led to the examination of the binding of deamino-oxytocin to
neurophysin. Stoufer, Hope and du V i g n e a ~ d [ ~observed
*]
that deamino-oxytocin, in contrast to oxytocin, does not associate itself with beef neurophysins if these are precipitated
at pH 3.9 with sodium chloride, nor does it accumulate in the
sac containing the neurophysins if these are dialyzed against
a solution containing the peptide.
These observations were confirmed by Breslow and
A b r ~ s h [ who
~ ~ I have shown that the side chain of the cysteine
in position 1 could be lengthened without disturbing the
binding since I-homocysteine oxytocin attaches itself to the
protein as well as the oxytocin. Subsequently, Breslow et
u1.[s4*ss1
observed that tripeptide analogues of the N-terminal
sequence, which contain S-methylcysteine or methionine in
position 1, were likewise bound, whereas the replacement by
glycine reduces the association constant thirty-fold. The nature of the amino acid in position 1 does not seem to be exactly defined, but the presence of methylene groups in the @and y-position, participating in hydrophobic bonds, and certainly that of an a-amino group, implicated in the electrostatic binding, seem necessary-It must be pointed out that
the deamino-hormones are active, but substitution of the first
residue causes inactivation.
Amino Acid Residue No. 2: Oxytocin containing the hydrophobic aliphatic isoleucine or glycine residue instead of tyrosine in position 2 is not bound whereas the 2-phenylalanine
oxytocin
The presence of one aromatic amino acid in
position 2 thus seems essential and this has been confirmed
with the aid of di- and t r i - p e p t i d e ~ ~ ~These
~ . ’ ~ ~data
. would
suggest the necessity of precise hydrophobic contacts and
perhaps the existence of T-Tinteractions. Finally, D-tyrosine
oxytocin is not bound[53].
854
Tyrosine-2 is present in all known neurohypophysial hormones and this constancy probably indicates a participation
in the association with the protein receptors of the cellular
membranes.
Amino Acid Residue No. 3: Residue No. 3 is isoleucine in
all known neurohypophysial hormones with the exception of
vasopressin in which phenylalanine occupies this position.
The oxytocin with glycine in position 3 has an affinity thirtyfold smaller[s31.Likewise, a glycine residue in position 3 of
tri- and tetrapeptides reduces the binding
It
would seem then that the presence of a hydrophobic aliphatic or aromatic amino acid in position 3 is necessary for binding with neurophysin.
Interestingly, the presence of isoleucine in position 3 seems
essential for a marked association of hormones with the uterine receptor, while phenylalanine favors the renal receptor.
The antidiuretic specialization of mammalian arginine vasopressin, derived from the arginine vasotocin of the other vertebrates, has come about during evolution precisely by this
Amino Acid Residues Nos. 4-9: Residues 4 to 9 do not appear to be essential for the binding with the neurophysins.
The 4-glycine oxytocin binds almost as well as o~ytocin[’~l,
and so does the 5-valine o ~ y t o c i n [ ~
Replacement
~].
of glycinamide in position 9 by glycine reduces the fixation by
50%[461.
Position 8 is occupied by leucine in oxytocin, and by
lysine or arginine in the vasopressins without giving rise to a
marked difference in binding power. Finally, the tripeptide
S-methyl-Cys-Tyr-PheNH,, an analogue of the sequence of
the first three residues of vasopressin, binds with mononitroneurophysin I1 of beef with two-thirds of the free energy of
oxytocin or lysine vasopressin[451.
Hence, it would seem that
fixation to neurophysins implicates essentially the first three
residues of the nonapeptides, whereas the activities of the
hormones tolerate only very slight variations of sequence,
and particularly no “truncation”. The zone of interaction between the peptide and the protein is thus extremely limited
in comparison with other known models of binary proteinprotein association. The weak association constants are in
agreement with a relatively small number of contacts, but location of the site of “anchorage” of the peptide is still not
possible because of a lack of direct structural data.
