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The composition of tunic from four species of ascidians
Comp. Biochem. Physiol., 1971, Vol. 40B,pp. 615 to 622. Pergamon Press. Printed in Great Britain
THE COMPOSITION OF TUNIC FROM FOUR SPECIES OF ASCIDIANS* M I C H A E L J. S M I T H 1 and PAUL A. D E H N E L z XDepartment of Zoology, University of Nebraska, Lincoln, Nebraska 68508; and 2Department of Zoology, University of British Columbia, Vancouver 8, Canada (Received 30 April 1971)
Abstract--1. Dry tunic from four species of ascidians contains between 3767% protein and 33-63% carbohydrate. 2. Tunicin from Corella willmeriana and Ascidia paratropa yields approximately 100 per cent of its dry weight as cellulose equivalents when determined by the phenol-sulfuric acid method, but Pyura haustor and Cnemidocarpa firrmarkiensis tunicin yields only about 50 per cent of its dry weight as cellulose equivalents. 3. Amino acid analyses of tunic from the four species reveal a striking similarity in relative amino acid composition with all four species displaying a high acidic amino acid tunic content. 4. Tunic amino acid analyses demonstrate the presence of hexosamine in the tunics of all four species. 5. No hydroxyproline is recovered in the amino acid analyses of tunic from these species. INTRODUCTION THE TUNIC of ascidians is an extracellular product, composed of protein and polysaccharide, which contains amoeboid cells that have migrated from the blood spaces. The carbohydrate portion of tunic is reportedly secreted by the epidermis (Deck et al., 1966; Smith, 1970a, b; Wardrop, 1970), although there is some disagreement with this (Endean, 1955, 1961). There is little evidence for the source of tunic protein. It is not unusual to find a protein-polysaccharide complex in supportive structures, e.g. collagen and glycosaminoglycans, chitin and associated protein in arthropod exoskeleton. The tunic of ascidians presents an unique system in that the carbohydrate portion is a cellulose-like polymer (tunicin) (reviewed by Hunt, 1970a) which may be linked to protein through serine (Smith & Dehnel, 1970). There is some disagreement concerning the protein fraction of tunic: Hall & Saxl (1961) report the presence of collagen, elastin and pseudoelastin in the tunic of Ascidiella aspersa, a member of the Order Phlebobranchia, * This study was supported by a University of Nebraska Research Council Junior Faculty Summer Research FeUowship to M. J. S. and by National Research Council of Canada Grant No. T-88 to P. A. D. This is publication No. 430 from the Department of Zoology, University of Nebraska. 615
616
MICHAEL J. SMITH AND PAUL A. DEHNEL
while Smith & Dehnel (1970) could find no hydroxyproline in hydrolyzates of tunic from Halocynthia aurantium, a m e m b e r of the Order Stolidobranchia. Ascidian tunic can range in consistency from gelatinous to a leathery fibrous system. Little is known of the relative protein and carbohydrate content as a function of tunic consistency. An attempt to understand the evolution and function of supportive systems has resulted in a series of comparative studies of extracellular structural proteins (Seifter & Gallop, 1966; Bailey, 1968; Hunt, 1970b; Meenakshi & Scheer, 1970; T h o m p s o n & T h o m p s o n , 1970) but information concerning ascidian tunic is limited (reviewed by H u n t , 1970a; Smith & Dehnel, 1970). T h i s study investigated the composition of ascidian tunic, and was designed to determine several basic facts: (1) the presence or absence of collagen as evidenced by hydroxyproline, (2) composition of tunic as a function of tunic consistency and (3) comparison of tunic composition with other extracellular structural products. Four species of ascidians were used, two from the Order Phlebobranchia, Corella willmeriana and Ascidia paratropa, which have gelatinous tunics and two from the Order Stolidobranchia, Pyura haustor and Cnemidocarpa finmarkiensis, which have leathery fibrous tunics. MATERIALS AND M E T H O D S The ascidians were collected by S.C.U.B.A. diving at depths between 10 and 20 m in Howe Sound, off Whytecliff Park in British Columbia. Ascidians were identified by M. J. S. based on the work of Van Name (1945). Tunics were removed from the ascidians, inner and outer layers of tunic were dissected away and discarded. The middle portion of the tunic was blotted dry and weighed to + 0-1 mg (wet wt.). Wet weighed tunic was washed six times with distilled water, ethanol, acetone and ether, placed over phosphorous pentoxide and sodium hydroxide in vacuo for 48 hr and then weighed to _+0-1 mg (dry wt.). This drying procedure was followed after all treatments of tunic to obtain pre- and post-treatment dry weights. Tunicin, the carbohydrate portion of tunic, was prepared by a method which parallels the method for the preparation of chitin (Hackman, 1954). Pronase digestion of tunic, acid hydrolyses of tunic and amino acid analyses of tunic hydrolyzates with a Spinco Model 120 C amino acid analyzer were done by methods described in a previous paper (Smith & Dehnel, 1970) except that pronase digestion was extended to 72 hr. Carbohydrate content of tunicin was estimated by the phenol-H,SO4 method (Dubois et al., 1956). The phenolH2SO 4 method depends upon the formation of a chromogen from phenol and the acid hydrolysis products of sugar. The strong acid conditions of this type of reaction make it suitable for determinations of monosaccharide content of polysaecharides, but polysaccharide hydrolysis products, in terms of chromogen formed, may yield different values than equivalent amounts of monosaccharides (Davidson, 1967). It is advisable, therefore, to determine standard curves for monosaccharides, in this study glucose, and polymeric forms which resemble closely the carbohydrate under investigation, plant cellulose, in the form of Whatman No. 1 filter paper. Under the reaction conditions, cellulose yields about one-half the amount of chromogen as an equivalent amount of glucose. Amino acid content is expressed as moles per 1000 moles of amino acid residues recovered. No corrections were made for the hydrolytic degradation of glucosamine, serine, threonine and tyrosine; consequently, reported values are minimal. Reagent grade chemicals were used throughout the study. Pronase is Streptomyces griseus protease from Sigma Biochemicals.
