A unique ligamentary system occurs in the polychaete Nephtys. Its fine structure and physico-chemical properties have been investigated.

The ligaments consist of alternating bands of elastic and inelastic elements, and they attach to the body by means of crystalloid attachment nodes. The nodes are probably glycoprotein in nature as they contain both protein and polysaccharide. They are not birefringent, and they are insoluble in comparison with the other components of the ligaments. Their structural stability is due mainly to strong ionic and hydrogen bonding. They resist enzymatic digestion.

The inelastic elements are bundles of thin, birefringent fibrils, 10 to 14 μ in length. The birefringence is positive with respect to the long axis of the fibrils and is primarily intrinsic in nature. The estimated coefficient of birefringence has a value between 10 and 20 × 10-3. The fibrils consist of a protein with an isoelectric point near pH 6 · 3. The visible structure of the fibrils is due to both hydrogen and ionic bonding, while the birefringence is more dependent on hydrogen bonding. The fibrils resist heating and peptic digestion, but are slowly digested by trypsin. Of all the fibrous proteins, they most resemble collagen.

The elastic elements are extremely fine, granular cross-membranes bearing delicate filaments to which the ends of the fibrils are attached. They are continuous with a bounding membrane surrounding the ligament, which also bears granules and is also elastic. Some of the granules appear to be folds of the membrane itself. The membranes consist of a protein which is quickly digested by trypsin but not digested by pepsin. Its isoelectric point lies near pH 5. Hydrogen bonding is more important than ionic bonding in the membranes, and Van der Waals forces may also contribute. Classification of this protein is not yet possible.

The polychaete Nephtys possesses a system of ligaments which is, so far as is known, unique. The ligaments are conspicuous, shining, broad straps of tissue overlying the nerve-cord and running from there to the bodywall and parapodia, but neither their nature nor their function is clear.

They were discovered in N. caeca (Fabricius) by Ehlers (1864—8), who described something of the anatomy of the system. He observed the characteristic sheen of the bands and their woven or plaited appearance, but formed no opinion about their nature or function. Langerhans (1880) found similar ligaments in N. hombergi and N. agilis and observed that they were crossed by zigzag lines between which there occurred a fine, narrow, longitudinal striping. He concluded that the system of ligaments was an internal skeleton. N. caeca was re-examined by Schack (1886), but he added nothing to the two previous accounts. Emery (1887) made a detailed study of the anatomy of the ligamentary system and of the microscopic structure of the ligaments. He found that the woven appearance was due to alternating light and dark bands. He observed that the dark bands were birefringent and concluded that these structures were striated muscles and that the coelomic epithelium investing them was a delicate sarcolemma. In fact, it has been shown by Hoyle (1957) that they are not muscles.

We have observed these structures in the following species: N. bucera Ehlers, N. caeca (Fabricius), N. caecoides Hartman, N. californiensis Hartman, N. cirrosa Ehlers, N. cornuta Berkeley and Berkeley, N. ferruginea Hartman, N. glabra Hartman, N. hombergi Lamarck, N. incisa Malmgren, N. longosetosa Oersted, N. magellanica Hartman, N. picta Ehlers, N. punctata Hartman, N. rickettsi Hartman, N. squamosa Hartman; and we know of no member of the family Nephtyidae that lacks them. We have not made a detailed examination of the ligamentary system in all these species, but it appears that its anatomy and structure are the same throughout the family.

The structure of the ligaments has been examined by phase contrast and polarizing microscopy, and their chemical and histochemical properties have been investigated.

Ligaments dissected from N. cirrosa and N. hombergi were used fresh, or fixed in 2·5% formalin, 10% formalin, or 35% alcohol as required.

