The composition of crystalline complexes of neurophysin-M with [8-arginine]-vasopressin and oxytocin

Neurophysin-M, a methionine-containing protein that is the major constituent of neurophysin, has been crystallized as complexes with [8-arginine]-vasopressin. Three moles of vasopressin alone or 2 moles of vasopressin together with 1 mole of oxytocin are bound/mole of protein. An amorphous complex of the protein with oxytocin alone contains 2 moles of the hormone/mole of protein. Deamino-[8-arginine]-vasopressin, a highly active basic analogue of vasopressin, is not bound by neurophysin. The primary amino group of both vasopressin and oxytocin is necessary for binding with neurophysin.     

Subcellular organization of neurophysins, oxytocin, [8-lysine]-vasopressin and adenosine triphosphatase in porcine posterior pituitary lobes

Posterior pituitary lobes from young pigs were fractionated by differential and sucrose-density-gradient centrifugation. The distributions of oxytocin and [8-lysine]-vasopressin were measured by bioassay and the distributions of neurophysin-I and -II by radioimmunoassays specific for each of these two proteins. Most of the hormone and neurophysin applied to the density gradient was localized in particles with the density expected of neurosecretory granules. However, the neurosecretory granules were separated into two bands (D and E). A close statistical correlation between the distributions of [8-lysine]-vasopressin and neurophysin-I, and of oxytocin and neurophysin-II on the gradients, suggested that in vivo porcine neurophysin-I binds [8-lysine]-vasopressin within one population of granules and porcine neurophysin-II binds oxytocin within another type of granule. However, there was no significant separation of oxytocin and vasopressin in fractions D and E. The molar ratios of hormones and neurophysins indicated that there was insufficient of either neurophysin to bind the [8-lysine]-vasopressin in the granule fractions or in the whole gland. Polyacrylamide-gel electrophoresis showed that only bands corresponding in mobility to porcine neurophysins-I, -II and -III were present in large amounts in the whole gland and in the granule fractions. The component with the mobility of neurophysin-III was, however, relatively enriched in whole young glands and granule fractions compared with adult gland extracts. It is suggested that the vasopressin that cannot be assigned to neurophysin-I may occur in (a) vesicles containing vasopressin but no neurophysin, (b) vesicles containing vasopressin and a protein that cannot be quantified by the radioimmunoassays used, such as porcine neurophysin-III, or (c) normal vasopressin–neurophysin granules which have accumulated extra vasopressin. Band E of the gradient was rich in adenosine triphosphatase activity, whereas band D possessed very little of this enzyme.     

The isolation of three neurophysins from porcine posterior pituitary lobes

1. Three neurophysins, proteins that bind the polypeptide hormones oxytocin and vasopressin, have been isolated from acetone-dried porcine posterior pituitary lobes. The proteins have been named porcine neurophysins-I, -II and -III in order of their electrophoretic mobilities at pH8.1. 2. Electrophoretic comparison of the purified proteins, which are homogeneous on starch-gel electrophoresis, with the soluble proteins of fresh porcine posterior pituitary lobes extracted in 0.1m-HCl and in buffer pH8.1 suggests that the isolated proteins are native to the fresh tissue. 3. Neurophysins-I and -II are present in similar amounts in the tissue, whereas neurophysin-III is present only in small quantities. Acetone-dried tissue also contains traces of other hormone-binding neurophysin components. 4. All the neurophysins can bind both oxytocin and [8-lysine]-vasopressin. 5. The apparent molecular weights of the neurophysins increase with increasing protein concentration as measured by equilibrium sedimentation in the ultracentrifuge. 6. Neurophysins-I and -III are of similar molecular dimensions, contain one residue of methionine per molecule and lack histidine. The minimum molecular weight of neurophysin-I obtained by amino acid analysis is 9360. Neurophysin-II is of larger molecular dimensions than neurophysins-I and -III and can be separated from these by gel filtration on Sephadex G-75. It contains no histidine or methionine, and its minimum molecular weight has been estimated as 14020 by amino acid analysis. 7. Each of the three neurophysins possesses N-terminal alanine. 8. The possible biological significance of the existence of several neurophysins within one species is discussed.     

