Characterization of two genetically distinct type I-like collagens from hagfish (Eptatretus burgeri)

Comp. Biochem. Physiol. Vol. 95B, No. 1, pp. 137-143, 1990 Printed in Great Britain

0305-049t/90 $3.00 + 0.00 Pergamon Press plc

C H A R A C T E R I Z A T I O N OF TWO GENETICALLY DISTINCT TYPE I-LIKE COLLAGENS FROM H A G F I S H

(EPTA TRETUS BURGERI) SHIGERU KIMURA a n d RISAKO MATSUI Laboratory of Food Biochemistry, Tokyo University of Fisheries, Konan-4, Minato, Tokyo 108, Japan

(Received 30 May 1989) Abstract--1. Soluble Type I-like collagens were isolated from the dermis and peritoneum of hagfish, a member of the cyclostomes. These tissue collagens had the typical heterotrimeric nature of (~ 1)2ct2, but were genetically distinct from each other. 2. Characterization of soluble and pepsin-solubilized Type l-like collagens from various tissues of hagfish revealed the tissue-specific existence of two different molecular species; one was designated skin collagen which existed in dermis and esophagus and the other was designated body collagen which was distributed in peritoneum, muscle and intestine. 3. The skin collagen had a strong resemblance to shark skin Type I collagen, while the body collagen was rather similar to invertebrate Type I-like collagens.

INTRODUCTION Type I collagen is the m a j o r fiber-forming collagen in skin, b o n e a n d m a n y o t h e r organs (Bornstein a n d Sage, 1980). A l t h o u g h Type I collagen was originally defined as the most a b u n d a n t a n d ubiquitous collagen o f higher vertebrates (Miller a n d M a t u k a s , 1969), recent advances in structural studies of lower vertebrate collagens have f o u n d proteins h o m o l o g o u s to Type I collagen. The molecular form of Type I collagen from cartilage fish, birds and m a m m a l s is a n [~ l (I)]2ct 2(I) heterotrimer c o m p o s e d of two identical I(I) chains a n d one ct2(I) chain. O n the other hand, most o f teleosts representative of lower vertebrates are unique in having Type I collagen which exists as a n ~ 1(I)~2(I)~3(I) heterotrimer (Piez, 1965; K i m u r a et al., 1987, 1988; K i m u r a a n d O h n o , 1987). As part of a c o n t i n u i n g p r o g r a m to o b t a i n phylogenetic d a t a on Type I collagen, soluble a n d pepsin-solubilized collagens from several tissues of hagfish (Eptatretus burgeri) were studied with respect to their molecular properties. Hagfish, as well as lamprey, is a m e m b e r of the cyclostomes which belong to the lowest class A g n a t h a o f Vertebrata. Thus its Type I collagen m a y provide a clue to the phylogeny of Type I collagen. However, little definitive i n f o r m a t i o n is available concerning hagfish collagen (Pikkarainen, 1968). In this paper, we report the tissue-specific existence of two genetically distinct Type l-like collagens in hagfish.

muscle and intestine of hagfish were dissected free of adherent tissues, washed extensively with distilled water and diced on ice. If necessary, tissue samples were defatted by extraction with a 1:1 mixture of ether and acetone at 4°C for 24 hr.

Isolation of soluble collagens and their ~ chains Soluble collagens were extracted with 0.5 M acetic acid for 24 hr from the dermal and peritoneal tissues of hagfish and purified by selective salt precipitation with NaC1; the majority of the soluble collagens were obtained in the Type I collagen fraction (Bornstein and Sage, 1980). The dermis collagen precipitated at 0.5 M NaC1 in 0.5 M acetic acid (pH 2.6) and at 2.6 M NaC1 in 0.05 M Tris-HCl (pH 7.5). Similarly, the major peritoneum collagen precipitated at 0.7 M NaCI (pH 2.6) and at 2.6 M NaC1 (pH 7.5). The collagen precipitates were dissolved in 0.5 M acetic acid, dialyzed against 0.1 M acetic acid and lyophilized. Soluble collagen was also isolated from the esophagus in the same manner as the dermis collagen. Soluble collagen ~ chains were isolated by chromatography of the denatured collagens on CM-cellulose (Whatman CM52), followed by gel filtration on Sepharose CL-4B (Pharmacia). Isolation of pepsin-solubilized collagens Insoluble collagenous tissues from the peritoneum, muscle and intestine of hagfish were rendered soluble by limited digestion with pepsin (Sigma, 2 x ) in 0.5 M acetic acid at 4°C for 24 hr at a collagen: pepsin weight ratio 100: 1. Pepsin-solubilized collagens thus obtained were precipitated by dialyzing against 0.02 M Na 2HPO 4 and then dissolved in 0.5 M acetic acid. The major collagen of each tissue was isolated by selective salt precipitation with 0.7 M NaC1 at acidic pH, followed by precipitation with 2.6 M NaCI at neutral pH.