The following histochemical methods were employed: biuret and ninhydrin tests for protein; Voisenet test for tryptophane; xanthoproteic reaction for phenols; nitroprusside test for sulphydryl and disulphide (all after Serra, 1946); lead acetate test for sulphur (Hawk, Oser, and Summerson, 1947); modified Thomas reaction for arginine (Liebman, 1951); periodic acid /Schiff (PAS) reaction after diastase treatment for polysaccharide other than glycogen; the standard Millon reaction for tyrosine; and saturated solution of Sudan black in 70% alcohol for lipid. For the arginine and PAS reactions, worms were fixed in Bouin and sectioned at 10 μ. Treatment with Sudan black was carried out on freshly dissected ligaments. In all other cases, dissected ligaments were fixed in 10% formalin and thoroughly rinsed in distilled water. In addition, the ninhydrin reaction was carried out on fresh ligaments as follows: (1) 0·2% ninhydrin in M/15 phosphate buffer, pH 6·98; (2) 0·1% ninhydrin in distilled water; (3) 0·1% ninhydrin in 2 M CaCl2; (4) 0·1% ninhydrin in 2 M NaCl. The reaction in each case was carried out over boiling water for 1 to 2 min.

The reactions of fresh ligaments to the following reagents were also tested: 0·2 N HC1; 2 M CaCl2; 6 M urea; saturated LiSCN; saturated Ca(SCN)2; 98% formamide; 0·5 M, 1 M, 2 M, and 5 M NaCl; 0·25% trypsin (B.D.H.), pH 7-5 and 8·0; 0·25% pepsin, pH 4·58.

The effect of increased temperature on dissected ligaments was observed by mounting the specimen in distilled water or 20% ethyl alcohol on a slide.

A coverslip was then sealed on with silicone grease. The slide was immersed in a water bath which was warmed by a heated copper plate placed on the microscope stage. Temperatures up to 85° C were obtainable. Changes in the length of small regions of the ligaments were followed with an ocular micrometer.

The solubility of the ligaments at varying pH was studied. Freshly dissected ligaments were immersed in buffers ranging from pH 1·45 to 12·00. For the pH range 1·45 to 9·20 a modified Michaelis acetate-barbiturate buffer (Pearse, 1953) was used, with 0·196 M sodium barbital substituted for the sodium barbiturate of the original. For the pH range 9·52 to 12·00, the Sörenson-Walbum glycine buffer (Pearse, 1953) was used. The pH of each solution was checked with an EEL direct-reading pH meter. In addition, the solubility of the ligaments was tested in 0·2 N HC1 (pH 0·7). All these tests were continued for 24 hours.

To determine the nature of the birefringence exhibited by these ligaments, specimens were studied with the polarizing microscope after immersion for 24 h or more in the following media (refractive indices shown in brackets): methylene iodide (1 · 756); 1-bromo-naphthalene (1 · 658); carbon disulphide (1 · 627); aniline (1 · 586); terpineol (1 · 483); castor oil (1 · 477); chloroform (1 · 466); 1-butanol (1 · 399); sea-water (1 · 34); distilled water (1 · 333). Measurements of retardation were made with a Berek compensator, in monochromatic light from a sodium lamp.

Microscopic appearance of the ligaments

The ligaments are flattened ribbons lying in the coelomic cavity. There is a paired, median ligament running in the midline on the dorsal surface of the ventral nerve-cord. At two points in each segment it gives off several pairs of lateral ligaments which run to the intersegmental body-wall and into the parapodia. The ligaments are attached both to the nerve-cord sheath and to the body-wall by means of small, hard ‘attachment nodes’.

The intact ligaments vary considerably in width, not only among animals of different size, but also within the same individual. The larger ones particularly have a silky, sometimes greenish, sheen in reflected light. In transmitted light the ligaments are colourless or brownish yellow.

The ligaments have a very unusual microscopic appearance. They are composed completely of extracellular, secreted material and are enclosed in a thin covering of coelomic epithelium. The attachment nodes have an irregular outline and a crystalline appearance, while the ligaments themselves are composed of alternating dark and light bands, which may cross the long axis perpendicularly or diagonally. These dark and light bands, just visible with the dissecting microscope, give the appearance of striations, and when, as often happens, the diagonal striping is irregular, a herring-bone or woven appearance results (see fig. 1).

FIG. 1.

A camera lucida drawing of the dorsal aspect of a ligament dissected from over the ventral nerve-cord, showing attachments to a node and the woven appearance of the ligaments.

FIG. 1.

