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/ . Biochem., 80, 405-407 (1976)

Connectin, an Elastic Protein from Myofibrils1 Koscak MARUYAMA Department of Biophysics, Faculty of Science, University of Kyoto, Sakyo-ku, Kyoto, Kyoto 606 Received for publication, May 26, 1976

The elastic protein isolated from myofibrils of chicken skeletal muscle was compared with extracellular non-collagenous reticulin prepared from chicken liver and skeletal muscle. The amino acid compositions of these proteins were similar except that their contents of Phe, Leu, Cys/2, and Hyp were different. The impregnations of the elastic protein and reticulin with silver were also different. The reticulin was not at all elastic. It also differed from reticulin in solubility and antigenicity. It is proposed to call the intracellular elastic protein connectin.

In a recent work from this laboratory (7), it -was observed that when skinned muscle fibers -were treated with dilute alkali to remove all the soluble structural proteins they still retained elasticity and an elastic protein was isolated from pure myofibrils free of muscle -fiber fragments. This elastic protein appeared to be a chemical entity of the so-called parallel elastic component of muscle (cf. 2). The amino acid composition of the myo-fibrillar elastic protein is entirely different from that of collagen or elastin (7), but it is similar to that of the non-collagenous component of reticulin (3), which is one of the constituents of extracellular basement membranes of the renal cortex and of stromal membranes of the liver (4). The reticulin Jias also been shown to be located in the endomysium, but not in the cytoplasm of muscle fibers, using fluorescent anti-kidney 1

This work was supported by grants from the Muscular Dystrophy Association, Inc., the Ministry of Health and Welfare, and the Ministry of Education, Science and Culture, of Japan. Vol. 80, No. 2, 1976

405

reticulin (4). Kefalides prepared similar proteins from basement membranes which had been treated with collagenase [EC 3.4. 24.3] and called them non-collagenous glycoproteins (5). In the present study, non-collagenous reticulin was isolated from chicken liver and skeletal muscle by the method of Pras and Glynn (3), and compared with the chicken myofibrillar elastic protein prepared as reported before (7). The yield of liver reticulin was approximately 110 mg per 100 g of liver, and the yield is in good agreement with reported values (3). On the other hand, less than 10 mg per 100 g of tissue were obtained from muscle, whereas the yield of elastic protein from muscle was more than 500 mg per 100 g of tissue. The amino acid composition of chicken muscle reticulin was found to be similar to that of liver reticulin and analytical values were in good accord with those reported for reticulins of pig liver (3) and dog kidney (5), except that the kidney protein had a high content of Cys/2 (Table I). However, the elastic protein differed significantly from

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406

TABLE I. Araino acid compositions of connectin and reticulin.

Number of residues per 1,000 residues.



Connectin

Reticulin

Chicken muscle

Rabbit* muscle

Chicken muscle

Chicken liver

Hyp

7

7

0

Asp

93

95

Thr

65

61

Ser

57

Glu

117

Pig liverb

Dog kidney0

0

0

0

96

89

82

91

55

54

59

59

61

55

63

66

73

130

123

102

110

112

Pro

59

63

43

50

58

55

Gly

86

89

72

82

90

93

Ala

80

82

93

88

95

79

Cys/2

2

2

5

6

7

22

Val

72

62

68

71

55

57

Met

24

24

28

24

21

12

He

59

53

57

56

39

48

Leu

70

73

88

100

98

89

Tyr

33

30

25

28

31

25

Phe

29

29

38

47

37

43

Lys

73

66

72

63

70

54

His

20

16

25

22

20

21

Arg

54

57

57

55

62

66

Maruyama et al. ( 7 ) ;

b

Pras & Glynn (3); •= Kefalides (5).

all the reported values for reticulin in its contents of Phe, Leu, Cys/2, and Hyp. Thus the intracellular elastic protein seems to be similar, but not identical to the extracellular reticulin. The elastic protein also differed from reticulin in the following properties. On silver impregnation the elastic protein stained grey, whereas reticulin stained black (Hosoi, Y., Maruyama, K., & Midorikawa, O., unpublished). The total sugar content of reticulin (4%) was more that of the elastic protein (0.5%) (Handa, S. & Maruyama, K., unpublished). The reticulin was freely soluble in dilute alkali and so must be removed when the muscle residue is extracted with 0.1 N NaOH (7). It was also partially soluble in hot phenol and the insoluble material after washing away the phenol was very fragile and not at all elastic. It is to be noted here that anti-reticulin did not react with the

elastic protein and antiserum against elastic protein also did not react with reticulin (Ohashi, K. &.Maruyama, K., unpublished). Reticulin was first denned as an extracellular insoluble protein of the connective tissue by Siegfried in 1892 (6). Histochemically, the reticulin has been identified by the silver impregnation method (cf. 3), and it is thought to be a protein component of connective tissue fibers. However, the basement membrane proteins are synthesized by epithelial cells of ectodermal origin as well as by endothelial cells of mesenchymal origin, so some investigators have called them collagenous or noncollagenous glycoproteins instead of reticulin (5). This work shows that the intracellular elastic protein of muscle is different from extracellular non-collagenous reticulin (glycoprotein). Hence the name connectin seems appropriate for the elastic protein. The / . Biochem.

CONNECTIN, AN ELASTIC PROTEIN FROM MYOFIBRILS relationship between connectin and reticulin is of special interest in relation to tension transmission at the myotendinous junction, where the basement membranes (external lamina) are considered to connect collagen fibers with muscle cells (cf. 7). Connectin has been isolated from skeletal muscles of the rat, rabbit, chicken, and frog, heart muscles of the cow, chicken, and frog, smooth muscles of the chicken, and tail and jaw muscles of the crayfish. Connectin preparations from different types of muscles have very similar physico-chemical properties. I am grateful to Professors T. Yamakawa and S. Ebashi (Univ. Tokyo) and I. Yamashina (Univ. Kyoto) for critical reading of the manuscript. I also thank President R. Natori (Jikei Univ.) and Professors S. Sano and O. Midorikawa (Univ. Kyoto), H. Noda (Univ. Tokyo), and T. Nakao (Akita Univ.) for helpful advice.

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REFERENCES 1. Maruyama, K., Natori, R., & Nonomura, Y. (1976) Nature 262, 58-59 2. Natori, R. (1954) Jikfdkai Med. J. 1, 18-28 3. Pras, M. & Glynn, L.E. (1973) Brit. J. Exp. Path. 54, 449-456 4. Pras, M., Johnson, G.D., Holborow, E.J., & Glynn, L.E. (1974) Immunol. 27, 469-478 5. Kefalides, N.A. (1973) in Internal. Rev. Connective Tissue Research (Hall, D.A. & Jackson, D.S., eds.) pp. 63-104, Academic Press, New York 6. Siegfried, M. (1892) Vber die chemischen Eigenschaften des reticulirten Gewebes Habilitationschrift, Leibzig, cited from Puchtler, H. (1964) / . Histochem. Cytochem. 12, 552 7. Nakao, T. (1976) Cell Tiss. Res. 1G6, 241-254

Connectin, an elastic protein from myofibrils.

The elastic protein isolated from myofibrils of chicken skeletal muscle was compared with extracellular non-collagenous reticulin prepared from chicke...
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