Endocrinology 1971: Proceedings of the Third International Symposium

Hormones

X-Ray Analysis and the Structure of Insulin

DOROTHY CROWFOOT HODGKIN,     Department of Zoology, University of Oxford

Publisher Summary

This chapter discusses the use of X-ray technique to study the structure of insulin. With the electron microscope, it is possible to see dense, apparently crystalline, aggregates in the β granules of many animals and within these, the compact particles are regularly arranged. The size of a single particle visible within the β granules is very similar to that of the hexamer of insulin molecules, 48 Å across, found in rhombohedral pig insulin crystals by X-ray analysis. Groups of atoms, such as benzene rings, appear as single peaks, while peptide chains are represented by strands with higher density in the neighborhood of, for example, carbonyl groups. The insulin molecules are linked in threes around them through the B 10 histidine residues. Though the linking of the histidine residues to the zinc ions is similar, it does not appear quite identical in the two triplets in the projected view along the three fold axis. Very possibly the C peptide provides both an additional template for chain support and a protective sheath over the active surface during transport of insulin to the β granule.

In. a symposium on endocrinology it seems proper to begin by looking at insulin as it exists in the β granules of the pancreas. There have been a number of observations in the past made with the light microscope of single crystals, probably of insulin, visible in the granules, particularly in the pancreas of the dog. With the electron microscope it is possible to see dense, apparently crystalline, aggregates in the β granules of many animals and within these, compact particles, regularly arranged. A very good example, photographed by Greider, Howell and Lacy (1969), shows roughly spherical particles in lines about 50 Å apart in a β granule in rat pancreas. It may well be that the actual formation of crystals is a consequence of partial drying of the granules on isolation or preparation for electron microscopy; the individual particles of insulin that appear would however be almost certainly normally present in the living tissue.

The size of a single particle visible within the β granules is very similar to that of the hexamer of insulin molecules, 48 Å across, found in rhombohedral pig insulin crystals by X-ray analysis. Hexamers are shown in Fig. 1 in projection along the three fold axis of the rhombohedral crystals; the view is not unlike that of the rat islet ‘crystal’ though this may well have a different structure in detail from pig insulin. The appearance of hexamers both in the crystals and in the islets would be expected from experiments on insulin in solutions which show that, in the presence of zinc, six insulin molecules aggregate around two zinc ions; in most creatures, except perhaps the guinea pig and coypu, zinc is present in the β granules.

Fig. 1 Projection of the atomic positions found in the crystal structure of rhombohedral insulin crystals along the threefold axis. The atoms (small circles) are grouped in four hexamer units at relative z heights, z, z + 1/3, z + 2/3.

Fig. 2 shows a single hexamer of insulin, again observed in projection along the three fold axis of a rhombohedral insulin crystal. All of the atoms, except hydrogen atoms, are shown by circles varying in size with atomic number in the order zinc, sulphur, oxygen, nitrogen and carbon. Their positions are not at all precisely defined. The experimental evidence on which they are based – our next order of observation on insulin – is an electron density map derived by calculation from the intensities of spectra obtained by diffraction of X rays passing through the insulin crystals (Adams et al., 1969, Blundell et al., 1971). The spectra extend to spacings of 2·8 Å and the electron density map calculated accordingly provides a very blurred representation of the atomic positions. Its character can be demonstrated by plotting to scale contours of equal calculated electron density on sheets of perspex and stacking the sheets together to cover the unit volume in the insulin crystal. Groups of atoms, such as benzene rings, appear as single peaks, while peptide chains are represented by strands with higher density in the neighbourhood of, for example, carbonyl groups. A few overlapping sections of the electron density map are shown in Fig. 3 while Fig. 4 indicates how a part of the chemical structure of insulin is fitted within the electron density outlines in a single section of the map.

Fig. 2 Projection along the three fold axis of the atomic positions in a single insulin hexamer. the atoms zinc (along the three fold axis), sulphur, oxygen, nitrogen and carbon are shown by circles in decreasing order of size and are joined by lines representing chemical bonds. Hydrogen atoms are omitted.

Fig. 3 Photograph of part of the electron density model showing sections between 10/48 and 5/48 in z. The contours are drawn on each sheet at intervals of 01 e/A³.