A camera lucida drawing of the dorsal aspect of a ligament dissected from over the ventral nerve-cord, showing attachments to a node and the woven appearance of the ligaments.

On closer examination, the ligament is seen to be made up of many fine, short fibrils which are grouped together side by side to form the dark bands. They connect at each end with an extremely fine, irregular, transverse membrane bearing small granulations, which occurs in the middle of the light bands. The fibrils are not attached to the membrane directly, but by means of a wavy filament which connects the fibril to a granule on the membrane. Evidence of these has been found on observing the torn ends of ligaments where frequently the fibrils retain these filaments and the granules from the cross-membranes. The cross-membranes are continuous with a limiting membrane which surrounds the whole ligament. In larger ligaments there may be internal longitudinal membranes as well as the limiting one, so that these ligaments are actually composed of several large parallel units. Such longitudinal membranes can only be detected under special conditions, as described below. The fine structure of the ligaments, for which evidence is given later, is shown diagrammatically in fig. 2.

FIG. 2.

A semi-diagrammatic representation of an enlarged portion of a ligament, showing the fine structure and relationships of the various components.

FIG. 2.

A semi-diagrammatic representation of an enlarged portion of a ligament, showing the fine structure and relationships of the various components.

Ligaments treated with dilute trypsin or ligaments from sexually mature animals are quite fragile and partially disintegrate, releasing individual fibrils. These measure 10 to 14 μ in length (though occasionally fibrils are 20 or 21 μ long) and about 0·3 to 0·5 μ in diameter. The latter figure can only be estimated with the light microscope, as diffraction lines make exact measurement impossible. The range of length of the fibrils is the same in large (132 mm) and small (45 mm) worms. The longer fibrils are slightly thicker than the short ones. All appear to taper at their ends. Isolated fibrils are usually straight, but some are sharply befit near one end or, rarely, in the middle. This is probably an artifact resulting from damage during dissection. When ligaments are torn, they invariably break cleanly at a cross-membrane, and when they are bent in handling they bend most readily at these points.

The ligaments are flexible and exhibit elastic properties. During dissection ligaments were often stretched as much as twice their resting length before breaking. When the distortion was small, as it is during normal movements of the animal, an extended ligament retracted to its initial length as soon as the distorting force was removed. The ligaments are not always under tension in the animal, however, since when the worm is contracted the longitudinal ligaments fall into shallow folds.

When the ligaments were manipulated, it was found that stretching takes place only in the light bands, but the complexity and small size of the structure involved made it impossible to detect exactly how this occurs. However, in partially torn ligaments, isolated regions of the limiting membrane were seen to stretch as much as two to three times before breaking, and this was followed by instantaneous contraction. The limiting membranes, like the cross-membranes, bear granules and some of these disappear on stretching and reappear on contraction, which may indicate that they are simply folds of the membrane. As will be shown below, no differences were found in the chemical nature of the bounding membranes on the one hand, and of the cross-membranes and the wavy filaments by which the fibrils attach to them, on the other. It is reasonable to conclude that the cross-membranes and their wavy filaments are also elastic, though it cannot be said whether the elasticity of the isotropic regions is primarily due to stretching of the wavy filaments or to distortion of the cross-membranes with unfolding of some of the granules.

Birefringence of the ligaments

A most striking property of the ligaments is their high degree of birefringence. In polarized light they show alternating bright and dark bands, corresponding to the dark and light bands, respectively, seen in non-polarized light (figs. 3 A, B). The fibrils are responsible for the birefringence, and they can just be seen individually in the polarizing microscope. The birefringence is positive with respect to the long axis of the fibrils. In favourable preparations, the interband membrane also shows an extremely faint birefringence.

FIG. 3.

(plate). A, the torn end of a ligament seen in non-polarized light. The fibrils and irregular cross-membranes are just visible. B, the same ligament as it appears in polarized light. Birefringence is associated with the fibrils. C, part of a ligament viewed with phase contrast. D, the same ligament immediately after treatment with buffer at pH 1 · 70. The fibrils have disappeared and the relationship of bounding and cross-membranes is apparent. A longitudinal membrane is also visible in the middle of the ligament. Granules can be seen on some of the membranes. Note lateral shrinkage of the ligament. E, ligaments after treatment for 24 h at pH 3 · 90. The fibrils are not completely dissolved but have swollen and distended the membranes.