TUNIC COMPOSITION
617
RESULTS AND DISCUSSION As would be expected, the dry weight-wet weight ratios (expressed as a percentage) of the gelatinous tunics from Corella and Ascidia are much less (0.6 and 0.8 per cent, respectively) than the ratios determined for the leathery tunics of Pyura and Cnemidocarpa (12.8 and 12.4 per cent, respectively). At the microscopic level, there is a greater condensation of fibrous material in leathery tunic than in gelatinous (Smith, unpublished observations). The range of relative amounts of protein and polysaccharide in tunic can be estimated by measures of weight loss due to tunicin preparative procedures and pronase digestion. Corella shows the least weight loss due to pronase treatment and the greatest weight loss due to tunicin preparative procedures (Table 1). Ascidia, on the other hand, has the greatest susceptibility to pronase digestion and the least to tunicin preparative TABLE 1 - - W E I G H T LOSS (PER CENT) IN TUNIC DUE TO TUNICIN PREPARATIVE PROCEDURES AND PRONASE DIGESTION
Ascidia (%) Mean S.E. n
49"0 1"1 3
Mean S.E. n
50.0 3"7 5
Corella Cnemidocarpa Pyura (%) (%) (%) Tunicin preparative procedure 67"1 59"5 61"4 1"3 0"8 1"6 3 3 3 Pronase digestion 37.2 45"0 1.1 2.8 5 5
40.9 2'8 5
procedures. Pyura and Cnemidocarpa are intermediate in weight loss due to these procedures (Table 1). Since both Corella and Ascidia have gelatinous tunics, this evidence does not indicate that tunic consistency is a function simply of carbohydrate-protein ratio. It is more likely that tunic consistency is a function of the structure or composition of either the protein fraction, the carbohydrate fraction or both. Carbohydrate analyses of tunicin by the phenol-sulfuric acid method indicate a difference between the tunicins from gelatinous and leathery tunics (Table 2). Corella and Ascidia tunicins yield approximately 100 per cent of their weight as cellulose-equivalents suggesting a resemblance between these tunicins and plant cellulose. The leathery tunics of Pyura and Cnemidocarpa have tunicins which yield only about half of their weight as cellulose-equivalents. The leathery tunics have a higher degree of fibrosity, which may reflect a difference in the chemical structure or the cohesive physical interactions of tunicin molecules accounting for the decreased yield in terms of cellulose equivalents. Recently, ultrastructural studies of Pyura stolonifera tunic indicate that tunicin microfibriUar structure is
~VlICHAELJ.
618
SMITH AND PAUL A . DEHNEL
'I'ABLE 2--DETERMINATIONS OF GLUCOSE AND CELLULOSE EQUIVALENTS IN TUNICIN
Tunicin weight as glucose equivalents (%)*
Tunicin weight as cellulose equivalents (%)t
59-3 53' 1 23"9 27.0
106.8 94"9 42"7 49.2
Ascidia CoreUa Cnemidocarpa Pyura
* Each per cent value is the mean of the three replicates. t Cellulose values are from a standard curve for Whatman No. 1 filter paper. significantly different than such structures in plant cellulose (Wardrop, 1970). Although there has been general agreement that tunicin is comparable to plant cellulose, there is some evidence for sugars other than glucose, and physical measurements reveal differences between animal and plant cellulose (reviewed by Hunt, 1970a). Amino acid composition of tunic from the four species is remarkably similar (Table 3) and comparable to the composition of tunic of H. aurantium (Smith & Dehnel, 1970). I n all four species investigated the acidic amino acids make up more than 25 per cent of the amino acids recovered. T h e absence of hydroxyproline in the hydrolyzates negates the possibility that collagen forms a major TABLE 3--THE AMINOACID COMPOSITIONOF ASCIDIANTUNIC*
Ascidia Aspartic Threonine Serine Proline Glutamic Glycine Alanine Valine Cystine (half) Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Hexosamine
139 58 65 53 104 94 75 71 40 16 42 59 24 36 39 17 39 30
Corella 158 71 65 68 108 96 59 73 41 9 42 67 16 43 15 8 44 16
Cnemidocarpa 142 52 62 59 104 111 64 68 44 17 36 45 28 32 53 16 59 4
Pyura 138 65 54 69 105 84 50 72 51 15 42 46 34 34 60 17 42 20
* Values reported are moles of amino acids per 1000 moles recovered. Reported values are the means of three replicates.
