Eur. J . Biochem. 112, 389-396 (1980) Q by FEBS 1980

Cockroach Collagen : Isolation, Biochemical and Biophysical Characterization Jean FRANCOIS, Daniel HERBAGE, and Simone JUNQUA

Laboratoire de Zoologie-Universite de Dijon, Laboratoire de Chimie Macromoleculaire-Universite de Lyon, and Laboratoire de Biochimie du Tissu Conjonctif-Universitk Paris Val de Marne

(Received July 15, 1980)

Collagen fibrils from the mesenteric connective sheath of the adult cockroach Periplaneta americana were extracted by enzymatic digestion with pepsin and were purified. Chromatographic studies and sodium dodecylsulfate electrophoresis revealed the presence of a single chain. It was demonstrated that the structure of this collagen could be represented by the formula (a)3. The amino acid compo- sition is typical of collagens (one-third glycine, and a high imino acid content) and similar to that of type 11. The carbohydrate content was high (8.8%), and the cyanogen bromide pattern was dif- ferent from that of known collagens. The chains were linked by the stable intermolecular bond dihydroxylysinonorleucine. The banding patterns of the segment-long-spacing crystallites and of the reconstituted fibrils were similar to type I collagen. The molecular weight ( M , 280000) and length (285 nm) were typical, but the denaturation temperature was high (38.5 "C). It was concluded that cockroach mesenteric collagen showed the characteristic features of invertebrate mesodermal colla- gens, except that of the thermal stability of the triple-helical structure.

The collagen molecule, which is one of the most fundamental constituents of connective tissue, pos- sesses a characteristic triple-helical structure. Its physical and chemical properties are now well established in Vertebrata (review in [l -4]), but not in Invertebrata [l, 5 - 71. The collagen from some phyla has been studied in detail (Porifera, Coelenterata, Annelida and Nematoda) but for the other groups our knowledge is very incomplete. In the arthropodian phylum, although the collagen of Crustacea was the object of recent works [8-121, that of Chelicerata, Myriapoda and Insecta has not been the subject of complete studies. Studies have been conducted on the cockroach Leucophaea [ 131, and recently the bio- chemistry of the collagen of the locust Locusta has been reported [14].

In this work we report the precise extraction, purification and characterization of an insect collagen. We used the mesenteric connective sheath of the adult cockroach Peripianeta americana; this tissue has the advantage of possessing a very large quantity of well- localized typical collagen fibrils [15].

MATERIALS AND METHODS

Mesenteric sheaths were obtained from adults of the cockroach Periplaneta americana (L.). They were

Enzyme. Pepsin (EC 3.4.23.1)

dissected in physiological medium [16] at 2-5 "C. About 500 mesenteron with the coeca were freed from the peritrophic membrane, the gut content, the adhering trachea and the fat body, and then stored at - 28 "C until used.

BIOCHEMICAL INVESTIGATIONS ON THE WHOLE TISSUE

Hydroxyproline Content

Woessner's [17] method of titration was used after hydrolysis of the tissue in 6 M HC1 at 110 "C for 24 h. These determinations of the hydroxyproline content were made on both the whole intestine and on the extracted residues to determine the quantity of extractable collagen.

EXTRACTION AND PURIFICATION OF COLLAGEN

Solubilizution with Pepsin

All the operations were performed at 2-5 "C except where otherwise stated.

The tissue was first extracted with a 4.0 M guani- dinium chloride solution in 0.05 M sodium acetate, pH 5.8, for 24 h [18], centrifuged (1 h at lOOOOxg), washed in ethanol, then vacuum-dried. This fraction was twice digested with pepsin (twice crystallized, Sigma) at 100 pg/ml in 0.5 M acetic acid to give a collagen/pepsin ratio of 10: 1 (w/w). After constant

390 Cockroach Collagen

stirring for 24 h at 10 "C, the digest was centrifuged (1 h at 1 5 000 x g), and soluble collagen was precipitated by dialysis against 0.02 M Na,HPO,, pH 9.4, for 2 days to inactivate the pepsin. The precipitate was collected by centrifugation (1 h at 20000xg). It was dissolved in 0.5 M acetic acid, centrifuged, reprecipitated by 2.5 M NaCl and recentrifuged. This process of dis- solution and precipitation was repeated twice. The final precipitates were dissolved in 0.1 M acetic acid, centrifuged ( 1 h at 20000 x g) , dialysed against 0.05 M acetic acid aiid freeze-dried.

