A sulfated polysaccharide from the sarcoplasmic reticulum of sea cucumber smooth muscle is an endogenous inhibitor of the Ca2+-ATPase

Ana M. Landeira-Fernandez2, Karin R. M. Aiello2,3, Rafael S. Aquino2, Luiz-Claudio F. Silva2,3, Leopoldo de Meis2 and Paulo A.S. Mourão1,2,3

2Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas and 3Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941–590, Brazil

Received on November 13, 1999; revised on February 18, 2000; accepted on February 28, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Vesicles derived from the endoplasmic reticulum of sea cucumber smooth muscle retain a membrane bound Ca2+-ATPase that is able to transport Ca2+ into the vesicles at the expense of ATP hydrolysis. In contrast with vesicles obtained from rabbit muscles, the activity of the Ca2+-dependent ATPase from sea cucumber is dependent on monovalent cations (K+>Na+>Li+). With the addition of highly sulfated polysaccharide to vesicle preparations from rabbit muscle, Ca2+ uptake decreases sharply and becomes highly sensitive to monovalent cations, as observed with vesicles from sea cucumber muscle. These results led us to investigate the possible occurrence of a highly sulfated polysaccharide on vesicles from the endoplasmic reticulum of sea cucumber smooth muscle, acting as an "endogenous" Ca2+-ATPase inhibitor. In fact, vesicles derived from the invertebrate, but not from rabbit muscle, contain a highly sulfated polysaccharide. This compound inhibits Ca2+ uptake in vesicles obtained from rabbit muscle and the inhibition is antagonized by monovalent cation. In addition, sea cucumber muscles contain high concentrations of another polysaccharide, which surrounds the muscle fibers, and was characterized as a fucosylated chondroitin sulfate. Possibly the occurrence of sulfated polysaccharides in the sea cucumber muscles is related with unique properties of the invertebrate body wall, which can rapidly and reversibly alter its mechanical properties, with change in length by more than 200%.

Key words: sulfated polysaccharides/heparin/smooth muscle/sarcoplasmic reticulum/Ca2+-ATPase/sea cucumber


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
In muscle cells the contraction–relaxation cycle is regulated by cytoplasmatic Ca2+ ions that activate the contractile proteins of the myofibrils. The Ca2+-ATPase from sarcoplasmic reticulum is responsible for the maintenance of low cytoplasmatic Ca2+ concentration (0.1 µM), thereby controlling muscle contraction. During its activity the enzyme pumps Ca2+ into the sarcoplasmic reticulum at the expense of ATP hydrolysis. The Ca2+-ATPase from sarcoplasmic reticulum belongs to the P-type family that includes the SERCA (sarco/endoplasmic reticulum Ca2+-ATPase) isoforms and the plasma membrane H+-ATPase found in plants and yeast (Pederson and Carafoli, 1987a,b; Serrano, 1989Go; Engelender and de Meis, 1996Go).

The sea cucumber (Echinodermata, Holoturoidea) has five bundles of smooth muscles attached to its body wall. These muscles are related with physiological features of the sea cucumber, which can rapidly and reversibly alter its body length by more than 200% (Trotter and Koob, 1989Go). The contraction of the sea cucumber muscles like all others is promoted by an increase of the cytosolic Ca2+ concentration. In contrast with the mammalian skeletal muscles, the sarcoplasmic reticulum of the echinoderm muscles is scarce and difficult to identify (Hill et al., 1982Go; Chen, 1986Go). The contraction–relaxation cycle of the longitudinal smooth muscle involves not only the movement of Ca2+ between the extracellular medium and the cytosol of the muscle cells but also the release and accumulation of Ca2+ from intracellular stores (Suzuki, 1982Go).

The study of these particular tissues of invertebrates may help to elucidate more general molecular mechanisms of muscle contraction, with possible implications in vertebrate tissues. This approach has already been employed in the case of connective tissues of some invertebrates (Vieira and Mourão, 1988Go; Vieira et al., 1991Go, 1993; Pavã et al., 1994Go).