TUNIC COMPOSITION
619
portion of tunic. It has been suggested that both collagen and elastin are present in tunic (Hall & Saxl, 1960, 1961), but the low glycine and imino acid content in the four species studied would make that proposition doubtful (Table 4). Tunic displays a high amount of hydroxylated amino acids; approximately 12 per cent of the total residues recovered. Both serine and threonine have been repeatedly implicated as the linkage between proteins and polysaccharides (reviewed by Neuberger et al., 1966; Wardi et al., 1969) and it has been suggested that serine may be involved in protein-polysaccharide linkages of H. aurantium tunic (Smith & Dehnel, 1970). Glycoproteins (Deck et al., 1966), Elson-Morgan positive material (Endean, 1955) and acid mucopolysaccharides (Endean, 1955; Bierbauer & Vagas, 1962; Godeaux, 1963) have been reported in ascidian tunic. Both glucosamine and galactosamine were found in the tunic of H. aurantium (Smith & Dehnel, 1970). In the four species investigated hexosamine is present in each tunic (Table 3). The method of analysis used in this study does not differentiate between glucosamine and galactosamine. The lowest level of hexosamine recovered was from Cnemidocarpa, but even this represents a minimal value. When tunic amino acid composition is compared with other invertebrate and vertebrate structural systems, it is convenient to group similar amino acids (Table 4). Tunic contains about one-third the amount of glycine as do resilin and elastin; and does not resemble these proteins in either small or non-polar amino acid content. There is some resemblance between tunic amino acid composition and the structural protein of the internal shell of Ariolimax (Meenakshi & Scheer, 1970), but the most striking similarity in grouped amino acid composition is between tunic and the acidic structural protein of rabbit or rat skin (Timpl et al., 1969). However, until more precise information concerning the components of tunic are available it would be premature to indicate relationships between the ascidians and higher groups by reference to structural systems. There is a great deal more information available concerning the physical and chemical nature of tunicin than the nitrogenous substances in tunic. There are apparently major differences in the quaternary structure of tunicin between species (Mishra & Colvin, 1969, 1970; reviewed by Hunt, 1970a; Wardrop, 1970). Electron microscopic studies of tunic suggest that the tunicin component resembles plant cellulose microfibrils (Mishra & Colvin, 1969, 1970) although there is evidence that this is not true for P. stolonifera (Wardrop, 1970). It would be of interest to know what, if any, relationship exists between tunicin quaternary structure and the nitrogenous components of tunic. This study indicates that the composition of the nitrogenous components is strikingly similar between gelatinous and leathery tunics. The differences in cellulose-equivalent yields of tunicin from gelatinous versus leathery tunics may indicate a difference in quaternary tunicin structure, but does not directly indicate that the physical structure of tunicin is responsible for consistency. The question of degree and type of molecular interactions between tunic protein and carbohydrate is still open. The answer to this question may resolve the problem of tunic consistency and morphology.
63 102
249 123 26
Imino Basic
Acidic Hydroxy Tyrosine
172 121 57
17 105
546 299 547
Buccinum ~
238 57 43
152 16 115
262 54 78
234 196 33
334 54 80 45 155
140 30
154 22
660 310 567
16 116 229
459 164 345
Prekeratin I1
Resilin * *
23 8
550 120 9 17
950 360
Elastin**
OF VARIOUS STRUCTURAL PROTEINS WITH THE
OF ASCIDIAN TUNIC*
Protocollagen¶
452 78
Ariolimax §
AMINO ACID COMPOSITION
OF GROUPED AMINO ACID COMPOSITION
112 34
213 40 98 253
453 68
A.S.P. ++++
* The non-polar group consists of proline, glycine, alanine, valine, leucine, isoleucine and phenylalanine. Small amino acids are glycine, alanine and serine; imino are proline and hydroxyproline, basic are arginine, lysine and histidine; acidic are glutamic and aspartic; and hydroxy are serine and threonine. Values are number of residues per thousand residues. Average of four species. ++Operculum protein (Hunt, 1970). § Internal shell protein (Meenakshi & Scheer, 1970)• ¶ Calculated for shrimp protocollagen (Thompson & Thompson, 1970). I1Rudall (1968). ** Seifter & Gallop (1966). ++Acidic structural protein of rabbit and rat skin, average of alkali soluble protein (Timpl et al., 1969).