FIG. 3.

(plate). A, the torn end of a ligament seen in non-polarized light. The fibrils and irregular cross-membranes are just visible. B, the same ligament as it appears in polarized light. Birefringence is associated with the fibrils. C, part of a ligament viewed with phase contrast. D, the same ligament immediately after treatment with buffer at pH 1 · 70. The fibrils have disappeared and the relationship of bounding and cross-membranes is apparent. A longitudinal membrane is also visible in the middle of the ligament. Granules can be seen on some of the membranes. Note lateral shrinkage of the ligament. E, ligaments after treatment for 24 h at pH 3 · 90. The fibrils are not completely dissolved but have swollen and distended the membranes.

An attempt was made to determine the degree of intrinsic and form birefringence by immersing ligaments fixed in 2 · 5% formalin in media of various refractive indices, and measuring the retardation with a Berek compensator while illuminating with monochromatic light. Thickness was measured by focusing. The effect of formalin fixation on the birefringence was found to be nil by measuring the retardation of the same area of a ligament before and after fixation.

Because the packing of the fibrils varied considerably in different specimens and could not be accurately estimated, no exact values were obtained of the relative amounts of intrinsic and form birefringence. Isolated fibrils, because of their fine width, give far too small retardations for accurate measurement by this method.

However, certain conclusions are possible. On immersion in various media with refractive indices between 1 · 333 and 1·756, the birefringence never disappeared nor did it ever become faint. In methyl salicylate (R.I. = 1 · 537) the fibrils lost their refractility, and their birefringence was slightly decreased. Thus, the fibrils have a refractive index of about 1 · 50 to 1 · 55, and their birefringence is largely intrinsic.

While measurements of the coefficient of birefringence (retardation/thickness) obtained here are at best only rough estimates, the values fall between 10 and 20 × 10−3, indicating a high degree of anisotropy for biological material.

According to Bear (1952), tannic acid treatment changes the sign of the intrinsic birefringence of collagen. This was tested on the ligaments, which were treated for 3 days with 10% tannic acid, and then immersed for 24 h in methyl salicylate. At this refractive index, the contribution of formbirefringence to total birefringence would be near its minimum. Unlike that of collagen, the birefringence of the ligaments remained strongly positive.

Histochemical characters of the ligaments

The ligaments consist of 4 components: (1) the birefringent fibrils; (2) the interband and bounding membranes; (3) the attachment nodes; (4) the ‘matrix’ in which the fibrils lie. Of these, all but the last are microscopically distinct. The interband and bounding membranes are very thin, and it is difficult to judge their reactions in most cases. This applies as well to the delicate coelomic epithelium surrounding the ligaments, the reactions of which will also be considered.

The results are summarized in table 1. The biuret and ninhydrin tests show that the fibrils, membranes, and attachment nodes all contain protein. The attachment nodes react only moderately with the biuret test but give a strong blue colour with 0 · 1% ninhydrin in distilled water. On the other hand, the fibrils react readily with the biuret test but require treatment with 2 M CaCl2 or NaCl before giving a moderate ninhydrin reaction. This suggests that ionic bonding of carbonyl and amide groups occurs in the fibrils, though not in the attachment nodes, and this prevents a reaction with ninhydrin. The interband and bounding membranes as well as the coelomic epithelium are biuret-positive and the matrix appears to be faintly positive also. However, the colour reactions of the matrix remain inconclusive since it is crossed in the interband region by the tapering ends of fibrils and by filaments from, and possibly folds of, the interband membranes, all of which are at or below the resolution of the light microscope.

TABLE 1.

Reaction of various components of the ligaments to histochemical tests

Reaction of various components of the ligaments to histochemical tests
Reaction of various components of the ligaments to histochemical tests

The strong positive birefringence of the fibrils seems to preclude the presence of significant amounts of lipid or nucleoprotein in them. Alcoholether extraction for 24 h has no observable effect on the ligaments, and treatment with Sudan black shows mild lipid granularity only in the coelomic epithelium. The presence of polysaccharide material can be demonstrated in the attachment nodes by using the PAS technique after diastase treatment. No reaction was observed elsewhere in the ligaments, though the coelomic epithelium is faintly PAS-positive.