We have previously described that heparin can, in vitro, inhibit and modulate the activity of different ATPases of the P-type family (de Meis and Suzano, 1994Go; Landeira-Fernandez et al., 1996Go; Rocha et al., 1996Go; Rocha et al., 1998Go). Thus, several SERCA isoforms derived from rabbit muscle, human platelets and rat brain are not dependent on KCl but, in the presence of heparin, they are inhibited and become highly sensitive to K+. The inhibition can be antagonized by K+>Na+>Li+ (de Meis and Suzano, 1994Go; de Meis, 1998Go). A unique fucosylated chondroitin sulfate isolated from the sea cucumber connective tissue can mimic the effect of heparin in these SERCA isoforms (de Meis and Suzano, 1994Go). However, the possible physiological implication of these results can be questioned since these highly sulfated polysaccharides (such as heparin and fucosylated chondroitin sulfate) are not normally found in mammalian tissues where these ATPases are located.

In this paper we concentrated our study on the muscles found on the body wall of the sea cucumber Ludwigothurea grisea. In contrast with the SERCA isoforms from mammalian tissues, the activity of the Ca2+-dependent ATPase from sea cucumber is dependent on monovalent cations. The echinoderm smooth muscle was found to have a high concentration of fucosylated chondroitin sulfate but as we purified vesicles derived from this muscle we found a new and highly sulfated polysaccharide. When added to vesicles obtained from rabbit muscle this unique compound inhibits the enzyme and mimics the dependence for monovalent cations found in the echinoderm ATPase. Thus, our results describe the isolation of a sulfated polysaccharide that modulates ATPase activity and coexists in the same cellular compartment as the enzyme.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
K+ and Na+ have a distinctive effect on Ca2+ transport by sarcoplasmic reticulum vesicles from mammal and echinoderm muscles
Figure 1 shows that Ca2+ transport by vesicles obtained from the sea cucumber muscle is highly activated by monovalent cations. K+ is more effective than Na+ while Li+ has almost no effect (open symbols in Figure 1A). In contrast, the activity of the Ca2+-ATPase from rabbit sarcoplasmic reticulum muscle is not affected by increasing concentrations of monovalent cations (open symbols in Figure 1B). In this case Ca2+ uptake is already very high in the absence of added monovalent cations. However, the monovalent cation dependence can be reproduced with rabbit vesicles when heparin is added to the medium (de Meis and Suzano, 1994Go). With the addition of 10 µg/ml heparin to the vesicle preparation from rabbit muscle, Ca2+ uptake decreases sharply and becomes highly sensitive to K+ and Na+, as in the case of vesicles from sea cucumber muscle, and again Li+ has a modest effect (solid symbols in Figure 1B).



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Fig. 1. Effect of monovalent cations on Ca2+ uptake by sea cucumber (A) and rabbit microsomes (B). After incubation for 40 min at 25°C for sea cucumber vesicles (A) or for 20 min at 35°C for rabbit vesicles (B), Ca2+ uptake was measured in the presence of increasing concentrations of either KCl (open circles), NaCl (open triangles), or LiCl (open squares). Solid symbols in (B) are results in the presence of 10 µg/ml heparin.

 
Sulfated polysaccharides are isolated from the sea cucumber vesicles
The observation that the distinctive effect of monovalent cations on Ca2+ transport by vesicles derived from echinoderm and rabbit muscles is abolished by the addition of heparin to the rabbit vesicles suggests that a highly sulfated polysaccharide (like heparin) may be present in the echinoderm and involved in the regulation of the event. In order to test this possibility we attempted to isolate sulfated polysaccharides from the entire muscle of the sea cucumber and from its sarcoplasmic reticulum vesicles at different stages of purification. The sulfated polysaccharides were extracted by protease digestion, purified by anion-exchange chromatography and analyzed by polyacrylamide gel electrophoresis. Both the longitudinal and transversal muscles of the sea cucumber body wall have a high content of sulfated polysaccharides (Table I).