425 96 220
Non-polar Glycine Small
Tunic t
TABLE 4---COMPARISON
>
~q
t~ to
TUNIC COMPOSITION
621
REFERENCES BAILEY A. J. (1968) The nature of collagen. In Comprehensive Biochemistry (Edited by FLORIN M. & STOTZ E.), Vol. 26B, pp. 297--424. Elsevier, New York. BIRaBAtma J. & VAGAS E. (1962) Histological and histochemical examination of tunic formation on the Ciona intestinalis. Acta Biol..4cad. Sci. Hungary 12 (Suppl. 4), 29. DAVIDSON E. A. (1967) Carbohydrate Chemistry. Holt, Rinehart and Winston, New York. DECK J. D., HAY E. D. & RBWL J. (1966) Fine structure and origin of the tunic of Perophora viridis, ft. Morph. 120, 267-280. DUBOIS M. D., GILLES K. A., HAMILTONJ. K., REBEaS P. A. & SMITH F. (1956) Colorimetric method for determination of sugars and related substances..4nalyt. Chem. 28, 350-356. ENDEAN R. (1955) Studies of the blood and tests of some Australian ascidians--II. The test of Pyura stolonifera. Aust. ft. Mar. Freshw. Res. 6, 139-156. END~-AN R. (1961) The test of the ascidian, Phallusia mammillata. Q. ffl microsc. Sd. 102, 107-117.
GOD~LrX J. E. A. (1963) Integument of tunicata. Proc. X V I Intern. Congr. Zool. 1, 16. HACKMAN R. H. (1954) Studies on chitin---I. Enzymic degradation of chitin and chitin esters. Aust..~. biol. Sci. 7, 168-178. HALL D. A. ~ SAXL H. (1960) Human and other animal cellulose. Nature, Lond. 187, 547-550. HALL O. A. ~/; SAXL H. (1961) Studies of human and tunicate cellulose and their relation to reticulin. Proc. R. Soc. B 155, 202-217. HUNT S. A. (1970a) Polysaccharide--protein Complexes in Invertebrates. Academic Press, New York. HUNT S. A. (1970b) Invertebrate structure proteins: characterization of the operculum of the gastropod mollusc Buccinum undatum. Biochim. biophys..4cta 207, 347-360. MI~a~rAKSHIV. R. & SCrI~R B. T. (1970) Chemical studies of the internal shell of the slug, ,4riolirnax columbianus (Gould) with special reference to the organic matrix. Corap. Biochem. Physiol. 34, 953-957. MISH~ A. K. & COLVINJ. R. (1969) The microscopic and submicroscopic structure of the tunic of two ascidians. Can. ft. Zool. 47, 659-663. MISHRA A. K. & COLVIN J. R. (1970) Scanning electron microscopy of the spines of the tunic of the ascidian Boltenia ovifera. Can..~. Zool. 48, 475-477. NEUBERGERA., GOTTSCHALKA. & MARSHALL R. D. (1966) Carbohydrate-peptide linkages in glycoproteins and methods for their elucidation. In Glycoproteins, their Composition, Structure and Function (Edited by GOTTSCHALKA.), pp. 273-296. Elsevier, New York. RUDALL K. M. (1968) Intracellular fibrous proteins and the keratins. In Comprehensive Biochemistry (Edited by FLORKIN M. & STOTZ E.), Vol. 26B, pp. 559-591. Elsevier, New York. SEIFTER S. & GALLOP P. M. (1966) The structure proteins. In The Proteins: Composition, Structure, and Function (Edited by NEtraATH H.), Vol. 4, pp. 153-458. Academic Press, New York. SMITH M. J. (1970a) The blood cells and tunic of the ascidian Halocynthia aurantium (Pallas)--I. Hematology, tunic morphology, and partition of cells between blood and tunic. Biol. Bull. 138, 354-378. SMITH M. J. (1970b) The blood cells and tunic of the ascidian Halocynthia aurantium (Pallas)--II. Histochemistry of the blood cells and tunic. Biol. Bull. 138, 379-388. SMITH M. J. & DEHNEL P. A. (1970) The chemical and enzymatic analyses of the tunic of the ascidian Halocynthia aurantium (Pallas). Camp. Biochem. Physiol. 35, 17-30. THOMPSON H. C. & THOMPSON M. H. (1970) Isolation and amino acid composition of the collagen of white shrimp (Penaeus setiferus)--II. Comp. Biochem. Physiol. 35, 471-477.
622
MICHAEL J. SMITH AND PAUL A. DEHNEL
TIMPL R., WOLFF I. & WEISER HI. (1969) Acidic structural proteins of connective tissue--I. Solubilization and preliminary chemical characterization. Biochim. biophys. Acta 194, 112-120. VAN NAME W. G. (1945) The North and South American Ascidians. Bull. Amer. Mus. Nat. Hist. 84, 1-476. WARDI A. H., ALLEN W. S., TURNER D. D. & STARYZ. (1969) Hyaluronate-peptide linkage group. Biochim. biophys. Acta 192, 151-154. WARDROP A. B. (1970) The structure and formation of the test of Pyura stolonifera (Tunicata). Protoplasma 70, 73-86.