The ligaments were then tested for the presence of various amino-acids. Both the alkaline lead acetate test for sulphur and the nitroprusside reaction for protein-bound —SH and S—S are negative, which indicates that no significant amounts of amino acids containing sulphur are present. Both tyrosine and arginine are present in the fibrils and the attachment nodes. The latter, presumably because of their denser nature, give a more intense reaction in each case. The fibrils give a negative reaction for tryptophane, though the attachment nodes are strongly positive. This further confirms that they contain different proteins, which was already suggested by their differing reaction to the biuret and ninhydrin tests. Both fibrils and attachment nodes give a positive xanthoproteic reaction of similar intensity. In view of the stronger tyrosine and tryptophane reactions in the nodes, this may possibly indicate a fair amount of phenylalanine in the fibrils, though no definite conclusion on this point can be drawn. (Hawk, Oser, and Summerson (1947) state that phenylalanine does not react under the usual conditions of the test, while Serra (1946) says that it does.)

Solubility at various hydrogen ion concentrations

The results of immersing freshly dissected ligaments in buffers ranging from pH 1 · 45 to 12 · 00 are given in table 2. The fibrils are insoluble over the pH range 4 · 9. The membranes have a wider range from 1 · 4 to 8-7, and the attachment nodes, which are the least soluble of the 3 in dilute acid and alkali, are insoluble between pH 1 · 4 and 9. As solubility is least at the pH of the isoelectric point (Cohn and Edsall, 1943) and increases nearly symmetrically on each side, the middle of the pH range of insolubility may be taken as an estimate of the isoelectric point. That of the fibrils is near pH 6 · 3, while the membranes and attachment nodes are more acidic, having isoelectric points near pH 5.

TABLE 2.

Effect of various hydrogen ion concentrations on components of the ligaments

Effect of various hydrogen ion concentrations on components of the ligaments
Effect of various hydrogen ion concentrations on components of the ligaments

Solution of the fibrils in dilute acid is accompanied by an initial lateral shrinkage followed by a slow lateral swelling. The latter is best seen after treatment for 24 h in the pH range 1 · 45 to 3 · 90, when the membranes remain intact. As swelling occurs, the membranes are stretched, and the ligament increases in width. At pH 3 · 90, the fibrils do not dissolve, but there is some swelling (fig. 3, E). This is accompanied by an almost complete loss of birefringence.

The differential solubility of the components of the ligaments allows their chemical dissection, giving a clearer picture of how they are constructed. Fig. 3, c, D shows a freshly dissected ligament in distilled water and the same ligament immediately after treatment with buffer pH 1 · 70. It can be seen that the fibrils have disappeared and the membrane system has become more apparent in D. This membrane system is composed of a continuous sheath surrounding the ligament, and of cross-membranes corresponding to the isotropic, interband regions of the untreated ligament. Fig. 3, E shows a ligament after treatment for 24 h in buffer pH 3 · 90.

Results of enzymic digestion

Pepsin at a concentration of 0·25% and at pH 4 · 58 has no effect on any component of the ligaments even after 24 h. On the other hand, 0·25% trypsin at pH 7 · 50 readily digests the coelomic epithelium and the entire membrane system, freeing the fibrils. The latter are digested only slowly, digestion and the loss of birefringence being nearly complete after 24 h. The attachment nodes remain intact.

Tryptic digestion, therefore, affords a method of isolating the fibrils from the other components of the ligament. Fig. 4, A, B shows the same ligament before and after digestion with 0·25% trypsin at pH 7 · 50 for 15 min. Loss of the membranes and partial digestion of a cell of the coelomic epithelium can be seen. Fig. 4, c shows the fibrils dispersed by pressure on the coverslip, and fig. 4, D shows rows of fibrils, without their membranes but still more or less in position, and it also shows them separated from the attachment nodes. This suggests that the fibrils are attached to the nodes by filaments or membranes similar to those elsewhere in the ligaments.