4.3. Binding Site of Neurophysins
The identification of certain amino acid residues of hormones associating with neurophysins has led to the search
for the protein residues that serve them as binding partners.
Thus, a carboxyl group implicated in ionic binding has received particular attention; in addition the single tyrosine
(tyrosine-49) present in all neurophysins has been studied in
detail.
4.3.1. Aspartic Acid-30, Glutamic Acids-31 and -47
The participation of at least one carboxyl group in the association is shown by the fact that blockage of the acidic residues of the neurophysins furnishes a derivative that no longer binds the hormones[321.
According to the degree of proton
displacement accompanying the binding of oxytocin or lysine vasopressin to beef neurophysin 11, this binding carboxAngew. Chem. Inf. Ed. Engl. IS, 846-860 (1979)
yl group can be assigned a pK value of 4.3[l7I.This would indicate binding at a side chain rather than at the free terminal
a-carboxy group of the protein (pK = 3-3.5). In fact, there
exist variations in length and sequence in the C-terminal region of the chaini34Jwhich make binding at this site unlikely.
Walter and H0ffman[~~1
announced that they were able to
couple the a-amino group of arginine vasopressin with a carboxy group of neurophysin I1 covalently, with the aid of a
water soluble carbodiimide, and to identify the residue involved as Asp-30 or Glu-31. Either one of these could, in the
non-covalent complex, be linked by ionic binding with the
amino group of the hormone. However, B r e ~ l o wsuggested
~~~~
the participation of Glu-47 on the basis of molecular models.
In fact, localization of the acidic residue intervening in the
electrostatic binding has not yet been definitely established.
4.3.2. Tyrosine-49
All neurophysins analyzed up to now possess a single tyrosine residue in position 49. The ease of detection of this amino acid by ultraviolet spectroscopy, circular dichroism, and
nuclear magnetic resonance, has focused attention on Tyr-49
as a residue playing an essential role in the binding of the
protein. The first observation supporting this participation
was made by Furth and
who transformed the residue
to mononitrotyrosine by treatment with tetranitromethane,
and showed that the absorption spectrum of the nitrotyrosine
changed during the binding of arginine vasopressin in a
manner which suggested a lowering of its pK, value. The
lowering of the pK was attributed to the approach of a positively charged group, the guanidine group in arginine-8, or
approach of a positively charged group of the protein following a change in the conformation. The authors also suggested
that the environment of the nitrotyrosine became more hydrophobic after the association. Breslow and Weid4’1 repeated this experiment using CD measurements and spectrophotometric titration. The results indicated that the arginine
vasopressin lowered the pK, of the nitrotyrosine from 7.45 to
6.85 and produced changes in the ellipticity of the tyrosine.
Since these alterations were brought about also by oxytocin
or by analogues involving the sequence of the first three residues of the hormones, they could not be due to arginine-8.
According to NMR investigations by Breslow el al. [58,591 the
ortho and meta protons of Tyr-49 are affected by the association with the tripeptide S-methyl-Cys-Phe-IleNH2,thus confirming that the environment of this residue is modified by
the binding. Application of the nuclear Overhauser effect to
this system established that the changes in the environment
of Tyr-49 on complex formation are attributable to the approach of the peptide residue in position P9].
In the case of
the above mentioned tripeptide, the intensity of the phenylalanine ring proton resonances decreases after irradiation in
the presence of neurophysin 11; this is not observed in the absence of neurophysin 11. The effect occurs also in the case of
hormones with tyrosine in position 2. It would appear that
the changes are not due to a stacking, one above the other, of
the phenol rings of Tyr-49 of the neurophysin and Tyr-2 of
the hormones, but rather to the fact that Tyr-49, masked in
the free protein perhaps by a phenylalanine residue, is displaced from this position, either directly by Tyr-2 or by a
more general change in conf~rmation[~~l.