Key Word Index--Ascidians; tunic; tunic composition; amino acids in tunic; tunicin.
THE COMPOSITION OF TUNIC FROM FOUR SPECIES OF ASCIDIANS* M I C H A E L J. S M I T H 1 and PAUL A. D E H N E L z XDepartment of Zoology, University of Nebraska, Lincoln, Nebraska 68508; and 2Department of Zoology, University of British Columbia, Vancouver 8, Canada (Received 30 April 1971)
Abstract--1. Dry tunic from four species of ascidians contains between 3767% protein and 33-63% carbohydrate. 2. Tunicin from Corella willmeriana and Ascidia paratropa yields approximately 100 per cent of its dry weight as cellulose equivalents when determined by the phenol-sulfuric acid method, but Pyura haustor and Cnemidocarpa firrmarkiensis tunicin yields only about 50 per cent of its dry weight as cellulose equivalents. 3. Amino acid analyses of tunic from the four species reveal a striking similarity in relative amino acid composition with all four species displaying a high acidic amino acid tunic content. 4. Tunic amino acid analyses demonstrate the presence of hexosamine in the tunics of all four species. 5. No hydroxyproline is recovered in the amino acid analyses of tunic from these species. INTRODUCTION THE TUNIC of ascidians is an extracellular product, composed of protein and polysaccharide, which contains amoeboid cells that have migrated from the blood spaces. The carbohydrate portion of tunic is reportedly secreted by the epidermis (Deck et al., 1966; Smith, 1970a, b; Wardrop, 1970), although there is some disagreement with this (Endean, 1955, 1961). There is little evidence for the source of tunic protein. It is not unusual to find a protein-polysaccharide complex in supportive structures, e.g. collagen and glycosaminoglycans, chitin and associated protein in arthropod exoskeleton. The tunic of ascidians presents an unique system in that the carbohydrate portion is a cellulose-like polymer (tunicin) (reviewed by Hunt, 1970a) which may be linked to protein through serine (Smith & Dehnel, 1970). There is some disagreement concerning the protein fraction of tunic: Hall & Saxl (1961) report the presence of collagen, elastin and pseudoelastin in the tunic of Ascidiella aspersa, a member of the Order Phlebobranchia, * This study was supported by a University of Nebraska Research Council Junior Faculty Summer Research FeUowship to M. J. S. and by National Research Council of Canada Grant No. T-88 to P. A. D. This is publication No. 430 from the Department of Zoology, University of Nebraska. 615
616
MICHAEL J. SMITH AND PAUL A. DEHNEL
while Smith & Dehnel (1970) could find no hydroxyproline in hydrolyzates of tunic from Halocynthia aurantium, a m e m b e r of the Order Stolidobranchia. Ascidian tunic can range in consistency from gelatinous to a leathery fibrous system. Little is known of the relative protein and carbohydrate content as a function of tunic consistency. An attempt to understand the evolution and function of supportive systems has resulted in a series of comparative studies of extracellular structural proteins (Seifter & Gallop, 1966; Bailey, 1968; Hunt, 1970b; Meenakshi & Scheer, 1970; T h o m p s o n & T h o m p s o n , 1970) but information concerning ascidian tunic is limited (reviewed by H u n t , 1970a; Smith & Dehnel, 1970). T h i s study investigated the composition of ascidian tunic, and was designed to determine several basic facts: (1) the presence or absence of collagen as evidenced by hydroxyproline, (2) composition of tunic as a function of tunic consistency and (3) comparison of tunic composition with other extracellular structural products. Four species of ascidians were used, two from the Order Phlebobranchia, Corella willmeriana and Ascidia paratropa, which have gelatinous tunics and two from the Order Stolidobranchia, Pyura haustor and Cnemidocarpa finmarkiensis, which have leathery fibrous tunics. MATERIALS AND M E T H O D S The ascidians were collected by S.C.U.B.A. diving at depths between 10 and 20 m in Howe Sound, off Whytecliff Park in British Columbia. Ascidians were identified by M. J. S. based on the work of Van Name (1945). Tunics were removed from the ascidians, inner and outer layers of tunic were dissected away and discarded. The middle portion of the tunic was blotted dry and weighed to + 0-1 mg (wet wt.). Wet weighed tunic was washed six times with distilled water, ethanol, acetone and ether, placed over phosphorous pentoxide and sodium hydroxide in vacuo for 48 hr and then weighed to _+0-1 mg (dry wt.). This drying procedure was followed after all treatments of tunic to obtain pre- and post-treatment dry weights. Tunicin, the carbohydrate portion of tunic, was prepared by a method which parallels the method for the preparation of chitin (Hackman, 1954). Pronase digestion of tunic, acid hydrolyses of tunic and amino acid analyses of tunic hydrolyzates with a Spinco Model 120 C amino acid analyzer were done by methods described in a previous paper (Smith & Dehnel, 1970) except that pronase digestion was extended to 72 hr. Carbohydrate content of tunicin was estimated by the phenol-H,SO4 method (Dubois et al., 1956). The phenolH2SO 4 method depends upon the formation of a chromogen from phenol and the acid hydrolysis products of sugar. The strong acid conditions of this type of reaction make it suitable for determinations of monosaccharide content of polysaecharides, but polysaccharide hydrolysis products, in terms of chromogen formed, may yield different values than equivalent amounts of monosaccharides (Davidson, 1967). It is advisable, therefore, to determine standard curves for monosaccharides, in this study glucose, and polymeric forms which resemble closely the carbohydrate under investigation, plant cellulose, in the form of Whatman No. 1 filter paper. Under the reaction conditions, cellulose yields about one-half the amount of chromogen as an equivalent amount of glucose. Amino acid content is expressed as moles per 1000 moles of amino acid residues recovered. No corrections were made for the hydrolytic degradation of glucosamine, serine, threonine and tyrosine; consequently, reported values are minimal. Reagent grade chemicals were used throughout the study. Pronase is Streptomyces griseus protease from Sigma Biochemicals.