The isolation of purified neurosecretory granules from bovine pituitary posterior lobes: Comparison of granule protein constituents with those of neurophysin

1. A procedure for the isolation of highly purified neurosecretory granules from the posterior lobe of the bovine pituitary gland is described. The preparation was free from contamination by the mitochondrial enzyme succinate dehydrogenase and the lysosomal enzyme cathepsin. 2. The biological activities of the neurosecretory granules were measured: the oxytocic activity was 11.61+/-1.30units and the pressor activity was 10.73+/-1.74units/mg. of protein. 3. A lysate of the isolated granules was shown to contain two proteins that appear to be identical with two of the constituents of neurophysin. 4. The constituents of neurophysin not present in neurosecretory granules could not be detected in any other subcellular fraction. It is suggested that the components of neurophysin not present in the neurosecretory granules arise as a result of the degradation of the two granular proteins.     

Complete amino acid sequence of bovine neurophysin-I. A major secretory product of the posterior pituitary

Bovine neurophysin-I (bNP-I) is the first neurophysin protein which contains histidine and possesses an acidic COOH-terminal segment for which the complete amino acid sequence is presented: NH2-Ala-Val-Leu-Asp-Leu-Asp-Val-Arg-Thr-Cys-Leu-Pro-Cys-Gly-Pro-Gly-Gly-Lys-Gly-Arg-Cys-Phe-Gly-Pro-Ser-Ile-Cys-Cys-Gly-Asp-Glu-Leu-Gly-Cys-Phe-Val-Gly-Thr-Ala-Glu-Ala-Leu-Arg- Cys-Gln-Glu-Glu-Asn-Tyr-Leu-Pro-Ser-Pro-Cys-Gln-SerGly-Gln-Lys-Pro-Cys-Gly-Ser- Gly-Gly-Arg-Cys-Ala-Ala-Ala-Gly-Ile-Cys-Cys-Ser-Pro-Asp-Gly-Cys-His-Glu-Asp-Pro-Ala-Cys-Asp-Pro-Glu-Ala-Ala-Phe-Ser-Leu-COOH. Determination of the structure was greatly facilitated by new procedures used for the isolation of bNP-I and of its tryptic peptide fragments. bNP-I isolated from freshly frozen bovine posterior pituitaries is composed of 93 residues, but some preparations contain neurophysin protein with NH2- and COOH-terminal truncated sequences. bNP-I differs from bovine neurophysin-II, the second major neurophysin of cow, in 20 residue positions, and several of the differences cannot be accounted for by single nucleotide replacements in the genes coding for these two neurophysin proteins. The results reported in this study support our earlier hypothesis that neurophysin-gene duplication preceded species divergence.     

The isolation of neurophysin-I and -II from bovine pituitary neurosecretory granules separated on a large scale from other subcellular organelles. Demonstration of slow equilibration of neurosecretory granules during centrifugation in a sucrose density gradient

1. An improved procedure for the isolation of neurosecretory granules from the posterior lobe of the bovine pituitary gland is described. 2. Of the total oxytocic and pressor activities present in the original tissue 80% was sedimentable. 3. The granules were separated from mitochondria by prolonged centrifugation in a sucrose density gradient. During a sedimentation period of 5hr. the granules moved progressively into denser regions of the gradient and the mitochondria remained at the top. 4. The biological activities of the granules were measured: the oxytocic activity was 11·56±1·63 and the pressor activity was 15·60±3·91 units/mg. of protein. 5. A protein was isolated from a lysate of granules prepared from 40 pituitary glands. Amino acid analysis showed that it consisted of a mixture of neurophysin-I and neurophysin-II in equal proportions. It accounted for 60% of the soluble granule protein and for 50% of the total granule protein. 6. The neurophysins present in the granules are associated with 19·1 units of oxytocic and 21·1 units of pressor activity/mg. of protein. 7. Starch-gel electrophoresis revealed the presence of both neurophysins in extracts of 15 pituitary glands studied individually. 8. We conclude that the polypeptide hormones, oxytocin and [8-arginine]-vasopressin, are normally closely associated with the two neurophysins within neurosecretory granules of the pituitary gland.     