MATERIALS AND METHODS Unless stated otherwise, details of the methods were the same as described previously (Kimura et al., 1981a,b).

Preparation of collagenous tissues Mature hagfish were caught at Misaki Bay in Kanagawa Prefecture, Japan, and immediately frozen at -20°C and then transported to our laboratory in Tokyo. Several collagenous tissues from the dermis, esophagus, peritoneum, ¢sP~B)

95it--J

V8 protease digestion and peptide mapping Collagens and their ~ chains, each 200/~ g, were dissolved in 100/d of 0.1 M phosphate buffer (pH7.2) containing 0.5% SDS. After the addition of 10 #1 of the same buffer containing 5 # g of Staphylococcus aureus V8 protease (Miles laboratories) to each of the protein solutions, the reaction mixtures were incubated at 37°C for 25 rain, boiled for 137

138

SH1GERU KIMURA a n d RISAKO MATSUI

3 min and resolved directly by SDS-polyacrylamide gel electrophoresis on 7% gels.

V8 protease digests

Collagen

Other analytical methods Analyses of amino acids and hydroxylysine glycosides, glucosylgalactosythydroxylysine and galactosylhydroxylysine, and SDS gel electrophoresis were performed as previously described.

8~

t

-"

D

Q

il

RESULTS

al~

Isolation o f two distinct soluble collagens

When extracted with 0.5 M acetic acid at 4°C, all the collagen fibers of dermis were virtually solubilized to give a viscous solution, while only 2% of those of peritoneum was obtained as soluble collagen. Both tissue collagens precipitated with NaCI, as expected for Type I collagen, at 0.5~).7 M NaC1 (pH 2.6) and at 2.6 M NaC1 (pH 7.5). These two collagens, however, are distinct from each other in their electrophoretic patterns as shown in Fig. 1, although both of them are composed of two different 7 chains, ~ 1 and :~2, together with their cross-linked components. The dermis collagen showed large differences in the relative mobilities of 71 and ~2 chains when compared with the peritoneum collagen. Figure 1 also suggests that peptide mapping of these two collagens using V8 protease reveals significant differences in their primary structure. Amino acid composition of the collagens is listed in Table l, indicating that the peritoneum collagen has higher contents of glutamic acid, leucine, hydroxylysine and histidine, and lower contents of alanine and valine than the dermis protein. Furthermore, carbohydrate-linked hydroxylysine was relatively rich in the peritoneum collagen, 1.8 residues/1000, while it was extremely low in the dermis collagen, 0.1 residues/1000.

i

:!~I¸

2

i

soluble collagens and their V8 protease digests. Soluble collagens and their V8 digests were resolved on 3.5 and 7% gels, respectively, in 0.1 M sodium phosphate (pH 7.2) containing 0.1% SDS and 3.5M urea at 8mA/tube for 4-5 hr and visualized with Coomassie Brilliant Blue. 1, Dermis collagen; 2, peritoneum collagen. conditions, it eluted as four peaks (Fig. 2). Several fractions as indicated by the numbers were analyzed by SDS-gel electrophoresis; two distinct c¢chains, ~ 1 and ~2, were evident in the first and fourth peaks, respectively. /3 chains seen in the second and third peaks were estimated to b e / 3 . and/312, respectively, on the basis of their elution positions (Piez et al., 1963). The relative abundance of ~1 and 312 was characteristic of this collagen. This chromatographic pattern had a strong resemblance to those of various Type I collagens previously reported (Piez et al., 1963; Kimura et al., 1987, 1988). The amounts o f ~ l

Characterization of soluble dermis collagen

When the soluble dermis collagen was subjected to CM-cellulose chromatography under denaturing