The finding that
Tyr-49 can be di-nitrated[451or a ~ e t y l a t e d ~without
~ ’ ~ effect
Angew. Chem. Inr. Ed. Engl. 18,846-860 (1979)
on the binding capacity, does not speak in favor of stacking
between Tyr-49 and Tyr-2. In fact, the Tyr-49 of neurophysin would not be in direct contact with the Tyr-2 of the hormone, but in its immediate vicinity. The essential conclusions of Breslow and her group have subsequently been confirmed by other investigators16’].
4.4. Conformational Changes during Binding
The fact that the association between hormones and neurophysins favors precipitation by sodium chloride[52]and
markedly increases the sedimentation c o n ~ t a n can
t ~ ~be~ in~
terpreted either in terms of a modification of the tertiary
structure of the protein, or of a displacement of the equilibrium towards dimerization. It is actually difficult to distinguish between the effects of the interaction between hormone and monomer and those of the interaction between
two monomers. It is also unclear whether these two interactions are independent of each other. If this were the case, the
protein would possess, aside from the binding site for the
hormone, a binding site for the second subunit (for the structure of the neurophysins cf. Sections 3.2 and 3.3).
The nature of the contacts and the possible conformational
changes resulting from the interaction between hormone and
neurophysin are a matter of speculation. Since the side
chains of residues 1 , 2 and 3 of the hormones are implicated,
one can suppose that the disulfide bridge 1-6 and the phenol ring of the invariant tyrosine-2 are affected. Bresattributes the effects measured by analysis of
1ow[17,43,45,611,
the circular dichroism spectra of free and associated bovine
neurophysin 11 to the following phenomena:
(1) the increase in ellipticity at 195 nm and 240 nm to a disturbance of the disulfide bonds in the course of association,
(2) the increase in ellipticity centered near 280 nm to a disturbance of the tyrosine of the hormone, with perhaps a
small contribution by the tyrosine of the
(3) the variations in the far-ultraviolet spectrum to changes
in the tertiary structure of the protein, or to direct disturbances of different chromophores by the bound peptide.
The shifts of the amino acid residues of the protein, with
the exception of the change in orientation of Tyr-49 (see Section 4.3.2), cannot actually be determined.
On the other hand, the possibility should be considered
that the decrease in binding affinity between pH 6 and pH 2
may be the consequence of a carboxyl-dependent change in
protein
From measurements of temperature-jump relaxation it has recently been deduced that oxytocin and vasopressin bind with greater affinity to the neurophysin dimer than the monomer and that the binding of oxytocin and vasopressin in neurophysin dimer at pH 7.4 is
nearly identical for each hormone[62ai.
5. The Possible Common Precursor of Neurophysins
and Neurohypophysial Hormones
’
The stoichiometric association of neurohypophysial hormones and neurophysins may be fortuitous, and the complexes isolated by extraction may be artefacts. Nevertheless,
there are a number of arguments indicating that the two
855
types of substances are already associated in the secretory
granules. One must consider the possibility that cleavage of
an inactive protein precursor furnishes one neurophysin and
one active peptide which remain associated. This happens
e. g. in the cleavage of ribonuclease into peptide S and truncated protein by subtilisin. However, this hypothesis, which
would account for a certain number of cytological and biochemical observations, has not yet been proven (Fig. 10).
ance of the two types of substances, as determined by their
biological activities and cytochemical reactions.
Sachs et al.l6*I
have studied the biosynthesis of vasopressin.
They injected cysteine labeled with s5S into the third ventricle of dogs and removed the hypothalamus 1.5 h later. One
half of the tissue, examined right away, contained proteins
but practically no vasopressin. The other half, after incubation for 4.5 h in a medium containing puromycin (which
blocks the synthesis) and unlabeled cystein, yielded a significant amount of labeled vasopressin. These experiments suggest the biosynthesis of an inactive macromolecular precursor which is subsequently split to yield active vasopressin.