Solid phase synthesis and biological activities of [Arg8]-vasopressin methylenedithioether.

Solid phase synthesis of [Arg8]-vasopressin methylenedithioether, an analog of vasopressin which contains an extra methylene group between the two sulfur atoms of Cys1 and Cys6, is described. Methylene insertion occurred easily when the thiol free peptide on a solid support was treated with tetrabutylammonium fluoride in dichloromethane at room temperature for 3 h. The uterotonic in vitro, pressor, and antidiuretic activities of the compound were reduced in comparison to [Arg8]-vasopressin by one order of magnitude.     

Resistance of 1-deamino-[8-D-arginine]-vasopressin to in vitro degradation as compared with arginine vasopressin

In order to elucidate the mechanism(s) responsible for the prolonged antidiuretic activity of 1-deamino-[8-D-arginine]-vasopressin (dDAVP), antidiuretic activities of dDAVP and arginine vasopressin (AVP) were determined in the rat following either oral administration or incubation with AVP-degrading enzymes and reagents. Oral administration of dDAVP to conscious water-loaded rats resulted in significant antidiuresis while AVP resulted in slight and transient antidiuresis. In the ethanol anesthetized water-loaded rats, antidiuretic activities of 136pg of AVP and 50pg of dDAVP, which were found to be equipotent, were compared after incubation with digestive enzymes (pepsin, trypsin, alpha-chymotrypsin), late pregnancy plasma, or sodium thioglycollate. The antidiuretic activity of AVP was completely destroyed by 30-min incubation with trypsin, alpha-chymotrypsin, or late pregnancy plasma and almost all AVP was inactivated by 0.2 M sodium thioglycollate. On the other hand, the antidiuretic activity of dDAVP was not destroyed by trypsin or pregnancy plasma but was partly destroyed by alpha-chymotrypsin and sodium thioglycollate. Neither the antidiuretic activity of AVP nor that of dDAVP was affected by pepsin. Thus, the antidiuresis observed after oral administration of dDAVP might be brought about by the resistance to digestive enzymes. Furthermore, the resistance of dDAVP to digestive enzymes, late pregnancy plasma and sodium thioglycollate might be responsible for the prolonged antidiuretic action of dDAVP in vivo.     

The isolation and characterization of the link proteins from proteoglycan aggregates of bovine nasal cartilage.

A preparation containing the link proteins may be obtained from bovine nasal cartilage by extraction with 4 M guanidine hydrochloride and by equilibrium density gradient centrifugations of the extract as commonly employed in the isolation of proteoglycan monomers. In the present paper, protein-rich proteoglycans have been removed from such a preparation to give purified link proteins by chromatography on Sepharose CL-6B in 1% sodium dodecyl sulfate. The individual link proteins, which in order of increasing electrophoretic mobility are termed link proteins 1, 2, and 3, have been separated and isolated in a subsequent preparative gel electrophoresis step. The link proteins present in largest amount, link proteins 1 and 2, have essentially the same amino acid compositions, and following partial digestion with the V8 protease from Staphylococcus aureus and analytical electrophoresis in sodium dodecyl sulfate, their peptide patterns closely resemble each other. Therefore,it is probable that link proteins 1 and 2 are structurally similar. Link protein 1 contains more carbohydrate than link protein 2 (9.5% and 3.0%, respectively) and it is suggested that the major difference between them is in carbohydrate content.     