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Table I. Total sulfated polysaccharide content in different tissues of the sea cucumber
 
On elution from a Mono Q-FPLC with a linear gradient of NaCl, the polysaccharide extracted from the entire sea cucumber longitudinal muscle eluted at ~1.8 M NaCl and showed a strong metachromasia as produced by sulfated polysaccharides with 1,9-dimethylmethylene blue (solid circles in Figure 2A). Analysis of this compound on polyacrylamide gel electrophoresis stained with toluidine blue (lane 1 in Figure 3) reveals a major component with an average molecular mass of 30 kDa as a typical "fucosylated chondroitin sulfate" when compared to that isolated from the sea cucumber connective tissue (lane 6) (Vieira et al., 1991Go; Mourão et al., 1996Go). The similarity between these two polysaccharides was confirmed by their chemical analysis, showing equimolar proportions of galactosamine, glucuronic acid, and fucose (Table II), and their electrophoretic mobility on agarose gel (not shown but similar as reported by Mourão et al., 1996Go). When these experiments were performed with the transversal muscle from the sea cucumber body wall, we observed the same results as reported in Figure 2A and Figure 3, lane 2, for the longitudinal muscle (not shown).



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Fig. 2. Purification of sulfated polysaccharides from entire muscle (A) and from microsome preparations at different stages of vesicle preparation of sea cucumber (B and C) or rabbit (C) on a Mono Q-FPLC. The DEAE-cellulose-purified sulfated polysaccharides from sea cucumber (solid circles) or rabbit (open circles) were applied to a Mono Q-FPLC and purified as described under Materials and methods. Fractions were assayed for metachromasia and NaCl concentration (dashed line). The fractions corresponding to fucosylated chondroitin sulfate (fucCS) or vesicle polysaccharides (VP), as indicated by horizontal bars, were pooled, dialyzed against distilled water, and lyophilized. The arrows in (A) indicate elution of heparin and chondroitin 6-sulfate (CS) standards on the same column.

 


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Fig. 3. Polyacrylamide gel electrophoresis of sulfated polysaccharides extracted from the sea cucumber smooth muscle. Sulfated polysaccharides extracted from the entire muscle of sea cucumber (lane 1), from preparations containing endoplasmic reticulum vesicles of this muscle at different stages of purification (lanes 2–4) and also from purified vesicles of rabbit muscle (lane 5) were analyzed by polyacrylamide gel electrophoresis. Standard sulfated polysaccharides are: fucosylated chondroitin sulfate (fucCS, lane 6), chondroitin 6-sulfate (C-6-S, lane 7) chondroitin 4-sulfate (C-4-S, lane 8) and low-molecular-mass dextran sulfate (Dex 8 kDa, lane 9).

 

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Table II. Chemical composition of sulfated polysaccharides isolated from the sea cucumber smooth muscle
 
We then prepared a homogenate of the sea cucumber longitudinal muscle and microsomes were obtained by sequential centrifugation. Sulfated polysaccharides were extracted from these preparations at different stages of purification. We observed a decrease in the proportion of the polysaccharide eluted at ~1.8 M NaCl and a simultaneous increase of another polysaccharide eluted at ~2.2 M NaCl (solid circles in Figure 2B,C). Notably, the polysaccharide from the sarcoplasmic reticulum vesicles is eluted from the Mono Q-FPLC at a higher NaCl concentration than mammalian heparin (~1.3 M). But, more interesting, polyacrylamide gel electrophoresis revealed a marked difference in the mobilities between the sulfated polysaccharide extracted from the entire muscle and from the purified vesicles. Instead of the 30 kDa polysaccharide we noticed a wide variety of metachromatic bands, but each one a very narrow band (lanes 3 and 4, Figure 3). This pattern of molecular mass distribution is totally unusual for sulfated polysaccharides (see electrophoretic mobilities of standard sulfated polysaccharides on Figure 3, lanes 6 to 9). An additional incubation of this sulfated polysaccharide with papain did not modify the pattern of electrophoretic mobility on polyacrylamide gel (not shown). Thus, this variety of narrow electrophoretic bands cannot be attributed to peptides linked to glycan chains. The sulfated polysaccharide isolated from the sea cucumber vesicles resists chondroitin ABC lyase digestion and deamination with nitrous acid, treatments that degrade most glycosaminoglycans. This polysaccharide has a very complex chemical composition, containing fucose, galactose, and equimolar proportions of galactosamine and glucosamine (Table II).