Sachs assumed that the precursor is formed on the ribosomes
of the perikarya of the hypothalamic nuclei and that the
“maturation” (cleavage) takes place in the secretory granules
during their axonal transport to the neurohypophysis. On the
other hand, Sachs et aL16s1demonstrated on the dog hypothalamus, after infusion of labeled cysteine, the presence of a
labeled neurophysin whose molecular weight (determined by
molecular sieving), electrophoretic mobility (on cellulose
acetate) and immunological characteristics were very similar
to those of bovine neurophysins.
During subcellular fractionation, the labeled protein followed the vasopressin; in the case of animals kept alive for
10-21 days with free access to water, the protein could be
recovered from the posterior lobe. Incubation of the latter in
vitro with a high content of K led to simultaneous secretion
of vasopressin and labeled protein. In vivo, simultaneous se+
n
Fig. 10. Neurohypophysial terminals abutting a capillary containing (upper
right) a red blood cell. The arrangement is conventional with the terminal regions of the neurons separated from the fenestrated capillary wall by the parenchymal and endothelial basement membranes. Terminals show evidence of secretory activity: paucity of large electron-dense neurosecretory granules and
abundance of microvesicles (synaptic vesicles) (after [SO]).
In the neurohypophysis of mammals, among which most
species have been studied from different points of view, one
finds generally two hormones, oxytocin and arginine vasopressin, which very often occur in roughly equal proportions.
It is reasonable to assume that the two putative protein precursors would have similar though only slightly different sequences, and consequently, if the neurophysins represent
inactive ballast, that they ought to exist in the gland in the
same numbers as the hormones and probably in equivalent
proportions. In fact, this argument is difficult to substantiate
because, on the one hand the actual number of distinct neurophysins can be established only by sequential studies, and
on the other hand the proportions can be affected by degradation or selective secretion.
5.1. Biosynthesis of Neurophysins and Hormones
There are a number of data proving that the neurohypophysial hormones and the neurophysins are synthesized in
cell bodies located in the hypothalamus. Destruction of the
supraoptic and paraventricular nuclei leads to the disappear856
Fig. 1 1 . A model for the biosynthesis, transport, and release of posterior pituitary
hormones and neurophysins. A: synthesis of precursor In rough endoplasmic reticulum; B: folding of precursor; C: formation of neurosecretory granules in the
Golgi apparatus: D: axonal transport (2-3 mm/h); hormone bound to neurophysin within granules; E: exocytosis; depolarization of the neuronal plasma
membrane by propagated action potential; release of hormone and of neurophysin (possible conformational change) (after 1161).
Angew. Chem. Inf Ed. Engl. I X , 846-860 (1979)
cretion of the two substances and their release into the circulation occurred in dogs subjected to a severe hemorrhage.
These experiments would indicate that the biosynthesis and
secretion of vasopressin and of one neurophysin are parallel
events.
Recently, Gainer et aZ.[63aJ
have shown that [35S]-cysteine
injected adjacent to the supraoptic nucleus of the rat is rapidly incorporated into a 20000-dalton protein which, in time
is converted into a 12000-dalton labeled protein, neurophysin, and several small peptides including possibly oxytocin
and vasopressin.
Brownstein et al.163b1have also shown that two putative
neurophysin precursors (PI 5.4 and 6.1) with molecular
weights of about 20000, and two “intermediates” (PI 5.1 and
5.6) with molecular weights of about 17000, extracted from
supraoptic nuclei, bind specifically to anti-rat neurophysin
antiserum.
The localization of the neurophysins and the neurohypophysial hormones within the neurons seems to confirm the
parallelism and favors the hypothesis of the common precursor (cf. Fig. 11).
dog: twenty hours after constriction of the stalk, he observed
an accumulation of immunofluorescent material in the proximal, but not in the distal portion[l61(Fig. 12). In the rat it
was even possible to estimate the speed of transport of a labeled protein similar to a neurophysin to be 2-3 ~nrn/h‘(’~].
The intracellular distribution of neurophysins in the hypothalamus and posterior lobe of normal rats (Long-Evans)
and of rats with a vasopressin deficiency and considerable
diabetes insipidus (Brattleboro strain) has been examined by
Sokol et al.[64a1 using the immunoperoxidase technique (Fig.