Table 1. Amino acid and carbohydrate composition of Type I-like collagens from hagfish Pepsin-solubilized collagen

Soluble collagen Dermis Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxylysine Lysine Histidine (NH3) Arginine Totals Glucosylgalactosylhydroxylysine Galactosylhydroxylysine

2

Fig. 1. S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f h a g f i s h

59 46 31 67 58 98 367 108 23 16 13 16 4 10 5 19 8 (48) 52 1000 0.1 0

Peritoneum Muscle (residues/1000 residues) 70 45 27 55 91 95 325 81 17 14 20 33 5 15 12 22 20 (77) 53 1000

73 42 25 54 92 95 337 82 15 14 19 31 3 14 11 21 19 (59) 53 1000

1.3

1.4

0.5

0.5

Hagfish collagens and a 2 were calculated by measuring the areas under each peak; the stoichiometry of ~ 1 and ~ 2 was found to be about 2:1, suggesting the molecular form of (~ 1)20~2. Isolation of the dermis collagen ~ chains was successfully achieved by Sepharose CL-4B gel filtration of proteins recovered from the respective peaks of CM-cellulose chromatography (data not shown). The mol. wts of ~ 1 and a2 were comparable to those of calf skin Type I collagen ~ chains on the basis of their elution positions from the calibrated Sepharose column. The amino acid composition of the isolated chains is given in Table 2; as a whole, the hagfish dermis collagen has a close similarity to shark skin Type I collagen (Piez et al., 1963; Lewis and Piez, 1964; Kimura et al., 1981a). Particularly, the ~2 chain was characterized by its lower contents of imino acids, hydroxyproline and proline, than the ~ 1 chain. Table 2 also shows that a 2:1 mixture of ~ 1 and u2 has an amino acid composition almost equivalent to

~

139

the original dermis collagen (Table 1). The structural differences of ~1 and ~2 were further elucidated by comparing their V8 protease digests; as depicted in Fig. 3, the non-identity of dermis ~1 and ~2 chains is apparent. From these combined results, the dermis collagen was suggested to exist as (al)2~2 heterotrimers.

Characterization of soluble peritoneum collagen A chromatographic pattern on CM-cellulose of the soluble peritoneum collagen is shown in Fig. 4. It should be noted that the peritoneum collagen required a linear gradient of 0-0.2 M NaCI to elute its subunit chains from the CM-cellulose column, contrasted with 0-0.1 M NaCI for the dermis collagen described above. The higher NaC1 concentration required tbr peritoneum collagen was ascribed to its high content of histidine (Table 1) (Piez et al., 1963). Electrophoretic analyses indicated that the first and third peaks contained mostly ~ chains, ~ 1 and ~2,

N

1

2

3

4

5

6

I

7

8

9

l

i0

ii

l

7

I

EcC~ t,th 0.6-

I_ld

23

0 oO

6

4

)

8

5 42

0 I

0

50

I

1 O0

I 150

ELUTION VOLUME (ml) Fig. 2. CM-cellulose chromatography of denatured hagflsh soluble dermis collagen. Soluble dermis collagen, 25 mg, was dissolved in 5 ml of 0.06 M sodium acetate (pH 4.8), denatured at 40°C for 10 min and applied to a column (0.9 × 10 cm) of CM-cellulose. Elution was achieved at 37°C with a linear gradient of 0-0.I M NaCI over a total volume of 200 ml at a flow rate of 40 ml/hr. Fractions indicated by the numbers were examined by SDS-gel electrophoresis.