When similar methodologies were applied to vesicles obtained from rabbit muscle we did not detect sulfated polysaccharides, as indicated by the absence of metachromasia on the Mono Q-FPLC (open circles in Figure 2C) and of metachromatic bands on polyacrylamide gel electrophoresis (lane 5, Figure 3).

Overall, these results indicate that sea cucumber muscles have a high content of fucosylated chondroitin sulfate, but vesicles derived from these muscles have, instead, a different sulfated polysaccharide, eluted from an anion exchange column at higher NaCl concentration and with a unique migration pattern on polyacrylamide gel electrophoresis. In contrast, vesicles derived from rabbit sarcoplasmic reticulum muscles have no detectable amounts of sulfated polysaccharides.

Sulfated polysaccharides from sea cucumber muscle modify Ca2+ uptake of rabbit vesicles
Both, the fucosylated chondroitin sulfate purified from the entire sea cucumber muscle and the unusual sulfated polysaccharide extracted from the vesicles inhibit Ca2+ transport by vesicles derived from rabbit sarcoplasmic reticulum (Figure 4A). The concentration required for half maximal inhibition of Ca2+ uptake was 10-fold lower for the sulfated polysaccharide extracted from sea cucumber vesicles than that for fucosylated chondroitin sulfate or heparin. In addition, the unusual polysaccharide from sea cucumber induces a monovalent cation dependence for Ca2+ uptake by rabbit vesicles (solid symbols in Figure 4B), as that already observed in the presence of heparin (solid symbols in Figure 1B).



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Fig. 4. Effect of different sulfated polysaccharides (A) and of K+ or Li+ (B) on Ca2+ uptake by vesicles from rabbit muscle. In the experiment of (A) increasing concentrations of heparin (open triangles), fucosylated chondroitin sulfate (solid triangles) or the sulfated polysaccharide extracted from the vesicles of sea cucumber muscle (VP, open circles) were incubated with microsomes from rabbit muscles and Ca2+ uptake was determined. In the experiment of (B), increasing concentrations of KCl (solid circles, open circles) or LiCl (solid squares, open squares) were incubated with rabbit microsomes in the absence (open symbols) or in the presence (solid symbols) of 2 µg/ml of the sulfated polysaccharide extracted from the sea cucumber vesicles.

 
Ca2+ uptake activity: dependence on molecular mass of the sulfated polysaccharide
When the sulfated polysaccharide extracted from the vesicles from sea cucumber muscles was chromatographed on a Superose 12-FPLC column, we observed a wide dispersion of molecular mass (Figure 5A), as already observed for the majority of sulfated polysaccharides. The fractions were pooled into three subfractions as indicated in the figure. Polyacrylamide gel electrophoresis (Figure 5B) confirmed that the molecular mass decreased from subfraction I to III. In addition, each subfraction is composed of a variety of very narrow electrophoretic bands, as already seen for the unfractionated polysaccharide (Figure 3). The three subfractions were able to inhibit the Ca2+ uptake by vesicles prepared from rabbit muscle and no marked difference was observed among their activities (not shown). Thus, the inhibitory activity of the sulfated polysaccharide on the Ca2+ uptake by vesicles derived from rabbit muscle seems independent of its molecular mass.




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Fig. 5. Fractionation of the sulfated polysaccharide from sea cucumber vesicles on a Superose-12 column. In (A) the Mono Q-purified sulfated polysaccharides were applied to a Superose-12 (HR 10/30) column, linked to a FPLC system. The various subfractions (I–III) were pooled, dialyzed against distilled water and lyophilized. In (B) the three subfractions were analyzed by polyacrylamide gel electrophoresis. A sample of unfractionated sulfated polysaccharides was included for comparison (Total).

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
The muscles of the sea cucumber body wall have a high content of sulfated polysaccharide
The longitudinal and transversal muscles of the sea cucumber body wall have a high content of sulfated polysaccharides. Protease digestion and purification by anion exchange chromatography followed by molecular mass and chemical composition determination showed that the preponderant polysaccharide present in this tissue is a fucosylated chondroitin sulfate.