13). In the homozygous Brattleboro rats, in contrast to nor-
5.2. Intracellular Localization of Neurophysins and Hormones
Fig. 13. Low power photomicrograph of mouse pituitary gland sectioned coronally. Immunoperoxidase stain using anti-human estrogen-stimulated neurophysin. There is heavy staining throughout the posterior lobe. The intermediate and
the large anterior lobes are essentially free of reaction products (after 182)).
5.2.1. Localization in the Neurons by Immunocytological
Methods
ma1 rats, about 50% of the magnocellular neurons of the supraoptic and paraventricular nuclei lacked immunoreactive
neurophysin. It was suggested that the neurons deficient in
vasopressin also lacked neurophysin, which once again
speaks in favor of a common biosynthesis.
The identification of neurophysins in the perikarya of the
supraoptic nucleus of the rat by immunofluorescence was accomplished by Hope[’61using anti-pig neurophysin 11. Furthermore, in dog with anti-pig neurophysin he could demonstrate by this technique the transport of the neurosecretory
material by axoplasmic flow in the hypophysial stalk of the
5.2.2. Localization within Neurosecretory Granules
Fig. 12. The distribution of neurophysin immunofluorescence in the neurohypophysial stalk both proximal and distal to the site of a constriction. Note accumulation of fluorescence proximal to the point of constriction (arrows), its absence
from axons in the tract immediately distal to the constriction, and its depletion in
the proximal neurohypophysis (after 1811).
After isolation of secretory granules from bovine posterior
lobes by prolonged ultracentrifugation in a density gradient
of sucrose it could be demonstrated by electrophoresis that
the two major components of the bovine neurophysin, as also
the two hormones, are present in about the same
Dean and Hope1651
found that the two neurophysins represent
about 50% of the “granular” proteins. Furthermore, the two
neurophysins were simultaneously present in each of the 15
glands individually examined, thus ruling out a genetic heterogeneity. The amino acid content of the crude “granular”
neurophysin corresponds to that of a mixture of neurophysins I and I1 in equal proportions[651.In addition, the distribution of oxytocin and vasopressin in the sucrose gradient
suggests that the two hormones are stored in separate granu l e ~ [ ~Moreover,
~’.
neurophysin I is presumably present in
the granules containing oxytocin, and neurophysin I1 in
those containing vasopressin[661.
The existence of two categories of granules each possessing one type of hormone and one
type of neurophysin also supports the hypothesis of a common precursor of the two substances. In the pig, lysine vasopressin is associated within the granules with neurophysin
I[671.This neurophysin (which is a truncated MSEL-neurophysin) is diminished selectively during the dehydration of
the animal which leads to the secretion of lysine vasopresAll of the available data suggests affiliation of the
MSEL-neurophysin with vasopressin, and it is interesting to
note that in the bovine fetus the excess of vasopressin (in re-
Angew. Chem. Int. Ed. Engl. I H , 846-860 (1979)
857
lation to oxytocin) in the posterior lobe is accompanied by an
excess of MSEL-ne~rophysin[’~].
The simultaneous release of
one hormone and one type of corresponding neurophysin
can be explained by the mechanism of exocytosi~[~~].
The fact
that the neurophysins of one species do not reveal a particular specificity of binding vis-a-vis the one or the other hormone leads one to think that the association does not have a
biological significance related to the transport of the material
but rather that it results from a juxtaposition due to the phenomenon of biosynthesis.