FIG. 4.

(plate). A, part of a ligament viewed with phase contrast. Granules can be seen on the cross-membranes. The nucleus of a cell of the coelomic epithelium is lying against the ligament. B, the same ligament immediately after treatment with 0 · 25% trypsin at pH 7 · 50. The cell of the coelomic epithelium and the membranes are partially digested, but the fibrils remain intact. c, a region from the same preparation as B after pressure on the cover slip has dispersed the fibrils. D, another region of c, showing separation of the fibrils from an attachment node (upper left).

FIG. 4.

(plate). A, part of a ligament viewed with phase contrast. Granules can be seen on the cross-membranes. The nucleus of a cell of the coelomic epithelium is lying against the ligament. B, the same ligament immediately after treatment with 0 · 25% trypsin at pH 7 · 50. The cell of the coelomic epithelium and the membranes are partially digested, but the fibrils remain intact. c, a region from the same preparation as B after pressure on the cover slip has dispersed the fibrils. D, another region of c, showing separation of the fibrils from an attachment node (upper left).

Results of heat treatment

The distance from one interband membrane to the next was measured with an ocular micrometer on a ligament mounted in a water bath on the stage of the microscope. There is no change in length during heating of the ligaments from room temperature to 80° C, or after maintaining them at the latter temperature for as long as 112. Ligaments heated while immersed in 20% alcohol also remain unchanged in length. There is no swelling in either water or alcohol.

After heating over a boiling-water bath 114h, the fibrils and attachment nodes remain unchanged, but the interband membranes partially dissolve. After only 40 min there is a marked decrease in the birefringence of the fibrils, which suggests a disruption at the submicroscopic level. This means that very little of the fibrillar birefringence can be due to their microscopically visible anisometry.

Effect of lyotropic agents

The various bonds stabilizing structural proteins may result from Van der Waals forces, from tanning agents, and from electrovalent and covalent linkages (Brown, 1950). An attempt has been made to determine the degree and type of interpeptide chain bonding in the various proteins of the ligaments, especially the fibrils.

The high resistance to thermal agitation suggests that Van der Waals forces are not primarily responsible for the stability of the fibrils and attachment nodes, though they may contribute to the birefringent properties of the fibrils and to the structure of the interband membranes. The ready solubility of the ligaments in 0·2 N HC1 rules out tanning agents, and indeed, fixation in formaldehyde confers resistance to acid treatment. The absence of positive results to sulphur tests and the solubility of the ligaments at moderate pH preclude any significant contribution from disulphide bonding. There remain hydrogen and ionic bonds to account for the structural stability of the fibrils and attachment nodes. Ionic bonds are broken by dilute acid and alkali and by solutions of strong electrolytes, whereas both ionic and hydrogen bonds are broken by lyotropic agents (Bear, 1952). Whether the action of a given salt in dissolving a protein is due to its effect on ionic or hydrogen bonds can only be determined by comparison with the actions of other salts which are more or less lyotropic (Gustavson, 1949). NaCl, which has little lyotropic activity, serves as a base-line for the action of more lyotropic salts, such as CaCl2, Ca(SCN)2, and LiSCN. Proteins attacked by the latter but unaffected by NaCl must depend for their structural stability primarily on hydrogen bonds. For this reason, the effect on the ligaments of NaCl at various concentrations as well as of more lyotropic agents has been tested.

The results are shown in table 3. At no concentration does NaCl affect the membranes. It has its maximum effect on the fibrils at 1 to 2 M and on the attachment nodes at 5 M. In no case does it cause solution, but the effect on the fibrils is striking and unmistakable. During swelling they lose much of their resolution when viewed in ordinary light, though not in polarized light. On the other hand, lyotropic agents of similar ionic strength dissolve both fibrils and membranes. The birefringence of the fibrils is more susceptible to lyotropic action than is their visible structure. The attachment nodes are most resistant, and only dissolve readily in 98% formamide.

TABLE 3.