TUNIC COMPOSITION
617
RESULTS AND DISCUSSION As would be expected, the dry weight-wet weight ratios (expressed as a percentage) of the gelatinous tunics from Corella and Ascidia are much less (0.6 and 0.8 per cent, respectively) than the ratios determined for the leathery tunics of Pyura and Cnemidocarpa (12.8 and 12.4 per cent, respectively). At the microscopic level, there is a greater condensation of fibrous material in leathery tunic than in gelatinous (Smith, unpublished observations). The range of relative amounts of protein and polysaccharide in tunic can be estimated by measures of weight loss due to tunicin preparative procedures and pronase digestion. Corella shows the least weight loss due to pronase treatment and the greatest weight loss due to tunicin preparative procedures (Table 1). Ascidia, on the other hand, has the greatest susceptibility to pronase digestion and the least to tunicin preparative TABLE 1 - - W E I G H T LOSS (PER CENT) IN TUNIC DUE TO TUNICIN PREPARATIVE PROCEDURES AND PRONASE DIGESTION
Ascidia (%) Mean S.E. n
49"0 1"1 3
Mean S.E. n
50.0 3"7 5
Corella Cnemidocarpa Pyura (%) (%) (%) Tunicin preparative procedure 67"1 59"5 61"4 1"3 0"8 1"6 3 3 3 Pronase digestion 37.2 45"0 1.1 2.8 5 5
40.9 2'8 5
procedures. Pyura and Cnemidocarpa are intermediate in weight loss due to these procedures (Table 1). Since both Corella and Ascidia have gelatinous tunics, this evidence does not indicate that tunic consistency is a function simply of carbohydrate-protein ratio. It is more likely that tunic consistency is a function of the structure or composition of either the protein fraction, the carbohydrate fraction or both. Carbohydrate analyses of tunicin by the phenol-sulfuric acid method indicate a difference between the tunicins from gelatinous and leathery tunics (Table 2). Corella and Ascidia tunicins yield approximately 100 per cent of their weight as cellulose-equivalents suggesting a resemblance between these tunicins and plant cellulose. The leathery tunics of Pyura and Cnemidocarpa have tunicins which yield only about half of their weight as cellulose-equivalents. The leathery tunics have a higher degree of fibrosity, which may reflect a difference in the chemical structure or the cohesive physical interactions of tunicin molecules accounting for the decreased yield in terms of cellulose equivalents. Recently, ultrastructural studies of Pyura stolonifera tunic indicate that tunicin microfibriUar structure is
~VlICHAELJ.
618
SMITH AND PAUL A . DEHNEL
'I'ABLE 2--DETERMINATIONS OF GLUCOSE AND CELLULOSE EQUIVALENTS IN TUNICIN
Tunicin weight as glucose equivalents (%)*
Tunicin weight as cellulose equivalents (%)t
59-3 53' 1 23"9 27.0
106.8 94"9 42"7 49.2
Ascidia CoreUa Cnemidocarpa Pyura
* Each per cent value is the mean of the three replicates. t Cellulose values are from a standard curve for Whatman No. 1 filter paper. significantly different than such structures in plant cellulose (Wardrop, 1970). Although there has been general agreement that tunicin is comparable to plant cellulose, there is some evidence for sugars other than glucose, and physical measurements reveal differences between animal and plant cellulose (reviewed by Hunt, 1970a). Amino acid composition of tunic from the four species is remarkably similar (Table 3) and comparable to the composition of tunic of H. aurantium (Smith & Dehnel, 1970). I n all four species investigated the acidic amino acids make up more than 25 per cent of the amino acids recovered. T h e absence of hydroxyproline in the hydrolyzates negates the possibility that collagen forms a major TABLE 3--THE AMINOACID COMPOSITIONOF ASCIDIANTUNIC*
Ascidia Aspartic Threonine Serine Proline Glutamic Glycine Alanine Valine Cystine (half) Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Hexosamine
139 58 65 53 104 94 75 71 40 16 42 59 24 36 39 17 39 30
Corella 158 71 65 68 108 96 59 73 41 9 42 67 16 43 15 8 44 16
Cnemidocarpa 142 52 62 59 104 111 64 68 44 17 36 45 28 32 53 16 59 4
Pyura 138 65 54 69 105 84 50 72 51 15 42 46 34 34 60 17 42 20
* Values reported are moles of amino acids per 1000 moles recovered. Reported values are the means of three replicates.