In the higher vertebrates the neurons terminating in the
neurohypophysis have their origins in two hypothalamic regions, the supraoptic and paraventricular nuclei, which made
it possible to assume a specialization whereby each of them
would synthesize one hormone. However, recent investigations do not speak in favor of such a s p e c i a l i ~ a t i o n [ ~This
~~~‘~.
does not weaken the hypothesis of “one neuron-one hormone”, since each nucleus may contain both types of neurons, a situation that is supported by the cytological analysis
of Brattleboro rats which, being afflicted by hereditary diabetes insipidus, show a dispersion of neurons with and without
5.3.3. Common Genetic Control of Vasopressin and One
Neurophysin
One mutant of rats known as “Brattleboro strain” presents
a hereditary diabetes insipidus caused by a deficiency either
in the biosynthesis of vasopressin or in its liberation from an
inactive p r e c ~ r s o r [ ~This
~ , ~ ~deficiency
].
affects only a single
gene, and the heterozygous animals manifest a control over
diuresis intermediary between that of normal animals and
homozygous mutants. It is, therefore, important to verify if
the absence of vasopressin is accompanied by a deficiency in
one neurophysin. In the normal rat, three neurophysins (I or
A, I1 or B, 111 or C) have been detected by means of polyacrylamide gel electroph~resis[~~.’~~.
By injection of [35S]Cysteine before sacrifice, Pickering et al. observed that the
ratio between the quantities of radioactivity incorporated in
neurophysins I and I1 is very close to that of the amounts of
vasopressin and oxytocin present in the gland[751.
On the other hand, in the homozygous Brattleboro rat, neurophysin I is
absent, and in the heterozygote its quantity is lower than that
of neurophysin 11. Pickering et al. 129*751 concluded from this
observation that vasopressin is synthesized with neurophysin
I (or A) and oxytocin with neurophysin I1 (or B). Pickering
attempted to determine the respective quantities of neurophysin I and I1 by using antiserums. Even though the specificity is imperfect-the antiserum for neurophysin I also precipitates 20% of neurophysin I1 - he concluded that vasopressin and neurophysin I (or A) are present in the gland in
approximately equivalent amounts. These data argue in favor of the existence of a provasopressin which, by cleavage,
gives rise to vasopressin and neurophysin I; this is in agreement with the conclusions of S u c h et ul. 162,631.
Sunde and Soko1[761also compared the neurophysins of the
normal rat with those of the Brattleboro homozygote by
polyacrylamide gel electrophoresis and likewise observed the
disappearance of neurophysin I (neurophysin A of Pickering)
in the strain manifesting diabetes insipidus. In spite of this,
according to their experimental results neurophysin I1 (B)
and neurophysin 111 (C) are present in equal proportions.
858
The authors have examined the molar ratios of vasopressin/
neurophysin I and oxytocin/neurophysin I1 + neurophysin
111 and have found the values to be 0.91 and 1.8 for the normal rat, and (0.0) and 1.77 for the Brattleboro homozygote.
Furthermore, in the dehydration experiments, where the
amount of vasopressin in the posterior lobe of the rat is considerably reduced, the molar ratio vasopressin/neurophysin I
is 1.37 for the control animal (free access to water) and 1.31
for the animal deprived of water for three days. Even if the
ratio between oxytocin and neurophysins I1 + I11 is debatable, that between vasopressin and neurophysin I appears, by
way of contrast, to be very constant. It remains open whether
rat neurophysin I belongs to the MSEL- or VLDV-family of
neurophysins.
The sum total of observations made on the Brattleboro rat
would indicate that a single gene controls the biosynthesis of
vasopressin and of one neurophysin and that the two substances could be fragments derived from a common protein
precursor.
6. Conclusions
The problem of the biosynthesis of the neurohypophysial
hormones extrapolates to the more general topic of the biosynthesis of polypeptide hormones, and in a still broader
sense to that of protein secretion. Since the work of Steiner et
uI.[~’]one knows that most of the polypeptide hormones are
synthesized in the form of an inactive precursor of larger
size: thus proinsulin, proparathormone, proglucagon, the
progastrins, etc., have been characterized, in small quantity,
in the secretory cells. One is dealing here with a general characteristic concerning the secretory proteins since one has
equally been able to identify a proalbumin, proenzymes, etc.