When the muscle was stained with toluidine blue we observed strong metachromasia surrounding the fibers (not shown). However, when the sea cucumber muscle was homogenized and the vesicles derived from its sarcoplasmic reticulum purified, the fucosylated chondroitin sulfate was no longer detected. Instead, we observed a sulfated polysaccharide which eluted from the anion-exchange column at higher salt concentration (Figure 2B,C). The observed pattern of molecular mass distribution is totally unusual for sulfated polysaccharides. Besides vesicles obtained from the sarcoplasmic reticulum of rabbit muscles did not contain any detectable sulfated polysaccharide (open symbols in Figure 2C).

We have not been able yet to determine the chemical structure of this polysaccharide due to scarce material and its very complex nature. Thus, 1H NMR spectrum at 600 MHz of the polysaccharide recorded at 60°C showed broader and poorly resolved signals (not shown), indicating a clearly heterogeneous chemical structure. It was not possible to assign the peaks even with the two dimension techniques. Partial acid hydrolysis (150 mM H2SO4 100°C for 30 min) removes most of the sulfated fucose from the polymer but not galactose and hexosamines. Thus, the sulfated fucose residues are apparently branched units. Other attempts to prepare small fragments using partial hydrolysis procedures were unfruitful. This polysaccharide differs from mammalian glycosaminoglycans since it resists to chondroitin lyase digestion and to nitrous acid deamination.

Thus, the sea cucumber muscles contain two different pools of sulfated polysaccharides. Fucosylated chondroitin sulfate is present at very high concentration (almost similar to those found in the connective tissue of the invertebrate) and is located surrounding the muscle fibers. Another unique sulfated polysaccharide, almost undetectable when extraction was performed in the entire muscle, is found in the vesicles derived from the sarcoplasmic reticulum. The co-location of this polysaccharide along with the Ca2+-ATPase led us to speculate on a possible physiological relationship between the two.

We suggested that the sulfated polysaccharide present in the sea cucumber vesicles could act as an "endogenous" Ca2+-ATPase inhibitor. In fact, the sulfated polysaccharide obtained from vesicles of the sarcoplasmic reticulum of the sea cucumber muscle inhibits Ca2+ uptake in vesicles obtained from rabbit muscles even at lower doses than heparin (Figure 4A) and restores monovalent cation dependence (Figure 4B).

The effect of sulfated polysaccharide on Ca2+-ATPase from sarcoplasmic reticulum
The different proteins found in sarcoplasmic reticulum vesicles isolated from rabbit and sea cucumber muscles can be identified readily by SDS gel electrophoresis (MacLennan, 1970Go; MacLennan et al., 1978Go) and both revealed a major band of 110 kDa. The major component is the Ca2+-ATPase, this is a single polypeptide chain with a molecular weight of 110 kDa and accounts for 70–80% of the vesicles’ total protein content. The Ca2+-ATPase from sarcoplasmic reticulum is responsible for the active transport of Ca2+ from the cytosolic to the lumen of the reticulum controlling the muscle excitation-contraction cycle (de Meis, 1981Go).

Sulfated polysaccharides have been shown to modify the activity of different E1-E2 transport ATPases and the inhibition is antagonized by monovalent cations in a manner similar to that observed with the sea cucumber Ca2+-ATPase. Kinetic evidence presented in previous reports indicate that the different sulfated polysaccharides including heparin stabilize the E2 conformation of the different E1-E2 ATPases (de Meis and Suzano, 1994Go; Landeira-Fernandez et al., 1996Go; Rocha et al., 1998Go). The antagonism of the inhibition does not appear to be due to neutralization of the negative charges of the sulfate residues. Lithium is the most electropositive ion among the alkali ions tested. Potassium, but not lithium, strongly antagonizes the effect of the different sulfated glycosaminoglycans in different preparations of sarcoplasmic reticulum Ca2+-ATPases. Thus, if the effect of the alkali ions were to neutralize the negative charges of heparin, then Li+ should be as effective as K+ or Na+.

It is unlikely that the calcium binding to the sulfated polysaccharides plays an important role for the effects of these compounds on the Ca2+-ATPase. The calcium affinity constant for fucosylated chondroitin sulfate (Ruggiero et al., 1994Go) and heparin (Mattai and Kwak, 1981Go) is ~50 µM while the affinity of calcium for the ATPase is in the range of 0.1 µM (de Meis, 1981Go). In addition, the concentration of sulfated polysaccharide used in the Ca2+ uptake experiments is very low (~10 µg/ml) to decrease the amount of free calcium in the medium.