Fig. 4 Electron density contours in the section z = 9/48 with superimposed the positions of the histidine, leucine and serine residues. Riled circles represent atoms close to section, open circles atoms within 1 Å of section.

It will be clear from the character of the electron density map that while our knowledge of the atomic positions is, in detail, very imprecise, certain features of the arrangement of the peptide chains within the insulin molecules and of the six insulin molecules within the hexamer are very clear. Our confidence that many of the details of atomic positions now deduced are also reasonably correct depends on the close correlation of the electron density map with the chemical structure of pig insulin derived by Ryle, Sanger, Smith and Kitai (1955), shown in Table 1. It is also helped by other evidence, particularly on the titration of insulin in the presence and absence of zinc, which made it very probable that one of the histidine residues was attached to the zinc ions.

Table 1

Variations in insulin sequences.

The electron density map shows that the two zinc ions are arranged along the three fold axis of the crystal and are about 17 Å apart. The insulin molecules are linked in threes around them through the B 10 histidine residues. Though the linking of the histidine residues to the zinc ions is similar, it does not appear quite identical in the two triplets in the projected view along the three fold axis. The molecules in one triplet are arranged relative to those in the second triplet nearly but again not quite exactly as required by two fold symmetry axes along the lines OP and OQ of Fig. 2 normal to the three fold axis. In certain regions the packing together of the six molecules in the hexamer is as close as is the packing of the amino acid residues and peptide chains within a single molecule. As a whole, accordingly the hexamer presents a compact spheroidal appearance to the external world, 48 Å in diameter, 35 Å in height. Viewed down the three-fold axis its circumference appears smoothly circular but the upper and lower surfaces of the spheroid are in fact pitted by deep grooves between projecting residues of the A chain loops.

Around one of the two fold axis, OP of Fig. 2, the contacts between the insulin molecules appear to be very close; it seems almost certain that these are the contacts responsible for the dimeric character frequently observed in molecular weight measurements of insulin in solutions in the absence of zinc. They are illustrated in Fig. 5 which shows a view of the atomic positions in the dimer along the ‘two’ fold axis. They include both non polar van der Waal’s interactions between, for example, the valine B 12 and phenyl alanine B 24 groups of the two molecules, and also hydrogen bonded contacts between the peptide carbonyl and NH groups, B 24 and B 26. The latter appear as part of a β-pleated sheet type of structure, formed by the antiparallel arrangement of the terminal residues of the B chain in the two molecules. In Fig. 5b, where this system is isolated, one can see that one reason for geometrical differences between the two molecules in the dimer may be the necessity for close packing of residues; thus the two phenyl alanine B 25 groups pack together, destroying the exactly symmetrical relation between them which might have appeared from the chain arrangement.

Fig. 5 (a) The atomic positions in the insulin dimer viewed along the ‘two’ fold axis, (b) The last eight residues of each molecule within the dimer in antiparalleled arrangement; hydrogen bonds dotted.

The two insulin molecules in the dimer are therefore very similar but not geometrically identical in every detail. They are illustrated in Fig. 6 in which they have been set side by side, for comparison. In each, the B chain starts out in an extended conformation from B 1 – B 8, turns sharply into an α helix from B 9 – B 20, and then through a U turn involving residues 21 – 23, ends in a further long extended region from 24 – 30. Within the rigid framework provided by these structures, the A chain forms a small compact unit. The exact conformation in certain regions has proved difficult to define; it follows a more involved course after an initial α helical turn, to make the loop bridged by an internal disulphide bond. From A 13 – A 19 it takes a loosely helical course, placing the long hydrophilic side chains on the outside of the molecule. It is held in position within the B chain cavity by the disulphide bridges to either end of the B chain α helix; there are strategic internal contacts, both polar and non polar, between the chains.

Fig. 6 Molecule II. The two molecules of insulin found in the crystal viewed in equivalent directions perpendicular to the three fold axis. Molecule I is seen in the orientation corresponding with Fig. 5; molecule II has been rotated around the two fold axis by180°.

If we knew the receptor system with which insulin interacts, it might now be possible to try experiments within the crystal structure to locate the precise region or regions in the molecule that are most closely involved in the biological activity. In our present state of ignorance about the nature of the reactions concerned, we have to follow, by more devious routes, whatever clues are available. These include various experiments on the inactivation of insulin under different circumstances and the variation of the residues in the insulin molecule with species.