Salf Fructionation

Pepsin-soluble collagen was fractionated by dif- ferential salt precipitation at neutral pH [19], with a stepwise addition of NaCl up to 4.5 NaC1. The single precipitate obtained was collected by centrifugation, dialysed against 0.05 M acetic acid and freeze-dried. This precipitate represented the purified collagen.

BIOCHEMICAL CHARACTERIZATION OF COLLAGEN

Amino Acid Composition

Collagen samples were hydrolysed for 18 h. with 6 M HCI in sealed tubes at 120 "C, and analysed by using a Jeol JLC 5 AH automatic amino acid analyzer.

Hexoses

Collagen was hydrolysed in 0.5 HCl for 4 h, with Dowex 50 ( 1 3 ' ) 200-400 mesh. The total neutral sugar content was estimated by the phenol sulfuric acid method [20], using glucose as a standard and expressing thc results as glucose equivalents.

Chroniutogruph?

a) CM-cellulose chromatography has been carried out with buffer containing urea [21]. To be desalted the fractions were dialysed against 0.05 M acetic acid and 1 yophilized.

b) Collagen wa\ fractionated by molecular-sieve chromatography on a column of agarose beads [22]. The fractions corresponding to the R , and jj chains were pooled, dialysed against 0.05 M acetic acid and lyophilized.

Electrophorr.\i\

Collagen was wbjected to sodium dodecylsulfate/ polyacrylamide disc gel electrophoresis [23], or the flat-bed technique [24] with or without 2-mercapto- ethanol, and the component composition determined by densitometric tracing of the band patterns at 560 nm using a DCD-16 Gelman densitometer.

Collagen Cleavage with Cyanogen Bromide

A collagen sample (5 mg) was dissolved in 70($ (v/v) formic acid (5 mg/ml), flushed with nitrogen, treated with cyanogen bromide (7.5 mg,ml) for 4 h at 40 "C, tenfold diluted with water and freeze-dried. The electrophoresis of the cyanogen bromide peptides was performed as described above for the subunits of collagen [23,24].

IdentlJication o j the Cross-Links

Mesenteric sheaths (approximately 300-mg sam- ples) were suspended in 0.9 M NaCI, 0.05 M bicar- bonate buffer, pH 7.4, and stirred with tritiated po- tassium borohydride at a collagen: KB3H, ratio of 30 : 1 (w/v) for 1 h. The excess of KBH, was destroyed by addition of acetic acid, The suspension was dialysed against water and, after hydrolysis in 6 HCI for 24 h, the cross-links were analysed by ion-exchange chroma- tography [25]. The radioactive peaks were accurately located by comparison with authentic standards.

PHYSICOCHEMICAL CHARACTERIZATION

Thermal Stability

u ) DIf72wnriuI Thermul Ancily.vi,\.. Samples of colla- gen (2 mg) in 0.1 ml 0.1 M acetic acid solution were studied in sealed cells in a DuPont thermal analyser. We obtained a thermogram with a linear heating rate of 10 "C/mn. The extrapolated onset temperature is the best estimate of transition temperature [26].

h) Mic.ropol~~ri~zc.rrl,. Collagen was dissolvccl at a concentration of 3 mg/ml in 0.1 M acetic acid. The change in optical rotation was measured at 560 nm in a Jouan micropolarimeter with an increase of temper- ature at a rate of 5 "Cih.

Moleculur Length

The molecular length was measured by electrical birefringence variation (Kerr effect) with an instru- ment built in the Biochemical Laboratory, Universitc C1. Bernard, Lyon (J. C. Bernengo and B. Roux). We studied collagen in 0.1 M acetic acid at 20 C. at a concentration of 0.35 mg/ml. From the birefringence decay curve, we obtained the relaxation time T of the collagen molecule. The molecular length is calculatcd from this time by adopting a cylindrical-rod model ~ 7 1 .