Is there any physiological role for the sulfated polysaccharides in sea cucumber muscles?
The body wall of the sea cucumber is composed by connective tissue and smooth muscle layers. It can rapidly and reversibly alter its mechanical properties. These alterations, which are thought to be neuronally controlled, allow the tissue to change its length by more than 200% (Trotter and Koob, 1989Go). The connective tissue of this echinoderm contains parallel collagen fibrils which are able to slide past one another during length changes but are inhibited from sliding when the tissue is in "catch." It has been suggested that sulfated polysaccharides (specifically the fucosylated chondroitin sulfate) is an important component of the stress-transfer matrix in echinoderms (Trotter and Koob, 1989Go).

In this paper we show that fucosylated chondroitin sulfate is not an exclusive component of this connective tissue but is also found in high concentrations surrounding the muscle fibers of the sea cucumber. The function of this polysaccharide in the echinoderm muscle is still not known. Perhaps its high negative charge density (Mourão et al., 1996Go) and consequent capacity to retain water in the extracellular matrix produces a "space" among muscle fibers allowing the intense change in length that may occur in this invertebrate (such as 200%) and are not observed in mammalian muscles.

Finally, the sulfated polysaccharide extracted from the sarcoplasmic reticulum vesicles has probably a totally different physiological role. The effect of this polysaccharide as an inhibitor of Ca2+ uptake by rabbit skeletal vesicles, producing its monovalent cation dependence, suggests that this compound may act as an "endogenous" Ca2+-ATPase regulator in sea cucumber smooth muscles. This possibility, however, requires future investigation.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Preparation of microsomes from the longitudinal muscle of the sea cucumber body
The sea cucumber L.grisea was collected in Rio de Janeiro, Brazil, and immediately brought to the laboratory immersed in sea water. The longitudinal muscles from the body wall of 20 specimens (~50 g, wet weight) were dissected, cut in small pieces, and homogenized in a warping blender with 160 ml of an ice cold buffer containing 10 mM MOPS-Tris buffer, (pH 7.0), 10% sucrose, 1 mM EDTA, 30 mM NaSO4, 450 mM NaCl, 10 mM KCl, and 1 mM phenylmethylsulfonyl fluoride. The microsomes were isolated as described by Eletr and Inesi (1972)Go. Briefly, the homogenate, which contains mostly intact muscle, was centrifuged at 14,000 x g for 20 min at 4°C. The supernatant was filtered through two layers of cheesecloth and centrifuged at 100,000 x g for 40 min at 4°C. The pellet was suspended in 30 ml of ice-cold buffer containing 10 mM MOPS-TRIS buffer (pH 7.0), 0.6 M KCl, 1 mM EDTA, and incubated on ice during 60 min and centrifuged at 15,000 x g for 20 min at 4°C. This procedure separates supernatant and pellet, the latter containing "vesicles and muscles contracting proteins." Finally, the supernatant was further centrifuged at 100,000 x g for 40 min at 4°C. The microsomes collected in the pellet, named "vesicle-enriched fraction," were suspended in 2 ml of ice-cold buffer containing 50 mM MOPS TRIS pH 7.0, 800 mM sucrose, 5 mM NaN3 and 1 mM EDTA, and stored in liquid nitrogen until use. Microsomes from skeletal muscle of rabbits were prepared following similar methodology, but only "vesicle-enriched fractions" were used throughout this study.

Ca2+ uptake by microsomes prepared from muscles of sea cucumber and rabbit
Ca2+ uptake was measured by the filtration method using 45Ca (Chiesi and Inesi, 1979Go). Briefly, microsomes (20 µg/ml, as protein) in 50 mM MOPS/TRIS (pH 7.0), 1 mM MgCl2, 50 µM 45Ca-CaCl2 (from Dupont, Wilmington, DE), 5 mM oxalate, 1mM ATP and increasing concentrations of either KCl, NaCl, LiCl, in the presence or absence of sulfated polysaccharides were incubated at 25°C for sea cucumber vesicles and at 35°C for rabbit vesicles. Ca2+ uptake was measured by filtration on Millipore filter. The filters were washed five times with 5 ml of 3 mM La(NO3)3 and the radioactivity remaining on the filters was counted on a liquid scintillation counter.