The isolation of large molecular mass procyanidins from grain sorghum and their interaction with [14C]methyl bovine serum albumins

1. Three protein-free procyanidin fractions with molecular masses of 4000, 8000 and 10,000-50,000 were isolated from a bird resistant sorghum grain. 2. The interaction between the largest procyanidin and 88 or 1% methylated bovine serum albumin (14C labelled) is stronger with the more methylated protein over a range of 0-2.75 ionic strength at pH 5. This indicates the involvement of apolar forces throughout the range though apolar forces were more pronounced from 0.7-2.75 ionic strength. 3. In the interaction between procyanidins of different molecular masses and 88% 14C-methylated bovine serum albumin the smaller procyanidins exhibited a more pronounced sigmoidal precipitation pattern than the larger with an increase in their concentrations.     

Distance distributions from the tyrosyl to disulfide residues in the oxytocin and [Arg8]-vasopressin measured using frequency-domain fluorescence resonance energy transfer

We have examined the fluorescence intensity decays of oxytocin and [Arg⁸]-vasopressin resulting from the single tyrosyl residue in each peptide, and the intensity decay of the Asu ^1,6-analogues in which the disulfide bridge is substituted by a CH₂-CH₂ bridge. Viscosity-dependent steady state and intensity decay measurements indicated that fluorescence resonance energy transfer (FRET) from tyrosyl phenol to the disulfide bridge is responsible for the decrease in fluorescence relative to the Asu-analogues. The frequency-domain phase and modulation data for the tyrosyl donor were interpreted in terms of fluorescence resonance energy transfer (FRET) to the weakly absorbing disulfide bridge and a distribution of donor-to-acceptor distances. Energy transfer efficiencies were determined from both time-resolved and steady-state measurements. Fitting the frequency-domain phase and modulation data to a Gaussian distance distribution indicated that the average inter-chromophoric distance (R_av) is similar in both compounds, R_av=7.94 Å for oxytocin and R_av = 8.00 Å for vasopressin. However, the width of the distance distribution is narrower for vasopression (hw =2.80 Å) than for oxytocin (hw =3.58 Å), which is consistent with restriction of the tyrosine phenol motion due to its stacking with the Phe³ side chain of vasopressin. Finally, the recovered distance distribution functions are compared with histograms describing the distance between the chromophores during the course of long, in vacuo, molecular dynamics runs using the computer program CHARMm and the QUANTA 3.0 parameters.Abbreviations AVP [Arg⁸]-vasopressin - FRET fluorescence resonance energy transfer - FD frequency-domain - D donor - A acceptor - DTT dithiothreitol Correspondence to: J. R. Lakowicz     

Conformation of [8-arginine]vasopressin and V1 antagonists in dimethyl sulfoxide solution derived from two-dimensional NMR spectroscopy and molecular dynamics simulation

Structural and dynamic properties of [8-arginine]vasopressin and a class of highly potent vasopressin V1 antagonists which contain 3-mercapto-3,3-cyclopentamethylene propionic acid (Mca) in position 1 of the vasopressin sequence have been determined. On the basis of two-dimensional NMR experiments in dimethyl sulfoxide solution, interproton distances were derived according to which model conformations were built and refined using molecular dynamics simulations. The conformation of vasopressin and the V1 antagonists differ mainly in the region of the mutated residue. The antagonistic property was found to be related to an inversed chirality of the disulfide bridge. In all investigated molecules, characteristic beta-turn structure elements were found for the backbone conformation of the endocyclic residues Tyr2-Asn5. For this portion of the peptide sequence, various conformational equilibria were detected which matched different time scales. For [Arg8]vasopressin, averaged NMR parameters were obtained which could be explained by rapid interconversion between different beta-turn geometries, whereas multiple slowly exchanging conformations were observed for the V1 antagonists. V1 antagonists containing sarcosine in position 7 exhibited multiple spectral patterns for the exocyclic part attributed to cis/trans isomerization. The X-ray structure of deamino-oxytocin [Wood, S. P., Tickle, I. J., Treharne, A. M., Pitts, J. E., Mascarenhas, Y., Li, J. Y., Husain, J., Cooper, S., Blundell, T. L., Hruby, V. J., Buku, A., Fischman, A. J. & Wyssbrod, H. R. (1986) Science 232, 633-636] was found to represent one sample of the conformational space covered by the multiple conformations found for [Mca1, Arg8]vasopressin.