So far sequences have been determined for some twenty different insulins (Smith, L.F., 1966) – not a great number, really, in view of the complexity of nature. They are summarized in Table 1. The residues that have up to now been found to be invariant are circled; one might suppose that most, if not all, of these are important to our understanding of the biological activity of the molecule; it is interesting to see how they are arranged geometrically in relation to the observed structures of insulin, as monomer, dimer and hexamer. From Fig. 7 one can see straight away that a large number, all the cystine residues, glycine B 8 and B 23, leucine B 6, B 11, B 15 and A 16, valine B 18 and isoleucine A 2, are concerned with composing the relative arrangement of the two insulin chains in space; both by chemically defining their junction, enabling particular geometrical turns to be made, and encouraging defined internal residue relations. Certain other groups, valine B 12, tyrosine B 16 and phenyl alanine B 24, play a similar role in the surface within the dimer, encouraging particular contacts between molecules. A few ‘invariant’ residues only, A 1 glycine, A 5 glutamine, A 19 tyrosine, and A 21 asparagine are close together in the same region of the surface in all states of aggregation of the molecule. Among the different reactions which inactivate insulin it is perhaps significant that removal of either glycine Al or asparagine A 21 leads to almost total inactivation (Carpenter, 1966).

Fig. 7 The peptide skeleton of molecule II with the invariant residues of Table 1 shown in filled circles.

So we have a possible picture emerging of particular residues held on a surface in an arrangement which is essential to activity but there are additional complexities in the situation which make us hesitate to say this is all that is required. The characteristics of the insulin dimer seem to be important in relation to the distribution of invariant residues. This might be either because the dimer presents an essential extended active surface or because the residues in the monomer monomer interface are themselves involved in interactions and are at some stage released. At the concentrations at which insulin circulates in the blood it is probably present as a mixture of monomers and dimers; with traces of zinc around even hexamers may occur, so that any of the three might well be the active species; it would certainly be interesting, as suggested on another occasion, to test the activity of dimers internally cross linked to inhibit dissociation to the monomer.

At present it seems most likely that the insulin hexamer provides a useful but not essential method of storing insulin in the β granules – not essential because the B 10 histidine, necessary for its construction, is absent in the guinea pig and coypu. Possibly ease of packing into crystals may assist the storage. And here the existence of proinsulin may be considered. The C peptide which in proinsulin joins A 30 to A 1 may well have functions additional to those of holding these two points together. They are, in the insulin molecule as we see it, very close, about 8 Å apart, a distance which could easily be spanned by two or three residues instead of the 32–35 provided by nature. Very possibly the C peptide provides both an additional template for chain support and a protective sheath over the active surface during transport of insulin to the β granule. It will be fascinating to see its structure as we may now hope to do, in the crystals recently isolated by Fullerton, Potter and Low (1970).

REFERENCES

Adams, M. J., Blundell, T. L., Dodson, E. J., Dodson, G. G., Vijayan, M., Baker, E. N., Harding, M. M., Hodgkin, D. C., Rimmer, B., Sheat, S. Structure of rhombohedral 2 zinc insulin crystals. Nature. 1969; 224:491.

Blundell, T. L., Cutfield, J. F., Dodson, E. J., Dodson, G. G., Hodgkin, D. C., Mercola, D. A., Vijayan, M. Atomic positions in rhombohedral 2 zinc insulin crystals. Nature. 1971; 231:506.

Blundell, T. L., Cutfield, J. F., Dodson, E. J., Dodson, G. G., Hodgkin, D. C., Mercola, D. A., Vijayan, M. Atomic positions in rhombohedral 2 zinc insulin crystals. Nature. 1971; 231:506.

Carpenter, F. H. Relationship of structure to biological activity of insulin as revealed by degradative studies. Am. J. Med. 1966; 40:750.

Fullerton, W. W., Potter, R., Low, B. W. Proinsulin: Crystallization and preliminary X-ray diffraction studies. Proc. Nat. Acad. Sci. U.S.A. 1970; 66:1213.