TUNIC COMPOSITION
619
portion of tunic. It has been suggested that both collagen and elastin are present in tunic (Hall & Saxl, 1960, 1961), but the low glycine and imino acid content in the four species studied would make that proposition doubtful (Table 4). Tunic displays a high amount of hydroxylated amino acids; approximately 12 per cent of the total residues recovered. Both serine and threonine have been repeatedly implicated as the linkage between proteins and polysaccharides (reviewed by Neuberger et al., 1966; Wardi et al., 1969) and it has been suggested that serine may be involved in protein-polysaccharide linkages of H. aurantium tunic (Smith & Dehnel, 1970). Glycoproteins (Deck et al., 1966), Elson-Morgan positive material (Endean, 1955) and acid mucopolysaccharides (Endean, 1955; Bierbauer & Vagas, 1962; Godeaux, 1963) have been reported in ascidian tunic. Both glucosamine and galactosamine were found in the tunic of H. aurantium (Smith & Dehnel, 1970). In the four species investigated hexosamine is present in each tunic (Table 3). The method of analysis used in this study does not differentiate between glucosamine and galactosamine. The lowest level of hexosamine recovered was from Cnemidocarpa, but even this represents a minimal value. When tunic amino acid composition is compared with other invertebrate and vertebrate structural systems, it is convenient to group similar amino acids (Table 4). Tunic contains about one-third the amount of glycine as do resilin and elastin; and does not resemble these proteins in either small or non-polar amino acid content. There is some resemblance between tunic amino acid composition and the structural protein of the internal shell of Ariolimax (Meenakshi & Scheer, 1970), but the most striking similarity in grouped amino acid composition is between tunic and the acidic structural protein of rabbit or rat skin (Timpl et al., 1969). However, until more precise information concerning the components of tunic are available it would be premature to indicate relationships between the ascidians and higher groups by reference to structural systems. There is a great deal more information available concerning the physical and chemical nature of tunicin than the nitrogenous substances in tunic. There are apparently major differences in the quaternary structure of tunicin between species (Mishra & Colvin, 1969, 1970; reviewed by Hunt, 1970a; Wardrop, 1970). Electron microscopic studies of tunic suggest that the tunicin component resembles plant cellulose microfibrils (Mishra & Colvin, 1969, 1970) although there is evidence that this is not true for P. stolonifera (Wardrop, 1970). It would be of interest to know what, if any, relationship exists between tunicin quaternary structure and the nitrogenous components of tunic. This study indicates that the composition of the nitrogenous components is strikingly similar between gelatinous and leathery tunics. The differences in cellulose-equivalent yields of tunicin from gelatinous versus leathery tunics may indicate a difference in quaternary tunicin structure, but does not directly indicate that the physical structure of tunicin is responsible for consistency. The question of degree and type of molecular interactions between tunic protein and carbohydrate is still open. The answer to this question may resolve the problem of tunic consistency and morphology.
63 102
249 123 26
Imino Basic
Acidic Hydroxy Tyrosine
172 121 57
17 105
546 299 547
Buccinum ~
238 57 43
152 16 115
262 54 78
234 196 33
334 54 80 45 155
140 30
154 22
660 310 567
16 116 229
459 164 345
Prekeratin I1
Resilin * *
23 8
550 120 9 17
950 360
Elastin**
OF VARIOUS STRUCTURAL PROTEINS WITH THE
OF ASCIDIAN TUNIC*
Protocollagen¶
452 78
Ariolimax §
AMINO ACID COMPOSITION
OF GROUPED AMINO ACID COMPOSITION
112 34
213 40 98 253
453 68
A.S.P. ++++
* The non-polar group consists of proline, glycine, alanine, valine, leucine, isoleucine and phenylalanine. Small amino acids are glycine, alanine and serine; imino are proline and hydroxyproline, basic are arginine, lysine and histidine; acidic are glutamic and aspartic; and hydroxy are serine and threonine. Values are number of residues per thousand residues. Average of four species. ++Operculum protein (Hunt, 1970). § Internal shell protein (Meenakshi & Scheer, 1970)• ¶ Calculated for shrimp protocollagen (Thompson & Thompson, 1970). I1Rudall (1968). ** Seifter & Gallop (1966). ++Acidic structural protein of rabbit and rat skin, average of alkali soluble protein (Timpl et al., 1969).