Furthermore, it has recently been observed that the proproteins are themselves cleavage products of very short-lived
chemical compounds which are called pre-proproteins. Hubener et al. [781-inspired by experiments on pre-proparathormone-have proposed a general scheme of the biosynthesis
and conversion. The initial protein chain, resulting from the
transcription of messenger RNA, leaves the ribosome at its
NH2-terminus and penetrates the membrane of a cistern of
the endoplasmic reticulum. An N-terminal sequence consisting of about 20 amino-acid residues, generally hydrophobic,
represents the “perforator” of the membrane which is itself
hydrophobic, and the rest of the chain can then penetrate the
canal thus formed. The N-terminal sequence, called pre-peptide, becomes detached from the chain after 1-2 min, probably by enzymatic action. The isolation of the preproprotein
and the characterization of the N-terminal sequence require
microtechniques (radioactive labeling, special degradation
procedures) that can be applied to quantities of the order of a
nanomol. It was in this way that the sequences of the prepeptides of pre-proparathorm~ne[’~~
and of pre-proin~ulin[~’~
were determined.
Inside the cistern the chains, once liberated from the ribosome, acquire the native conformation of the prohormone.
At the ends of the cisterns, vesicles detach themselves which
then become fused to form secretory granules. Inside these
granules a second proteolytic cleavage converts the prohormone into the hormone. Despite the fact that the enzymes responsible for these cleavages have not yet been isolated, they
Angew. Chem. Int. Ed. Engl IR, 846-860 (1979)
seem to have a “trypsin-like” specificity. The life span of the
prohormone in the granule is of the order of 1-2 h; in the
case of insulin, for example, 95% of the proinsulin is converted into the active hormone whereby a peptide (peptide
C) is liberated. The secretory granules reach the secretory
pole of the cell and probably their entire content is discharged from the cell by the phenomenon of exocytosis (Fig.
14).
c
*
Fig. 14. Scheme depicting exocytosis-vesiculation sequence suggested to operate
in neurohypophysial terminals. Contents of neurosecretory granules (A) are
emptied directly into extracellular space by exocytosis (B). Granule membrane is
retrieved from terminal surface by micropinocytosis-like activity (vesiculation),
producing coated caveolae (C) w h c h pinch off as coated microvesicles (D) and
then shed coat fragments (E) to become partially coated microvesicles (F) and,
finally. smooth (synaptic) microvesicles ( G ) .These in turn are incorporated in lysosomal bodies (H) (after [SO]).
This general scheme can also be accepted in the special
case of neurosecretion; the neurosecretory neurons function
like gland cells[801.However, here the axoplasmic pathway
for the transport of the granules from the cell body in the hypothalamus to the axon terminal in the neurohypophysis is
particularly long and the transformations which the proteins
undergo within the granules (“maturation”) can be especially important. It seems reasonable to assume that the hormonal nonapeptides of the neurohypophysis are synthesized in
the form of much longer chains, and that a sequence of
cleavages, similar to those observed in several other polypeptide hormones, leads to active stable compounds which one
can isolate in relatively important amounts. The simultaneous presence of hormones and neurophysins in the perikarya of the neurosecretory neurons, as revealed by the immunocytological technique; their close association within the
granules, proved by the isolation of the latter and by chemical characterization; the indirect demonstration, by the incorporation of labeled amino acids, of the existence of a macromolecular precursor of vasopressin; the simultaneous disappearance of vasopressin and one neurophysin as a consequence of a unique mutation in the rat: all these observations
provide a very strong argument in favor of the existence of a
Angew. Chem. I n / . Ed. Engl. 18, 846-860 (1979)
common precursor yielding one hormone and one neurophysin by cleavage.
The author is indebted to ProJ Bertha Scharrer for her help
in the composition of the manuscript.
Received: February 1978 [A 294 IE]
German version: Angew. Chem. 91, 905 (1979)
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Angew. Chem. Int. Ed. Engl. IX, 846-860 (1979)
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molecular, aspects, cellular, neurophysin
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