Greider, M. H., Howell, S. L., Lacy, P. E. Isolation and properties of secretory granules from rat islets of Longerhaus. J. Cell. Biol. 1969; 41:162.

Ryle, A. P., Sanger, F., Smith, L. F., Kitai, R. The disulphide bonds of insulin. Biochem. J. 1955; 60:541.

Smith, L. F. Species variation in the amino acid sequence of insulin. Am. J. Med. 1966; 40:662.

The Meaning of ‘structure’ and Activity for Peptide Hormones

J. RUDINGER,     Institute of Molecular Biology and Biophysics, Swiss Federal Institute of Technology, Zurich, Switzerland

Publisher Summary

This chapter discusses the chemical characterization and meaning of structure and activity for peptide hormones. The term structure evidently refers to that arrangement of the atoms of the active molecule, which it takes up for, or during, interaction with a biological receptor site. A topochemical entity may include the portions of amino acids linked vicinally in a single sequence or the portions of amino acids widely separated in the sequence and brought together conformationally to form a coherent functional unit. It is a crucial question whether these topochemical regions whose features are recognized by the receptor or other partner molecules are already preformed in the crystal or in aqueous solution or if they develop only during interaction with the partner molecule. Now as the concentration of peptide in the vicinity of the receptors in the tissue is given by a steady state determined by the rate of accession and of disposition a decreased rate of disposition will, other factors being equal increase the steady-state concentration at the receptors and thereby the response.

Structure

In classical chemistry, ‘structure’ denoted the way in which the atoms of a molecule were covalently linked together and the term ‘chemical structure’ is still often used in this sense. Increasingly the word has come to have stereochemical connotations and to many chemists the three-dimensional picture of the molecule, as determined most commonly by x-ray crystallography, has come to be the ultimate in ‘structure.’ The work reported at this Symposium by Dorothy Hodgkinshows the power and the beauty of the method asapplied to a protein hormone, insulin.

On the other hand, in the juxtaposition of ‘structure-activity relations’ the term ‘structure’ evidently refers to that arrangement of the atoms of the active molecule which it takes up for, or during, interaction with a biological receptor site (or with other sites or molecules encountered during its life-time in the biological system). It is useful to think of such arrangements in termsof their topochemistry – the spatial distribution of bulk, charge, polarity, lipophilicity etc. over the interacting surface of the molecule (Hofmann and Katsoyannis, 1963; Schwyzer,1963; Rudinger and Jost, 1964; Shemyakin, Ovchinnikov and Ivanov, 1969). This topochemistry is, of course, a consequence in turn of the amino-acid sequence (‘primary structure’) as moulded by the conformation (‘secondary’ and ‘tertiary structure’). A topochemical entity may include portions of amino acids linked vicinally in a single sequence, or portions of amino acids widely separated in the sequence and brought together conformationally to form a coherent functional unit. These two types of information-holding regions have been referred to as ‘continuate’ and ‘discontinuate words’ (Schwyzer, 1970); immunochemistry makes a similar distinction between ‘sequential’ and ‘conformational’ determinants (Sela, 1969).

It is a crucial question whether these topochemical regions whose features are ‘recognized’ by the receptor or other partner molecules are already preformed in the crystal or in aqueous solution, or if they developonly during interaction with the partner molecule; in other words, do peptides have the same conformation in the crystal, in solution, and onthe receptor? On this point, opinions are divided and direct evidence sparse.

It is perhaps somewhat paradoxical that the very large peptide (or small protein) hormone insulin is the only one whose three-dimensional structure has so farbeen determined. For molecules of this size theconformation in free aqueous solution may well resemble that in the (largely also aqueous) crystal. Since the sum of the conformation-holdingforces may be taken as very roughly proportional to the bulk of the molecule whereas the intermolecular interactions should be proportional to its surface area one might predict in generalterms that a high volume-to-surface ratio – as in a large molecule – would favour retention of the solution conformation also in the crystal, but this need not be true for the smaller peptide molecules. Moreover, the smaller peptide hormones such as oxytocin or angiotensin still elude x-ray analysis, if only for thetrivial reason that they do not afford satisfactory crystals. On the other hand, promising work has been done recently on the conformation of these peptides in solution, mainly by proton magnetic resonance spectroscopy. The results suggest that oxytocin (Urry and Walter, 1971), lysine vasopressin (Von Dreele, Brewster, Scheraga, Ferger and du Vigneaud, 1971), angiotensin (Jogensen and Weinkam, 1971) and perhaps also calcitonin (Wuthrich, Masson and Donzell, 1971) haverelatively stable, or statistically predominant, conformations in polar solvents whereas no evidence for a stabilized conformation has been found in the case of corticotropin derivatives (see Schiller and Schwyzer, 1971).