The effect of lyotropic agents on the ligaments

The effect of lyotropic agents on the ligaments
The effect of lyotropic agents on the ligaments

Of the four components of the ligamentary system of Nephtys, only the fibrils, attachment nodes, and membranes have been sufficiently characterized to warrant further discussion. Whether the matrix surrounding the fibrils is a dilute protein solution or is protein-free is not clear.

All three components are composed primarily of protein, though the attachment nodes also contain polysaccharide. The protein constituent of the attachment nodes differs from the proteins of both the fibrils and membranes not only in its resistance to solvents and to tryptic digestion, which may only be due to inaccessibility of its molecules through closer packing or to the presence of the polysaccharide, but also to its possession of a considerable amount of tryptophane, which is absent from the other two structures. The toughness of the nodes noted in handling, their crystalloidal appearance, and their relative insolubility suggest a close packing of the molecules, resulting from strong ionic and hydrogen bonds which are broken only by fairly strong acids and highly active lyotropic agents. The absence of detectable amounts of sulphur precludes any significant disulphide bonding. The absence of birefringence in the nodes probably means that the molecular packing is randomly oriented or that the micelles are nearly isometric. No conclusions have been drawn regarding the classification of this secreted, non-fibrous protein, though it seems possible that it is a glycoprotein.

The protein of the membranes differs from that of the fibrils in several respects, although the membranes are too small to show any differences with the usual histochemical tests. They are more readily susceptible to tryptic digestion. They also are stretchable and have been observed to possess elastic properties at distorting forces which have no effect on the fibrils. Indeed, no evidence has been found that the fibrils are elastic or even irreversibly extensible. The membrane protein has a much lower isoelectric point than has that of the fibrils, which indicates the presence of more acidic residues. It is also insoluble over a much wider pH range than the fibrils are, but is affected similarly by lyotropic agents. The membranes are unaffected by alcohol, but are partially destroyed after prolonged boiling, which has no effect on the visible structure of the fibrils though it decreases their birefringence.

The solubility of a protein depends on two opposing forces: (1) the lattice energy of the solid protein and (2) the energy of attraction between the dissolved protein and the solvent (Cohn and Edsall, 1943; Edsall, 1947). Three types of bonds contributing to the lattice energy must be considered here: ionic, hydrogen, and Van der Waals. As mentioned above, dilute acids and alkalis and solutions of strong electrolytes attack ionic bonds (Bear, 1952). The ions interact with the dipolar protein, producing hydration due to a Donnan equilibrium effect (Hermans, 1949), which results in a decrease in the activity coefficient of the protein and, consequently, in solution (Cohn and Edsall, 1943). On the other hand, both hydrogen bonds and ionic bonds are broken by lyotropic agents. The lyotropic action of salts can be estimated from their position in the Hofmeister series (Hermans, 1949). Other lyotropic agents include urea, formamide, &c. (Gustavson, 1949; Brown, 1950; Bear, 1952). Their loss of free energy on dilution is less than that of the protein concerned and consequently water passes from salt solution to the protein (Hermans, 1949). Van der Waals bonding is due to attractions between non-polar residues and is disrupted by organic solvents, which, however, decrease solubility of electrovalent bonds by decreasing the dielectric constant of the medium (Cohn and Edsall, 1943).

In the case of the membranes, insolubility over a wide pH range and in solutions of NaCl of varying ionic strength indicates either a high lattice energy of the ionic bonds or the presence of many hydrogen and/or Van der Waals bonds. That some hydrogen bonding is present is shown by the partial solution of the membranes in 2 M CaCl2 (ionic strength = 6) but not in 5 M NaCl (ionic strength = 5) and by their total solution in 6 M urea. If Van der Waals forces from non-polar residues play a significant part in the lateral bonding of the membrane proteins, as they do in vertebrate elastin (Bear, 1952), there must be sufficient polar residues, giving rise to electrovalent bonds, to prevent their solution in organic solvents. Whether the small granules on the membranes are the same or a different protein is not clear. It seems most probable that at least some of them are simply regions where the membrane is folded, and that the decreased granularity when the membranes are stretched is the result of an unfolding process.