Electron Microscope Studies

u) Segment- Long- Spucing Crj's tdlr tes . Segment - long-spacing crystallites were precipitated by dialysis of collagen solution (1 mg/ml) against 0.4':, adenosine triphosphate (ATP, Sigma), and positively stained

J. Francois, I). Herbage, and S. Junqua 391

with 0.25% phosphotungstic acid and 1 % uranyl ace- tate at pH 4.2.

h) Reconstituied Fibrils. After dissolving collagen in 0.1 M acetic acid, the solution was dialysed against water for 4 days or against 0.02 M Na,HPO,, pH 9.4, for 3 days. The fibril? were stained as above. The electron microscopic preparations were examined in a Hitachi HU 11 E electron microscope operating at 100 kV.

RESULTS CHARACTERIZATION OF THE WHOLE TISSUE

The analysis showed that the mesenteric sheath of adult cockroaches contains 2.1 pg/mg hydroxyproline. From this value for the hydroxyproline content, the collagen content was calculated by multiplying it by 7.3. Thus we may assume that collagen represents l.50/, of the dry weight of the whole tissue.

SOLUBILITY OF COLLAGEN

The extraction of collagen with 0.5 M acetic acid did not yield a sufficient quantity of collagen. Ap- proximately 10% was extracted. To overcome this problem we used limited pepsin digestion, which ex- cised terminal non-helical collagen peptides. With this method we extracted 44% of the total collagen. How- ever, the first determinations of the amino acid com- position showed a contamination by non-collagenous components. Therefore, prior to enzymatic digestion, an extraction with 4 M guanidinium chloride was per- formed as described for vertebrate tissues [18,28]. This pretreatment extracted 3% of the total collagen, but increased the quantity of extractable pepsin collagen up to 66% of the total.

BIOCHEMICAL CHARACTERIZATION OF COLLAGEN

Fractional Salt Precipitation

The collagen extracted by pepsin digestion was precipitated by a stepwise increase of NaCl concentra- tion. A single precipitation occurred at 4.010.2 M NaC1. No additional collagen was precipitated when the 4.0 M NaCl supernatant was dialysed against 0.02 M Na,HPO, or when solid (HN,),SO, was added to a 20%) concentration.

Amino Acid Composition

The values obtained are presented in Table 1 and compared with the composition of collagen rat tail (type I) and bovine articular cartilage (type 11) [28]. The analysis exhibits all the characteristic features of a collagenous protein : glycine typically makes up al- most one-third of all the amino acid residues, and the imino acids (proline and hydroxyproline) are in rel- atively high concentration (23 %). The amount of

Table 1. Amino acid composition of cockroach collagen, rat tail collagen (type I ) and bovine articular collagen (type II ) [28]

~ ~~~

Amino acid Cockroach Rat tail Bovine articular cartilage

(type 1) (type 11)

3 HYP 4 HYP

ASP Thr Ser Glu Pro

Ala Val Met Ile Leu TY r Phe HY 1 LYS

Arg Amide N Hexoses

GlY

His

(% dry wt)

Residues/lOOO residues

n.d. 107 50 24 39 91

120 320 78 22

n.d. 16 35

n.d. 13 16 20 4

45

(125) 8.6

1 92 46 20 43 71

121 331 106 22

8 11 24

3 12 6

28 5

50

0.7

97

48 23 28 92

115 330 98 18 9

10 26 1

14 20 18 2

51

4

glycine+imino acids makes up 54% of the total residues. The high hydroxylysine content is note- worthy, as its value is twice as high as that of type I. The average ratio of hydroxyproline to proline was 0.89, and the hydroxylysine/lysine ratio was high at 0.8.

Hexose Content

Large amounts of carbohydrates were found in this invertebrate collagen. The result shows that ap- proximately 8.6% of the total dry weight of collagen was neutral sugar.

Subunit Composition

The denatured collagen was characterized by CM- cellulose chromatography. This method shows (Fig. 1) the presence of one wide peak, which elutes in approxi- mately the same volume as that of b12 and a2 from type I collagen.

Three migration bands were observed on sodium do- decylsulfate/polyacrylamide gel electrophoresis, with the mobility of a 1, p 11 and y type I (Fig. 2A, B). The electrophoretic patterns did not show any significant differences when electrophoresis was performed in the presence or in the absence of reducing agents (Fig. 2B, C).