Though such work is, of course, exceedingly important, relevant, and stimulating to further research there are a priori considerations as well as some indirect experimental evidence which caution against the assumptionthat the conformation in solution is necessarily the same as that involved in the biologicallyfunctional interaction. A priori, it maybe reasoned that the intermolecular forces involved in bindingthe peptide, e.g. to a receptor, are of the same kind and magnitude as the intramolecular conformation-holding forces; the binding process may therefore more than compensate for a departure from the optimal conformation of the isolatedmolecule. Experimental evidence from model systems also indicates that peptide-protein bindingmay involve conformational changes in one or both partner molecules. Thus in the ribonuclease-S system, which may be regarded as a useful model for peptide hormone action, the S-peptide shows no evidence of a-helical structure in aqueous solution (Scatturin, Tamburro, Rocchi and Scoff one, 1967) but becomes largely helical whenbound to the S-protein (Wyckoff, Hardmann, Allewell, Inagami, Johnson and Richards, 1967). Again, when oxytocin forms a complex with the carrier protein neurophysin II there is a marked change in the circular dichroism in the absorption region of the disulphide group (Breslow, 1970) which most probably signifies a conformational change in one or both partners in the complex.

A similar direct approach to studying the conformation of peptide hormones interacting with their receptors will probably require improved and refined physical techniques, and this aim is being vigorously pursued in a number of laboratories.

Meanwhile the classical approach by structure-activity relations still remains a useful tool for the indirect exploration of the structural requirements for biological action, as well as being a source of analogues of theoretical and practical interest. Here again, however, it is necessary to be clear about the precisemeaning of ‘structure’ and to define as far as possible the various repercussions of a particular molecular variation. Preferably such considerations shouldalready enter into the design of hormone analogues (Rudinger, 1971). The individual factors ofstructure, both steric (bulk, bonding geometry) and chemically functional in the widest sense (reactivity, polarity and polarisability, hydrogen-bonding capacity, hydrophilic-hydrophobic properties) should as far as possible be varied separately so as to identify the particular factors of significance for the biologically active’structure.’ This is, indeed, a counsel of perfection but some examples taken fromour work may illustrate this endeavour.

In oxytocin or its desamino analogue, replacement of the disulphide bridge by a sterically similar thioether or dimethylene bridge (Table 1) gives biologically active analogues, showing that neither the reactivity nor any other specifically chemical properties of the disulphide group are necessary for the biological actions of the hormone (Rudinger and Jost, 1964; Schwartz, Rasmussen and Rudinger, 1964). In fact, it has recently been shown (Jost and Sorm, 1971) that these ‘carba’ analogues include the most potently uterotonic peptides known. On the other hand, the acyclic peptides [1,6-di-alanine]- and [1,6-di-serine] -oxytocin, in which the bridge element is lacking altogether, are less active than oxytocin by five or six orders of magnitude (Polácek, Krejcí, Nesvadba and Rudinger, 1970). This finding demonstrates the importance of the bridge for the biologically active conformation of the molecule.

Table 1

Effect on the uterotonic activity of replacement or omission of the disulphide bridge in oxytocin or desamino-oxytocin

aRat uterus; in IU./mg.

bFerrier, Jarvis and du Vigneaud, 1965.

cSaameli, 1964; cat uterus in situ.

dJost and Sorm, 1971.

eYamanaka, Hase, Sakakibara, Schwarts, Dubois and Walter, 1970.

fPolácek, Krejcí, Nesvadba and Rudinger, 1970.