It would be premature to attempt to relate this protein with other types of fibrous proteins. The membranes are too fine to give definite colour reactions to histochemical tests, and for this reason the standard tests for vertebrate elastin have not been tried. Even in vertebrate tissues, these may give equivocal results (Hall, 1957). The membranes are less resistant than vertebrate elastin to acid treatment and to lyotropic agents (Hall, 1957; Partridge, Davis, and Adair, 1957). Digestion by trypsin used to be considered characteristic of elastin (Hawk, Oser, and Summerson, 1947), but it has since been shown that recrystallized trypsin does not digest elastic fibres (Lansing, 1952) and that digestion by crude trypsin preparations is due to the presence of pancreatic elastase (Baló and Banga, 1950). The trypsin used in our experiments undoubtedly contained both enzymes, and it cannot be said which of the two digested the membranes. Aside from the claims of Bouillon and Vandermeerssche (1957) that mesogloeal fibres in medusae are composed of elastin, this protein has not been reported in invertebrates. On the other hand, secreted fibres with elastic properties are not unknown (e.g. the byssus threads of Mytilus (Brown, 1952)), and although collagen fibres are not usually very extensible (Bear, 1952), collagen-like fibres may be (Picken, 1940; Brown, 1952). There is even some evidence of elastin being produced during life from collagen in vertebrates (Hall, 1957), and elastin seems more and more to be a member of the collagen group (Burton and others, 1955; Astbury, 1957). However, if the elastic membranes of the ligaments in Nephtys resemble collagen, they must have a very different organization from the fibrils.

With regard to the protein of the fibrils, there are two factors to consider: the structure giving rise to the birefringence, and the visible structure. The presence of much arginine in the fibrils coupled with their slightly acidic isoelectric point means that they possess sufficient acidic amino-acids to balance the basic arginine, and therefore they probably have considerable ionic bonding. This conclusion is supported by their solution on fairly small shifts in pH (±2 · 5 units) from the isoelectric point, their swelling and loss of resolution in 1 · 2 M NaCl, and the necessity of treating them with NaCl or CaCl2 to obtain a satisfactory ninhydrin reaction. That the birefringence is hardly affected by NaCl but decreases sharply after treatment with the more lyotropic CaCl2 and urea suggests that the birefringence is more dependent on hydrogen bonding, while the visible structure is due to both types of bonds. Van der Waals forces may play some part in producing the anisotropy, since prolonged treatment in boiling water decreases the birefringence.

The properties of the fibrillar protein so far described resemble those of collagens more than those of other fibrous proteins. It differs from most collagens in not being soluble in boiling water and in being digested slowly by trypsin. However, ichthyocol, a collagen from teleost skin, is also digested by trypsin (Gustavson, 1949), and elasmobranch collagen, elastoidin, is not transformed to gelatin on heating (Picken, 1940; Bear, 1952). Its positive birefringence, like that of collagen, is characteristic of all fibrous proteins. However, the sign of the intrinsic birefringence is not reversed by tannic-acid fixation, as occurs with some collagens (Bear, 1952).

Collagen-like proteins are known to occur in many invertebrates, and they have been reported in all three major annelid classes. Bradbury (1957, 1958) has described collagenous connective-tissue fibres in the Hirudinea, and the cuticles of both oligochaetes (Lumbricidae) and polychaetes (Aphrodite) yield the X-ray diffraction pattern of collagens (Rudall, 1955). However, the protein of earthworm cuticle differs from vertebrate collagen in having low thermal stability and in not being striated in electron micrographs, and, despite its collagen-like, wide-angle X-ray diagram, Gustavson (1957) is doubtful whether it should be considered a member of the collagen family. Smith (1957) has reported the presence of circumferential fibres originating from the sheath of the ventral nerve-cord and running around the body-wall in Nereis, but these appear not to be collagenous. Thus, while a type of collagen may occur in polychaetes, the ligaments of Nephtys are the only connective-tissue elements that are known which contain a probable collagen not directly associated with the cuticle or the sub-epidermal basement membrane.

The ligaments of Nephtys are also unique in being composed of alternating elastic and inelastic elements, the membranes and the fibrils respectively. The functional significance of such a structural organization will be considered in a following paper.

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