The presence of a, p and y components was con- firmed by chromatography on an agarose column

392

0.5 A 0.4 1 a1 P11 P12 a 2

Cockroach Collagen

" 0 50 100 150

Elution volume (mi)

Fig. 1. CM-cellulose chromatogram of denatured ( A ) c a y skin ( fype I ) and (BJ cockroach collagens. The column (1.5 x 12 cm) was equilibrated at 40 "C with acetate buffer (0.04 M, pH 4.8) contain- ing 1.0 M urea, and eluted with a linear 0-0.1 M NaCl gradient at a flow rate of 50 ml/h (total volume 200 mi)

0

Ln P

z o P 100 200 300 O u--_- 0.2 i-

I U P a

0 100 200 300 " 100 200 300

Elu t i on volume (ml)

Fig. 3. Molecular sieve chromatography of ( A ) c a y skin collagen (type I ) and ( B ) cockroach collagen. The column ( 1 . 5 ~ 100 cm) of Bio-Gel A15m (200/400 mesh) was equilibrated and eluted with 1.0 M CaC1, in Tris/HCI buffer (0.05 M. pH 7.5). The fractions were collected at a flow rate of 25 mlih

812 alCB8

0.5 1 .0 1.5 a 2.0

Migra t i on (cm)

Fig. 2. Densitomrtric tracing of sodium dodecylsuljate/70/, polyacryl- umide gel electrophoresis of ( A ) calf skin collagen (type I ) , ( B ) cockroach collugen withoul 2-mercaptoethanol; ( C ) Cockroach col- lagen with 2-mercuptoeihanol. The proteins were denatured before the electrophoretic run in 52, sodium dodecylsulfate at 60 "C

(Fig. 3). The molecular weight of the a fraction, as calculated by comparison with the elution of CI, a and 1' components of type 1 collagen, was 94000.

Cyanogen Bromide Pattern

Fig. 4 shows a comparison between the peptide pattern of the well-known type I collagen and that of cockroach collagen. The CNBr cleavage of cockroach collagen (Fig. 4 B) gave a distinct peptide pattern, which was very different from the pattern obtained by the CNBr cleavage of type I collagen (Fig. 4A). The den- sitometric curve (Fig. 4B) shows 12 distinct peaks.

Cross-Link.,

Peaks were identified on the basis of their elution position. The major radioactive peak (Fig. 5, peak 8)

t x P

1 2 3 4 5 6

Fig. 4. Densitometric tracing of sodium dodecyl sulfate/7 polyacryl- amide gels following electrophoresis of' cyunogen bromide peptides of ( A ) type I collagen and (B) cockroach collagen. The well-known cyanogen-bromide-derived peptides of type I are identified in (A)

6 1 a I'

6.- 200 400 600

L e u Phe Tyr Hyi Lys Elution volume (mi)

OO

Fig. 5. Chromatogram showing the radioactiiw components from an acid hydrolysate of' cockroach collagen ajter reduction with tritiated borohydride

J. Francois, D. Herbage, and S. Junqua 393

0.2 1 \, I k Y-.-h+,. , 0

32 34 36 38 40 42 Temperature ("C)

Fig. 6. Denaturation curves (@) calfskin (type I ) and (A) cockroach collagens as measured by optical rotation. t,: denaturation tempera- ture of cockroach collagen; solvent: 0.1 M acetic acid; @=degree of helicity= [(a)t-(a)42]/[(a)32 -(a)42]

is the reduced intermolecular cross-link dihydroxy- lysinonorleucine. The reduced cross-link hydroxy- lysinonorleucine is not present in this collagen. The mesenteric sheath also revealed small amounts of cross- links precursors (peaks 1 and 2), intramolecular aldols (peak 3), hexosyl-lysines and hexosyl-hydroxylysines (peaks 5, 6, 7) and lysinonorleucine (peak 9).

PHY SICOCHEMICAL CHARACTERIZATION

Thermal Stability

The thermal denaturation curve for cockroach col- lagen, determined by the change in optical rotation as a function of increasing temperature, is presented in Fig. 6. The mid-point (t,) of the transition phase from collagen to gelatin was 38.5 "C. The differential ther- mal analysis indicated 38.7 "C.