Systematic replacement of isoleucine has been carried out in position 3 of oxytocin (see Rudinger, 1968) and in position 5 of angiotensin II (Jorgensen and Weinkam, 1971). A comparison of the results (Fig. 1) is particularly revealing. Whereas in angiotensin the lipophilicity of the side-chain seemsto be of overriding importance and β-branching the only significant steric feature, in oxytocin much more precisesteric requirements are superimposed on a general effect of lipophilicity. This is particularly clearly brought out by the relative positionsof the isoleucine and alloisoleucine analogues in the two schemes. In these two hormones different structural features of the same side-chainare therefore important for activity.

Fig. 1 Effect of replacing isoleucine in position 5 of angiotensin II (left) and in position 3 of oxytocin (right) on biological activity. Ordinate: rat pressor activity (angiotensin) or activity on the rat uterus in vitro (oxytocin) as percentage of the activity of the parent peptide, logarithmic scale. Abscissa: lipophilicity parameter Π for the appropriate amino acids (E. C. Jorgensen, personal communication). Circles:β-branched side-chains, triangles: unbranched or γ-branched side-chains. Values for angiotensin analogues from Jorgensen and Weinkam (1971), for oxytocin analogues from Rudinger (1968).

It may be noted in passing that this phenomenon – the functional non-equivalence of identical amino acids in different peptides, or in different positions of the same peptide – is to be anticipated from the conceptof topochemistry and is, indeed, trivial to theprotein chemist. It must severely limit any attempt to correlate biological activity with overall molecular properties of a peptide, or with the same structural parameter for all constituent amino acids (e.g. Sneath, 1966; Kaurov and Martynov, 1970).

‘Activity’

Hormone action is a very complex process. Even though the scheme in Fig. 2 may be simplified when exogenous peptide is used and the biological system chosen is simple, e.g. an isolated tissue, the peptide may still interact with numerous molecules or sites such as transport barriers, enzymes, or binding proteins as well as the specific receptors at which the response is initiated. The response to a given dose or concentration of peptide is a complex function of all these interactions, and since a structural change may affect any or all of them the recorded ‘activity’ is obviously a complex result, to be interpreted with due caution (Hofmann and Katsoyannis, 1963; Rudinger and Jost, 1964). The relevance of a particular ‘activity’ will depend on the purpose of the investigation. If it is the molecular processes at the receptor which are of primary interest then the response of the simplest available tissue, cellular, or subcellular system will presumably be the most informative; whereas from, e.g. the physician’s point of view, the information obtained under near-physiological or pathophysiological conditions is generally the most interesting. Unfortunately most of the work with synthetic peptide analogues has been done with standard pharmacological assay systems. Such preparations are chosen to meet criteria such as reproducibility, accuracy and sensitivity and are often quite complex without necessarily being physiologically relevant (e.g. the avian depressor assay for oxytocin or the adrenal ascorbic acid assay for corticotropin). There is a priori no reason why such bioassay preparations should be particularly suitable for investigating hormone analogues. Yet only rarely have biological preparations been designed or adapted specifically to study the effects of structural variations on particular factors of activity. Again, examples from our work with oxytocin analogues may serve to illustrate such efforts.

Fig. 2 Summary of factors in hormone action.

Oxytocin is known to be susceptible to degradation by aminopeptidases and this is thought to be an important pathway of physiological elimination of the hormone. Desamino-oxytocin, the highly active analogue (Table 1) synthesized by du Vigneaud and his co-workers (Hope, Murti and du Vigneaud, 1962; Ferrier et al., 1965) is resistant to such enzymes (Golubow and du Vigneaud, 1963). The analogue does not show a protracted effect in vivo and this throws some doubt on the obvious hypothesis that its high potency is related to its enzyme resistance. In order to study the physiologically meaningful inactivation of desamino-oxytocin and other analogues by the uterus itself we have adapted the simple and elegant oil-bath technique of Kalsner and Nickerson (1968). In our experiments the excised rat uterus, depolarized to abolish the rhythmic contractions, was contracted by exposure to oxytocin or the analogue in an aqueous medium and the medium was then replaced by mineral oil. Under these conditions the rate of relaxation of the muscle strip is a measure of the disappearance (‘disposition’) of the active substance (Kalsner and Nickerson, 1968). By this method we were able to show (J. Furrer, V. Pliska and J. Rudinger, unpublished results) that the half-time of relaxation after contractions induced by desamino-oxytocin was about 5–8 times longer than the corresponding time after oxytocin-induced contraction; similar results were also obtained with desamino-1-carba-oxytocin (Fig. 3). Now since the concentration of peptide in the vicinity of the receptors in the tissue is given by a steady state determined by the rate of accession (by diffusion from the bulk solution) and of disposition (presumably by enzymic inactivation) a decreased rate of disposition will, other factors being equal, increase the steady state concentration at the receptors and thereby the response. Whether or to what degree the observed differences in disposition do in fact contribute to the high potency of desamino-oxytocin and its ‘carba’ analogues must be determined by further experiments and calculations.