Extraction of sulfated polysaccharides from intact muscles and from microsome preparations at different stages of vesicle purification
Sea cucumber muscles were cut into small pieces, immersed immediately in acetone, and kept for 24 h at 4°C. The sulfated polysaccharides were extracted from the dried tissue (~2 g) by papain digestion and partially purified by cetylpyridinium chloride and ethanol precipitation (Vieira et al., 1991Go). In the case of microsome preparations we employed a more simplified procedure and the cetylpyridinium precipitation step was omitted (Vieira and Mourão, 1988Go). Approximately 4.0 ml of the microsome preparation was mixed with 4 ml of 0.1 M sodium acetate buffer (pH 6.0) containing 5 mg of papain, 5 mM EDTA and 5 mM cysteine. The incubation mixture was then centrifuged (2000 x g for 15 min at 10°C), and the sulfated polysaccharides in the supernatant were precipitated with 5 volumes of ethanol. After 24 h at 4°C, the precipitate formed was collected by centrifugation (2000 x g for 15 min at 10°C), washed with 10 ml of 80% ethanol and once with 95% ethanol. The final precipitate was dried at 60°C for 24 h (~3 mg, dry weight), dissolved in 5 ml of distilled water, and lyophilized.

Purification of the sulfated polysaccharides
Anion exchange chromatography. Sulfated polysaccharides extracted from the muscles or from the microsome preparations (~10 mg of each) were applied to a DEAE-cellulose column (7 x 2 cm) equilibrated with 0.05 M sodium acetate buffer (pH 5.0) and washed with 100 ml of the same buffer. The sulfated polysaccharides were eluted stepwise with 20 ml of 1.0 M NaCl in the same buffer. Eluates were dialyzed against distilled water and lyophilized. The DEAE-cellulose-purified polysaccharides were applied to a Mono Q-FPLC column (HR 5/5) from Amersham Pharmacia Biotech, equilibrated with 20 mM Tris:HCl (pH 8.0). The column was developed by a linear gradient of 0–3 M NaCl in the same buffer. The flow rate of the column was 0.5 ml/min, and fractions of 0.5 ml were collected. The fractions were assayed for metachromatic property using 1,9-dimethylmethylene blue (Farndale et al., 1986Go). The NaCl concentration in the fractions was estimated by conductivity. The fractions containing the sulfated polysaccharides were pooled, dialyzed against distilled water, and lyophilized.

Gel filtration chromatography. The Mono Q-purified polysaccharides were applied on a Superose-12 (HR 10/30) column, linked to a FPLC system from Amersham Pharmacia Biotech, equilibrated with 0.1 M Tris:HCl and 1 mM EDTA (pH 8.0). The column was eluted with the same solution, at a flow rate of 0.5 ml/min and fractions of 0.5 ml were collected and assayed by metachromasia using 1,9-dimethylmethylene blue (Farndale et al., 1986Go). The various subfractions were pooled, dialyzed against distilled water, and lyophilized.

Polyacrylamide gel electrophoresis. Sulfated polysaccharides (~10 µg) were applied to a 1-mm–thick 6% polyacrylamide slab gel and after electrophoresis at 100 V for ~1 h in 0.06 M sodium barbital (pH 8.6), the gel was stained with 0.1% toluidine blue in 1% acetic acid. After staining, the gel was washed for ~6 h in 1% acetic acid.

Chemical analyses.
After acid hydrolysis of the polysaccharide (5.0 M trifluoroacetic acid for 5 h at 100°C), sulfate was measured by the BaCl2/gelatin method (Saito et al., 1968Go). The percentages of hexoses, 6-deoxyhexoses and hexosamines were estimated by gas-liquid chromatography of derived alditol acetates (Kircher, 1960Go). Hexuronic acid was estimated by the carbazole reaction (Dische, 1947Go).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq: FNDCT, PADCT and PRONEX), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Financiadora de Estudos e Projetos (FINEP). We are grateful to Adriana A.Piquet for technical assistance.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
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