This t, temperature of an insect collagen is higher than that of calf skin type I collagen (36 "C, Fig. 6).

Molecular Length

The variation in the time over which a collagen solution became birefringent, under the influence of an electrical field, allowed the measurement of its molec- ular length [27]. The plot of the log birefringence against time gives a straight line. From the slope of this line, the calculation gives a relaxation time z= 215 ~ s , and a molecular length of 285 nm.

Electron Microscopic Studies

Segment-Long-Spacing Crystallites. The examina- tion of the segment-long-spacing crystallites revealed

Fig. I . Segment-long-spacing crystallites positively stained from ( A ) calf skin (type I ) collagen, and ( B ) cockroach collagen. Note the NH, (N) and COOH (C) terminals. Numbering of the bands ac- cording to Bruns and Gross's notation [29]. Magnification x 198000

Fig. 8. Reconstitutedfibrils positively stainedfrom ( A ) rat tail colla- gen (type I ) and (B) cockroach collagen. The notation ofthe banding is the one used by Schmitt and Gross [30]. Magnification x I80000

that cockroach collagen molecules exhibit a charge distribution pattern indistinguishable from that of type I collagen, as shown by the similarity in the cross- striation pattern. In Fig. 7 segment-long-spacing crystallites are compared. They show the same num- ber of light and dark bands, and the intensity and disposition of the striations appear to be similar. Using the recently introduced band number designation [29], the cockroach collagen is similar to type I: bands 36,39 - 40 and 46 - 48 appear as a prominent striation. The length between bands 1 - 57 is 290 nm.

Reconstituted Fibrils. Fig. 8 illustrates the banding pattern of reconstituted fibrils with a 63-nm-length periodicity. A comparison of the cross-striation pat- tern using Schmitt and Gross's notation [30] does not indicate any significant differences between type I and cockroach collagens.

394 Cockroach Collagen

DISCUSSION

The cockroach mesenteric collagen, like that of Verte- brata, is characterized by its relative insolubility in acetic acid (lo%)), whereas that of Locusta [14] is very insoluble in this solution. However, pretreatment of the cockroach mesenteric sheath with guanidinium chlo- ride increased its solubility by 2270 and allowed us to avoid using [I-aminoproprionitrile [14]. It is probable, as in the case of Vertebrata, that the guanidinium chloride fraction, which most likely contains proteo- glycans, helps to make Periplaneta collagen insoluble [18,28].

The pepsin-dissolved cockroach collagen is preci- pitable, by differential salt precipitation with 4.0 M NaCI, as type I trimer, type 11 and type V [4, 31, 321. For this feature it is similar to the pepsin-soluble frac- tion of Locusfa collagen [14], except that it was not possible for us to isolate a 2.4 M NaCl fraction with properties similar to type 1V collagen [14].

The principal characteristics of the amino acid composition of a typical collagen are observed: it contains one-third glycyl residues, a high content of pyrrolidine residues (23 2)); hydroxyproline and hy- droxylysine are present, and it has a proline/hydroxy- proline ratio greater than 1.

In comparison with the composition of the well- known vertebrate collagens, the amino acid composi- tion of Per@/aneta collagen more closely resembles the composition of the type TI [l, 4, 281 than that of types I, 111, IV, AB and type I trimer [l, 4, 31,321. The amino acid composition of the collagen of the cock- roach Leucophaea has been studied by Harper et al. [13], who found that the characteristic amino acids were present, but that the amounts of glycine, hydroxy- proline and proline were very low. It is worth noting that in Locustu [I41 the amino acid composition of the 4.0 M NaCl collagen chains is rather similar to that of Periplunetu and type I1 vertebrate collagens. The crustaceans collagens which have been studied [8 - 121 have an amino acid composition similar to that of type IV vertebrate collagen [12].

The insect collagens of Locusta and Periplaneta do, however. have some features in common with those reported for other Invertebrata [ l , 331: they show a similar higher content of hydroxylysine and acidic amino acids. and a lower content of alanine, but the cysteic acid IS absent. Based on the system of classi- fication developed by Matsumura [34] for coflagens, we can put the cockroach and locust collagens in the 'S' range occupied by classic mesodermal col- lagens.