Fig. 3 Time course of the tension in a rat uterine muscle after contraction with oxytocin (OT) and with desamino-1-carba-oxytocin (DCOT) (added at P) and replacement of the aqueous medium with mineral oil (at O). Aqueous medium: Ca-containing K2SO4−Ringer (Schild, 1969) with added MgCl2(0·5 mM.). Tension recorded isometrically. The crosses mark relaxation to one-half of the plateau tension and the time required to reach this.

The second example demonstrates that even events following the peptide-receptor interaction, in which the peptide no longer participates, can affect the ‘activity’ of the hormone analogue quantitatively or even qualitatively. It was noted some time ago that the action of [2-0-methyltyrosine]-oxytocin on the isolated rat uterus under standard conditions varied from a normal, contractile effect to inhibition of the response to oxytocin, through intermediate stages resembling ‘partial agonism’ (Krejcí, Polácek and Rudinger, 1967). It was found that these changes could be deliberately brought about by varying the experimental conditions from those of the standard bioassay procedure; in particular, a lowering of the temperature and a decrease in the calcium concentration of the medium favoured inhibition. Similar properties were shown to varying degrees by other oxytocin analogues modified in sequence position 2 (see Rudinger and Krejcí, 1968). These results prompted us to reconsider pharmacological receptor models with a threshold and receptor reserve (e.g. Stephenson, 1956; Furchgott, 1966) and in particular the effect of the stimulus-effect coupling on the character of the dose-response relations in such systems. It turned out that a change in the efficiency of the stimulus-effectcoupling could, in fact, cause alterations in the response such as we had observed in the work with [2-0-methyltyrosine] -oxytocin and related analogues (Krejcí, Pliska and Rudinger, 1970). Similar conclusions were reached by Eggena, Schwartz and Walter (1970) from studies on the effect of the same group of analogues on the water permeability of the toad bladder – a system which has the additional merit that the stimulus-effectcoupling can here be biochemically identified in part with the adenyl cyclase – 3′,5′-AMP system.

It is an interesting corollary of this interdependence of ‘activity’ and stimulus-effect coupling that the observed responses can be used to obtain information about the processes involved in translating the primary stimulus into the final effect.

These examples should suffice to urge the need for, and the potentialities of, analysing ‘activity’ and ’structure’ in detail, separately and in relation to each other, if we wish to gain an understanding of what lies behind the glib phrase ’structure-activity relationships.’

Acknowledgement

The unpublished work reported in this paper was supported by grants No. 3.372.70 and 3.424.70 SR from the Swiss National Foundation for Scientific Research.

References

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Furchgott, R. F. The use of β-haloalkylamines in the differentiation of receptors and in the determination of dissocation constants of receptor-agonist complexes. Adv. Drug Res. 1966; 3:21.

Golubow, J., du Vigneaud, V. Comparison of susceptibility of oxytocin and desamino-oxytocin to inactivation by leucine aminopeptidase and α-chymotrypsin. Proc. Soc. Exptl. Biol. Med. 1963; 112:218.

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Krejcí, I., Polácek, I., Rudinger, J. The action of 2–0-methyltyrosine-oxytocin on the rat and rabbit uterus: effect of some experimental conditions on change from agonism to antagonism. Brit. J. Pharmacol. 1967; 30:506.

Polácek, I., Krejcí, I., Nesvadba, H., Rudinger, J. Action of [1,6-di-alanine]-oxytocin and [1,6-di-serine]-oxytocin on the rat uterus and mammary gland in vitro. European J. Pharmacol. 1970; 9:239.