140

SHIGERU KIMURA a n d RISAKO MATSUI

respectively. The amount of cross-linked fl was very small; the trailing edge of the first peak contained ft. and the second one fin. The weight ratio of :~1 and ~2 appeared to be about 2:1. Each ~ chain of the major peak was isolated by gel filtration (data not show) and analyzed for its chemical composition. As listed in Table 2, a 2:1 mixture of ~1 and ~2 is found to be consistent with the original peritoneum collagen (Table 1) in its composition. Table 2 also demonstrates that the relation between the ~ 1 and :~2 chains of peritoneum collagen follows a pattern similar to that observed for the dermis collagen; both ~2 chains are characterized by increased contents of serine, histidine and hydrophobic amino acids such as valine, isoleucine and leucine, and decreased contents of hydroxyproline and proline. Peptide mapping using V8 protease obviously indicated a difference in primary structure between the :t I and ~ 2 chains of peritoneum collagen. Figure 3 compares the peptide maps from the :~1 and ~2 chains of peritoneum and dermis collagens. The non-identity of all ~ chains was evident, suggesting that these ~ chains were the products of different genes. From these results, the peritoneum collagen is thought to have the molecular form of (~ 1)2~2, but is genetically distinct from the dermis collagen; we shall refer to both tissue collagens as Type I-like collagens in this paper. Tissue distribution Ofltwo distinct Type l-like collagens Several collagenous tissues were prepared from the esophagus, peritoneum, muscle and intestine of hagfish and subjected to acid extraction and subsequently pepsin digestion at 4 C . By extraction with 0.5 M acetic acid, more than 60% of the total esophagus collagen was rendered soluble. Other tissue collagens remained virtually intact at acidic pH, and only 2% of the total peritoneum collagen was obtained in its soluble collagen form as men-

(P)

(D)

,,.

a Q D

~i

~2

~i

~2

Fig. 3. Peptide mapping of V8 protease digests of ~ chains from hagfish soluble collagens. (D), Soluble dermis collagen; (P), soluble peritoneum collagen. tioned above. These acid insoluble collagens were partly solubilized by limited digestion with pepsin; the yield of pepsin-solubilized collagen to acid insoluble collagen was 7% for muscle, 20% for intestine and 60% for peritoneum. The major components of soluble and pepsin-solubilized collagens were obtained by selective salt precipitation with NaC1 in the Type I collagen fraction. These tissue collagens were then subjected to SDS gel electrophoresis after V8 protease digestion and compared with the soluble dermis and peritoneum collagens. As shown in Fig. 5, electrophoretic analyses revealed that they are classified into two groups. One was the collagen of dermis and esophagus which was designated skin collagen because of its abundance in the dermis. The other was the collagen of

Table 2. Amino acid and carbohydrate composition of :~ chains from soluble Type l-like collagens of hagfish Dermis

Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxylysine Lysine Histidine (NH3) Arginine Totals

Peritoneum molecule* ~1 (residues/1000 residues)

~1

:~2

69 49 28 59 59 108 367 107 18 14 10 15 3 11 4 20 7 (47) 52

41 38 38 81 52 8I 367 112 34 20 18 19 7 6 7 16 10 (58) 53

60 45 3I 66 57 99 367 109 23 16 13 16 4 I0 5 19 8 (51) 52

1000

1000

1000

tr. 0

tr. 0

tr. 0

Glucosylgalactosylhydroxylysine Galactosylhydroxylysine *Calculated as the molecular form of (e 1)2e2.

,:~2

molecule*

86 37 24 49 95 113 338 83 10 13 11 25 2 15 13 22 16 (73) 48

51 48 28 68 79 66 347 82 23 15 31 36 4 11 11 15 28 (76) 57

74 41 25 55 90 97 341 83 14 14 18 29 3 13 12 20 20 (74) 51

1000

1000

1000

1.1 0.3

1.6 0.3

1.3 0.3

141

Hagfish collagens peritoneum, muscle and intestine which was designated body collagen because of its wide distribution in the inner body. For comparison, chemical composition of the pepsin-solubilized muscle collagen is given in Table 1, indicating the strong resemblance to that of the soluble peritoneum collagen.

DISCUSSION The present experiments provide evidence for the tissue-specific existence of two genetically distinct Type I-like collagens in the hagfish; one is skin collagen and the other is body collagen. Both collagens are composed of the typical heterotrimer molecules of (~ 1)2~2, but skin ~ 1 and ~2 chains are different in their primary structure from body ~ 1 and ~2 chains, respectively. According to the current classification of vertebrate collagen types (Bornstein and Sage, 1980), the skin and body collagens of

0~N

hagfish should be assigned to different types, because collagen types are, by definition, products of different genetic loci. However, the precipitation properties with NaC1, subunit composition and tissue distribution of both hagfish collagens are compatible with those required for Type I collagen. This finding is at first surprising, because the existence in a given animal of two genetically distinct soluble collagens similar to Type I has not been reported so far. In fact, our recent study of Type I collagens from bonyfish (Kimura et al., 1988) have indicated that body collagens from their muscular tissues are genetically identical to skin collagens of the respective fish species. The hagfish skin collagen closely resembles shark skin Type I collagens (Piez et al,, 1963; Lewis and Piez, 1964; Kimura et al., 1981a) in more respects that any other Type I collagens. One of the compositional features common to hagfish and shark skin collagens is a low level of imino acids in their ~2 chains; the percent of ~2 to ~ 1 imino acid contents in skin

I

e

2

3

4

5

I

6

7

8

!