The high carbohydrate content of cockroach colla- gen (8.6'~);)) is similar to that reported for many in- vertebrate collagens [ I , 351.

Chromatography and electrophoresis clearly dem- onstrate that cockroach collagen has three identical

LX chains, and can be represented as having the con- formation of the type ( x ) 3 . This \tructure. which is found in most Vertebrata and in the lower Vertebrata [l, 12, 14, 33, 36-42], is considered to be primitive [ l , 6, 7, 12, 33, 36-39, 421. The vertebrate types 11, I11 and IV with the (x)3 chain composition are con- sidered to retain a 'primitive structure'. In Cephalo- poda and Crustacea x 2 chains are present in the col- lagen [12], whereas Echinodermata show another type of 3 3 as well as type of (ctl)2(m2) [41,43]. These ex- amples suggest that the differentiation of the m 2 chain might have occurred independently in the different phyla during their evolution.

The distribution of cyanogen bromide peptides from the cockroach collagen is different from that of the locust collagen [14] and from the known vertebrate collagens [I, 41. CNBr peptides have been studied in the sea-anemone [40], but the number and molecular weight of the isolated peptides prohibit any useful comparison.

The electrophoretic migration of the chains re- mained unaffected after reduction, which demonstrates that the collagen molecules were not held by disulphide bonds, as in types I11 and IV. Reduction with boro- hydride demonstrated that the dihydroxylysinonor- leucine (syndesine) was the important binding com- ponent of the sample. It was shown that this compo- nent was capable of a spontaneous Amadori rearrange- ment of the aldimine to give more stable hydroxy- lysino-5-ketonorleucine [44]. This may help to explain the relative insolubility of cockroach and locust colla- gens [14]. This type of cross-link was found in Coelen- terata, Annelida and Echinodermata [42, 45, 461, in the bones and the cartilage of Vertebrata [44], whereas Porifera [42, 45, 461 and Locusta [I 41 have cross-links of both hydroxylysinonorleucine and dihydroxylysino- norleucine types. We can conclude that in insect collagens the system of cross-links appears to be based on the same system as that previously described in Invertebrata and Vertebrata [l, 7, 14, 42, 44-461.

We have compared the cross-striation patterns in segment-long-spacing crystallites with those of locust collagen [14] and other invertebrate and vertebrate collagens [36, 37, 471. It seems that in segment-long- spacing crystallites the alternation of clusters of char- ged and non-charged residues is very similar. This similarity between these collagens indicates that the distribution of polar and apolar amino acid side- chains along the polypeptide chains has remained constant in each phylum. Probably little change has taken place in the quaternary structure of the colla- gens during evolution.

A comparison between reconstituted fibrils from Periplaneta, Locusta [14] and type I collagens does not reveal any differences. We can infer that both insect and type I collagen molecules aggregate in a similar fashion during fiber formation. Similar collagen fibrils

J. Francois, D. Herbage, and S. Junqua 395

have been described in various Insecta (review in [48,49]). in Myriapoda [50], Arachnida [51] and Crus- tacea [52], and in various invertebrate phyla [37, 38,

The length of the molecule (285 nm) studied by electrical birefringence and by measurements of the segment-long-spacing crystallites is the same as one found for other vertebrate and invertebrate meso- dermal collagens.

The molecular weight of 94 000, determined by molecular sieve chromatography and by electropho- resis agrees with the molecular weight of the CI chains from mesodermal collagens [l -41.

Cockroach collagen has a high value for the de- naturation temperature (t, = 38.5 "C), compared to that in other Invertebrata (t,=22- 36.5 "C), except in Nematoda (t,=40 "C) (data in [l]). A study of colla- gens has shown a linear relationship [57-581 be- tween the t, values and the total pyrrolidine residue content of the molecules. The point obtained for Periplaneta collagen agrees quite well with the plots of the values presented in literature [l, 37,57,58]. We can conclude that the biophysical characters of cock- roach collagen show the classic pattern.

Finally, in the mesenteric connective sheath of the adult cockroach Periplaneta americana, collagen shows the main characteristics of other invertebrate meso- dermal collagens: hexose content, amino acid com- position, three identical CI chains, but differs from them in having a high denaturation temperature.

53 - 561.