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CALCITONIN

Outline

Chapter 3: Studies of the Effects of Human Synthetic Calcitonin in Experimental Animals and Man

Chapter 4: Ultimobranchial Function in Non-mammals

Chapter 5: Plasma Calcitonin Levels in Birds and Fish

Chapter 6: Ultimobranchial Calcitonin of Anguilla Japonica

Chapter 7: Chicken Calcitonin: Isolation and Biological Properties

Chapter 8: Stimulation of Secretion of Pig Thyrocalcitonin by Gastrin and by Gastrointestinal Secretagogues

Chapter 9: Synthesis of Highly Active Analogues of Salmon Calcitonin

Chapter 10: Metabolic Fate of Human Calcitonin in the Dog

Chapter 11: Ultrastructural and Cellular Changes at the Costochondral Junction Following in vivo Treatment with Calcitonin or Calcium Chloride in the Rabbit

Chapter 12: Radiommunoassays for Calcitonins: Clinical and Experimental Studies

Chapter 13: Physiological Function of Thyrocalcitonin

Studies of the Effects of Human Synthetic Calcitonin in Experimental Animals and Man

I. MACINTYRE

P.G.H. BYFIELD

L. Galante

E.W. MATTHEWS

J.M. MOSELEY

N.J.Y. WOODHOUSE,     Wellcome Unit of Endocrinology, Royal Postgraduate Medical School, Ducane Road, London W12 OHS

Publisher Summary

This chapter discusses the embryological origin of the C cells, the purification and properties of dogfish calcination, and the therapeutic effects of calcination M in Paget’s disease. Dogfish calcitonin is extremely natriuretic in the rat. Calcitonin is extremely effective in suppressing excessive osteoclastic activity in man. This appears to be true for the porcine and salmon materials as well as the human hormone, but when these hormones are given over a prolonged period, only the human hormone has so far been found to cause complete return to normal of most of the indices of bone resorption. The human hormone has an extremely feeble action in comparison with the dogfish material. It is observed that (1) the C cells are of neural crest and not ultimobranchial origin; (2) dogfish calcitonin material is similar to the salmon hormone in having a powerful natriuretic effect; and (3) human calcitonin is effective in Paget’s disease and further careful trials are warranted.

We summarize here some of our recent experimental and clinical work with calcitonin at the Royal Postgraduate Medical School. We shall discuss: the embryological origin of the C cells; the purification and properties of dogfish calcitonin; and the therapeutic effects of calcitonin M in Paget’s disease.

Neural crest origin of the C cells

Pearse and Carvalhiera (1967) showed that the rat C cells migrated to the thyroid with cells from the ultimobranchial body. Pearse was careful to deduce only this fact and considered that it was possible that the C cells were in fact ultimately derived from elsewhere. The ultimobranchial origin origin was apparently confirmed when Tauber (1967), Copp, Cockcroft and Kueh (1967) and Moseley, Matthews, Breed, Galante, Tse and MacIntyre (1968) showed that calcitonin could be extracted from the ultimobranchial bodies of birds, fish and reptiles. But there was evidence which did not fit with these supposedly conclusive results.

1. In the pigeon the thyroid contains many C cells even although a separate ultimobranchial body exists in this species. This probably means that C cells reach the thyroid from some source other than the ultimobranchial body. Calcitonin can be extracted both from pigeon and chicken thyroid (Moseley et al., 1968; MacIntyre, 1970).

2. Ultimobranchial extracts are very rich in activity but in the case of the very potent salmon material this represents quite a small amount of hormone. The likely explanation is that most ultimobranchial cells in the salmon are not C cells, because otherwise the amount of activity would have been even higher.

We have felt uneasy about these discrepancies although some workers attempted to show that they did not exist. It is now quite clear from the beautiful experimental work of Le Douarin and Le Lièvre (1970) from France and Pearse and Polak (1971) of the Royal Postgraduate Medical School that the C cells are not derived from the ultimobranchial body at all. They are in fact neuro-ectodermal cells from the neural crest which may end up either in the thyroid or in the ultimobranchial body, or in both organs. I must not anticipate the scientific evidence for this statement which will be presented elsewhere in the symposium but it is convincing (this symposium). Calcitonin is therefore a hormone derived from the cells of the neural crest and not from cells of entodermal origin such as the ultimobranchial