E coN'l ¢xl v0.3 1,1

D

3

7

Z evCD CO

0

0

I

I

I

25

50

75

,

ELUTION VOLUME (m!) Fig. 4. CM-cellulose chromatography of denatured hagfish soluble peritoneum collagen. Soluble peritoneum collagen, 5 mg, was dissolved in ! ml of 0.06 M sodium acetate (pH 4.8), denatured at 40°C for 10 min and applied to a column (0.9 × 5 cm) of CM-cellulose. Elution was achieved at 37°C with a linear gradient of Oq).2 M NaCI over a total volume of 100 ml at a flow rate of 20 ml/hr. Fractions indicated by the numbers were examined by SDS-gel electrophoresis.

142

SHIGERUKIMURAand RISAKOMATSUI

collagens is 69% for hagfish, 76-77% for sharks and 89% or more for carp (Kimura, 1983), chick (Kang et al., 1969) and human (Burgeson et al., 1976). Also, the extremely low content of carbohydrate-linked hydroxylysine, 0.1 residues/1000, in hagfish skin collagen is comparable to 0.3 residues/1000 in shark skin collagen (Kimura et al., 1981a), and is different from about 1.0 residue/1000 in bonyfish skin collagens (Kimura, 1972). Comparison of the hagfish skin collagen with its body collagen shows the latter to be unusual, because of the extremely low solubility of native fibers at acidic pH, the extremely low ratio of ~2 to ~ 1 in the imino acid content (58%) and the relatively high contents of hydroxylysine and its glycoside, 12 and 1.8 residues/1000, respectively. As for cyclostome collagen, the occurrence in lamprey of Type I-like collagens has already been reported. We isolated soluble Type I-like collagen from the skin (dermis) of a lamprey Entosphenus j a p o n i e u s (Kimura et al., 1981a). This skin collagen has the molecular form of (~ 1)2~2; the ~ 1 chains are entirely present a s / ~ dimers. Its ~ 1 and ~2 chains are genetically distinct, as judged by their amino acid composition and cyanogen bromide-peptide maps (Kimura et al., 1981a; Kimura, 1983), but are closely related to each other when compared to those of typical Type I collagens. Further, the close similarity of lamprey skin ~ 1 (Kimura et al., 1981a) and hagfish skin ~1 (Table 2) in their composition implies homology of both cyclostome skin ~1 chains. Most probably, the molecular properties of lamprey skin collagen reflect its primordial nature as a Type I collagen (Kimura, 1985). On the other hand, Kelly et al. (1988) have isolated soluble dermis collagen, from a different lamprey species, which has only ~ l(I)-like chains. In addition, they have obtained another Type I-like collagen, called major body wall collagen, by limited pepsin digestion of the subcutaneous layer of lamprey skin. This body wall collagen contains three different ~ chains, ~1, ~1' and ~2, but its molecular form is not yet established. More recently, Sato et al. (1989) have reported that the major muscle collagen of lamprey, which was solubilized by pepsin, is found by SDS gel electrophoresis to comprise two distinct ~ chains, ~1 and ~2, and has an amino acid composition different from that of its skin collagen. It is uncertain why the hagfish, a contemporary representative of cyclostomes, possesses two distinct Type I-like collagens; this animal species is quite interesting from the phylogenetic aspect of Type I collagen. From our present study, it can be postulated that the skin collagen of hagfish is a typical Type I collagen resembling that of shark. On the other hand, the body collagen of hagfish is assumed to be rather similar to invertebrate Type I-like collagens isolated from octopus (Kimura et al., 1981b), squid (Kimura and Karasawa, 1985) and echiuroid (Kimura et al., 1983). This assumption is mainly based on the findings that in contrast to fish Type I collagen, the hagfish body collagen as well as the invertebrate Type I-like collagens is characterized by the high insolubility of its native fibers in dilute acid, the relatively high contents of hydroxylysine glycoside and glutamic acid and the low content of alanine.