This work was supported by the Centre National de la Recherche Scicntifiquc (ERA no. 231). We thank Mrs Buffevant and Mr Flandin (Lyon) and Mrs Mathelin (Dijon) for their valuable technical as- sistance. We are also indebted to Dr Robert (Creteil) for his interest in this study.

REFERENCES

1.

2.

3.

4. 5.

6. 7. 8.

9.

10.

11. 12.

Mathews, M. B. (1975) Connective Tissue, pp. 1-318, Springer- Verlag, Berlin, Heidelberg, New York.

Piez, K. A. (1976) in Biochemistry of Collagen (Ramachandran, G. N. & Reddi, A. H., eds) pp. 1 -44, Plenum Press, New York.

Ramachandran, G. N. & Ramakrishnan, C. (1976) in Bio- chemistry of Collagen (Ramachandran, G. N. & Reddi, A. H., eds) pp. 45-84, Plenum Press, New York.

Miller, E. J. (1976) Mol. Cell. Biochem. 13, 165-192. Piez, K. A. & Gross, J. (1959) Biochim. Biophys. Acta, 34,

Bairati, A. (1972) Boll, Zool. 39, 205 - 248. Adams, E. (1 978) Sc,iencc ( Wash. D C ) 202, 591 - 598. Thompson, H. C. & Thompson, M. H. (1968) Comp. Biochem.

Thompson, H. C . & Thompson, M. (1970) Comp. Biochem.

Kimura, S., Nagaoka, Y. & Kubota, M. (1969) Bull. Jap. SOC.

Kimura, S. (1972) Bull. Jap. SOC. Sci. Fish. 38, 1153-1161. Kimura, S. & Matsumura, F. (1974) J. Biochem. (Tokyo) 75,

24 - 29.

Physiol. 27, 127- 132.

Physiol. 35,471 -477.

Sci. Fish. 35, 743 - 748.

1231 - 1240.

13. Harper, E., Seifter, S. & Scharrer, B. (1967) J. Cell Biol. 33,

14. Ashhurst, D. E. & Bailey, A. J. (1980) Eur. J . Biochem. 103,

15. Francois, J. (1978) Cell Tissue Res. 189, 91 - 107. 16. Yeager, J. & Hager, A. (1934) Iowa State J. Sci. 8, 391 -395. 17. Woessner Jr, J. F. (1976) in Methodology of Connective Tissue

Research (Hall, D. A., ed.) pp. 227 -233, Joynson-Bruvvers, Oxford.

18. Herbage, D., Lucas, J. M. & Huc, A. (1974) Biochim. Biophys.

19. Chung, E. & Miller, E. J. (1974) Science (Wash. DC) 183,

20. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. &

21. Miller, E. J. (1971) Biochemistry, 10, 1652-1659. 22. Piez, K. A. (1968) Anal. Biochem. 26, 305-312. 23. Clark, C. C. (1976) in Methodology of Connective Tissue Re-

search (Hall, D. A., ed.) pp. 37 - 52, Joynson-Bruvvers, Oxford.

385 - 394.

75-83.

A c ~ u , 336, 108 - 116.

1200-1201.

Smith, F. (1956) Anal. Chem. 28, 350-356.

24. Sykes, B. & Bailey, A. J. (1971) Environ. Res. 4, 340-345. 25. Robins, S. P. (1976) in Methodology of Connective Tissue Re-

search (Hall, D. A,, ed.) pp. 37 - 52, Joynson-Bruvvers, Ox- ford.

26. McClain, P. E. & Wiley, E. R. (1972) J. Biol. Chem. 247,

27. Bernengo, J. C., Roux, B. & Herbage, D. (1976) Ber. Bunsen

28. Herbage, D., Bouillet, J. & Bernengo, J. C. (1977) Biochem. .I.

29. Bruns, R. R. & Gross, J. (1973) Biochemistry, 12, 808-815. 30. Schmitt, F. 0. & Gross, J. (1948) J. Am. Leather Ass. 43,

31. Uitto, J. (1979) Arch. Biochem. Biophys. 192, 371 -379. 32. Burgeson, R. E., El Adli, F. A., Kaitila, I. I. & Hollister, D. W.