425 96 220
Non-polar Glycine Small
Tunic t
TABLE 4---COMPARISON
>
~q
t~ to
TUNIC COMPOSITION
621
REFERENCES BAILEY A. J. (1968) The nature of collagen. In Comprehensive Biochemistry (Edited by FLORIN M. & STOTZ E.), Vol. 26B, pp. 297--424. Elsevier, New York. BIRaBAtma J. & VAGAS E. (1962) Histological and histochemical examination of tunic formation on the Ciona intestinalis. Acta Biol..4cad. Sci. Hungary 12 (Suppl. 4), 29. DAVIDSON E. A. (1967) Carbohydrate Chemistry. Holt, Rinehart and Winston, New York. DECK J. D., HAY E. D. & RBWL J. (1966) Fine structure and origin of the tunic of Perophora viridis, ft. Morph. 120, 267-280. DUBOIS M. D., GILLES K. A., HAMILTONJ. K., REBEaS P. A. & SMITH F. (1956) Colorimetric method for determination of sugars and related substances..4nalyt. Chem. 28, 350-356. ENDEAN R. (1955) Studies of the blood and tests of some Australian ascidians--II. The test of Pyura stolonifera. Aust. ft. Mar. Freshw. Res. 6, 139-156. END~-AN R. (1961) The test of the ascidian, Phallusia mammillata. Q. ffl microsc. Sd. 102, 107-117.
GOD~LrX J. E. A. (1963) Integument of tunicata. Proc. X V I Intern. Congr. Zool. 1, 16. HACKMAN R. H. (1954) Studies on chitin---I. Enzymic degradation of chitin and chitin esters. Aust..~. biol. Sci. 7, 168-178. HALL D. A. ~ SAXL H. (1960) Human and other animal cellulose. Nature, Lond. 187, 547-550. HALL O. A. ~/; SAXL H. (1961) Studies of human and tunicate cellulose and their relation to reticulin. Proc. R. Soc. B 155, 202-217. HUNT S. A. (1970a) Polysaccharide--protein Complexes in Invertebrates. Academic Press, New York. HUNT S. A. (1970b) Invertebrate structure proteins: characterization of the operculum of the gastropod mollusc Buccinum undatum. Biochim. biophys..4cta 207, 347-360. MI~a~rAKSHIV. R. & SCrI~R B. T. (1970) Chemical studies of the internal shell of the slug, ,4riolirnax columbianus (Gould) with special reference to the organic matrix. Corap. Biochem. Physiol. 34, 953-957. MISH~ A. K. & COLVINJ. R. (1969) The microscopic and submicroscopic structure of the tunic of two ascidians. Can. ft. Zool. 47, 659-663. MISHRA A. K. & COLVIN J. R. (1970) Scanning electron microscopy of the spines of the tunic of the ascidian Boltenia ovifera. Can..~. Zool. 48, 475-477. NEUBERGERA., GOTTSCHALKA. & MARSHALL R. D. (1966) Carbohydrate-peptide linkages in glycoproteins and methods for their elucidation. In Glycoproteins, their Composition, Structure and Function (Edited by GOTTSCHALKA.), pp. 273-296. Elsevier, New York. RUDALL K. M. (1968) Intracellular fibrous proteins and the keratins. In Comprehensive Biochemistry (Edited by FLORKIN M. & STOTZ E.), Vol. 26B, pp. 559-591. Elsevier, New York. SEIFTER S. & GALLOP P. M. (1966) The structure proteins. In The Proteins: Composition, Structure, and Function (Edited by NEtraATH H.), Vol. 4, pp. 153-458. Academic Press, New York. SMITH M. J. (1970a) The blood cells and tunic of the ascidian Halocynthia aurantium (Pallas)--I. Hematology, tunic morphology, and partition of cells between blood and tunic. Biol. Bull. 138, 354-378. SMITH M. J. (1970b) The blood cells and tunic of the ascidian Halocynthia aurantium (Pallas)--II. Histochemistry of the blood cells and tunic. Biol. Bull. 138, 379-388. SMITH M. J. & DEHNEL P. A. (1970) The chemical and enzymatic analyses of the tunic of the ascidian Halocynthia aurantium (Pallas). Camp. Biochem. Physiol. 35, 17-30. THOMPSON H. C. & THOMPSON M. H. (1970) Isolation and amino acid composition of the collagen of white shrimp (Penaeus setiferus)--II. Comp. Biochem. Physiol. 35, 471-477.
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MICHAEL J. SMITH AND PAUL A. DEHNEL
TIMPL R., WOLFF I. & WEISER HI. (1969) Acidic structural proteins of connective tissue--I. Solubilization and preliminary chemical characterization. Biochim. biophys. Acta 194, 112-120. VAN NAME W. G. (1945) The North and South American Ascidians. Bull. Amer. Mus. Nat. Hist. 84, 1-476. WARDI A. H., ALLEN W. S., TURNER D. D. & STARYZ. (1969) Hyaluronate-peptide linkage group. Biochim. biophys. Acta 192, 151-154. WARDROP A. B. (1970) The structure and formation of the test of Pyura stolonifera (Tunicata). Protoplasma 70, 73-86.
Key Word Index--Ascidians; tunic; tunic composition; amino acids in tunic; tunicin.