(S)

1

2

(B)

3

4

5

6

Fig. 5. Peptide mapping of V8 protease digests from several tissue collagens of hagfish. (S), Skin collagen; (B), body collagen; 1, soluble dermis collagen; 2, soluble esophagus collagen; 3, soluble peritoneum collagen; 4, pepsinsolubilized peritoneum collagen; 5, pepsin-solubilized muscle collagen; 6, pepsin-solubilized intestine collagen. REFERENCES

Bornstein P. and Sage H. (1980) Structurally distinct collagen types. A. Rev. Biochem. 49, 957 1003. Burgeson R. E., E1 Adli F. A., Kaitila I. I. and Hollister D. W. (1976) Fetal membrane collagens: identification of two new collagen alpha chains. Proc. natn. Acad. Sci. USA 73, 2579 2583. Kang A. H., Piez K. A. and Gross J. (1969) Characterization of the ~ chains of chick skin collagen and the nature of the NH2-terminal cross-link region. Biochemistry 8, 3648-3655. Kelly J., Tanaka S., Hardt T., Eikenberry E. F. and Brodsky B. (1988) Fibril-forming collagens in lamprey. J. biol. Chem. 263, 980-987. Kimura S. (1972) Determinationof the glycosylatedhydroxylysines in several invertebrate collagens. J. Biochem. 71, 367--370. Kimura S. (1983) Vertebrate skin Type I collagen: comparison of bony fishes with lamprey and calf. Comp. Biochem. Physiol 74B, 525 528. Kimura S. (1985) The interstitial collagens of fish. In Biology o f Invertebrate and Lower Vertebrate Collagens

(Edited by Bairati A. and Garrone R.), pp. 397~408. Plenum Press, New York. Kimura S., Kamimura T., Takema Y. and Kubota M. (1981a) Lower vertebrate collagen: evidence for Type I-like collagen in the skin of lamprey and shark. Biochim. biophys. Acta 669, 251 257. Kimura S. and Karasawa K. (1985) Squid cartilage collagen: isolation of Type I collagen rich in carbohydrate. Comp. Biochem. Physiol. 81B, 361-365. Kimura S. and Ohno Y. (1987) Fish Type I collagen: tissue-specifc existence of two molecular forms, (~ 1)2~2 and ~ 1~2~3, in Alaska pollack. Comp. Biochem. Physiol. 88B, 409413. Kimura S., Ohno Y., Miyauchi Y. and Uchida N. (1987) Fish skin Type I collagen: wide distribution of an ~3 subunit in teleosts. Comp. Biochem. Physiol. 88B, 27-34. Kimura S., Takema Y. and Kubota M. (1981b) Octopus skin collagen: isolation and characterization of collagen comprising two distinct ~ chains. J. biol. Chem. 256, 13,230-13,234.

Hagfish collagens Kimura S., Tanaka H. and Park Y.-H. (1983) Annelid skin collagen: occurrence of collagen with structure of (ct 1)2a 2 in Urechis unicinctus. Comp. Biochem. Physiol. 75B, 681 4584. Kimura S., Zhu X.-P. Matsui R., Shijoh M. and Takamizawa S. (1988) Characterization of fish muscle Type I collagen. J. Food Sci. 53, 1315-1318. Lewis M. S. and Piez K. A. (1964) The characterization of collagen from the skin of the dogfish shark, Squalus acanthias. J. biol. Chem. 239, 3336-3340. Miller E. J. and Matukas V. J. (1969) Chick cartilage collagen: a new type of ct I chain not present in bone or skin of the species. Proc. natn. Acad. Sci. USA 64, 1264-1268.

143

Piez K. A. (1965) Characterization of a collagen from codfish skin containing three chromatographically different ~ chains. Biochemistry 4, 2590-2596. Piez K. A., Eigner E. A. and Lewis M. S. (1963) The chromatographic separation and amino acid composition of the subunits of several collagens. Biochemistry 2, 58456. Pikkarainen J. (1968) The molecular structures of vertebrate skin collagens: a comparative study. Acta physiol, scand. Suppl. 309. Sato K., Yoshinaka R., Itoh Y. and Sato M. (1989) Molecular species of collagen in the intramuscular connective tissue of fish. Comp. Biochem. Physiol. 92B, 87-9 I.