(1976) Proc. Natl Acad. Sci. USA, 73, 2578-2583. 33. Kulonen, E. & Pikkarainen, J. (1970) in Chemistry and Molec-

ular Biology of the Intercellular Matrix (Balazs, E. A,, ed.) pp. 81 -97, Academic Press, London and New York.

34. Matsumura, T. (1972) Int. J . Biochem. 3, 265-274. 35. Gross, J. (1963) in Comparative Biochemistry (Florkin, M. &

Mason, H. S., eds) pp. 307 - 346, Academic Press, New York. 36. Pikkarainen, J., Rautanen, J., VastamBki, M., Lampihaho, K.,

Kari, A. & Kulonen, E. (1968) Eur. J. Biochem. 4, 555-560. 37. Nordwig, A. & Hayduk, U. (1969) J . Mol. Biol. 44, 161-

172. 38. Nordwig, A,, Rogall, E. & Hayduk, U. (1970) in Chemistry and

Molecular Biology of the Intercellular Matrix (Balazs, E. A., ed.) pp. 27-41, Academic Press, London and New York.

39. Nordwig, A., Nowak, H. & Hieber-Rogall, E. (1973) J . Mol.

40. Nowak, H. & Nordwig, A. (1974) Eur. J. Biochem. 45,333 - 342. 41. Minafra, S., Pucci-Minafra, I., Casano, C. & Gianguzza, F.

(1975) Boll. Zool. 42, 205, 208. 42. Junqua, S. (1979) Les constituants mole'culaires de la matrice

intercellulaire des Spongiaires, pp. 1 - 129, Thesis, Lille. 43. Pucci-Minafra, I., Galante, R. & Minafra, S. (1978) J. Sub-

microsc. Cytol. 10, 53 -63. 44. Robins, S. P. & Bailey, A. J. (1963) in Frontiers of Matrix Bio-

logy (Robert, L., ed.) vol. 1, pp. 130-156, Karger, Basel. 45. Eyre, D. R. & Glimcher, M. J. (1973) Proc. SOC. Exp. Biol.

Med. 144,400 - 403. 46. Bailey, A. J. (1971) FEBS Lett. 18, 154-158. 47. Stark, M., Miller, E. J. & Kiihn, K. (1972) Eur. J. Biochem. 27,

48. Ashhurst, D. E. (1968) Annu. Rev. Entomol. 13, 45-74. 49. Francois, J. (1976) in Traite' de Zoologie (Grass&, P. P., ed.)

50. Fuller, H. & Ude, J. (1969) 2. Wiss. 2001. 180, 21 -33.

692 - 697.

Ges. Phys. Chem. 80, 246 - 249.

161, 303 - 312.

658-675.

E v o l ~ t . 2, 175-180.

192 - 196.

vol. 8, pp. 491 - 516, Masson, Paris.

396 J. Francois, D. Herbage, and S. Junqua: Cockroach Collagen

51. Sichel, G. & Alicata, P. (1968) Boll. 2001. 35, 143- 146. 52. Bacceti, B. & Bigliardi, E. (1969) Z . Zelljorsch. Mikrosk. Anat.

53. Baccetti. B. (1967) J . Cell Biol. 34, 885-891. 54. Baccetti, B. (1967) Monit. 2001. Ital. I . 201 -206. 55. Hermans, C. 0. (1970) J . Ultrastruct. Res. 30, 255-261.

56. Franc, S., Franc, J. M. & Garrone, R. (1976) in Frontiers of Matrix Biology (Robert, L., ed.) vol. 3, pp. 143 - 156, Kar-

57. Josse, J. & Harrington, W. F. (1964) J. Mol. Biol. 9, 269-287. 58. von Hippel, P. H. & Wong, K. (1963) Biochemistry, 2, 1387-

99,25 - 36. ger, Basel.

1398.

J. Francois, Laboratoire de Zoologie, Faculte des Sciences, Universite de Dijon, 6 Boulevard Gabriel, F-21100 Dijon, France

D. Herbage, Laboratoire de Chimie Macromoleculaire, Universite Claude-Bernard, F-69622 Villeurbanne, France

S. Junqua, Laboratoire de Biochimie du Tissu Conjonctif, Faculte de Medecine, Universite Paris Val de Marne, 6 rue du General Sarrail, F-94000 Creteil, France