Hyaluronic Acid: A Component of the Aggregation Factor Secreted by the Marine Sponge,Microciona proliferaAuthor(s): William J. Kuhns, Max M. Burger and Eva TurleySource: Biological Bulletin, Vol. 197, No. 2, Centennial Issue: October, 1899-1999 (Oct., 1999),pp. 277-279Published by: Marine Biological LaboratoryStable URL: http://www.jstor.org/stable/1542650 .

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COMPARATIVE BIOCHEMISTRY COMPARATIVE BIOCHEMISTRY

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Hemolysis appears to result from the insertion of the hemo-

lytic protein into the plasma membrane of the target red cell to establish hydrophilic pores that mediate osmotic lysis of the cell. We draw this conclusion because the macromolecular osmolites dextran-4 (Mr 4-6 kDa) and dextran-8 (Mr 8-12 kDa) blocked

hemolysis completely. In this situation, the dextrans appear to act as osmolites that are too large to enter the red cell through the

presumed hydrophilic channels and thus act to balance the osmotic

pressure gradient across the red cell membrane and thus to protect the cell against osmotic damage (6). The molecular size of dex- tran-4 is approximately 1.7 nm (7), indicating that the effective

pore size is smaller than this value. We suggest that Busycon has a cytolytic system in the plasma

that functions to destroy invading pathogens. Hemocyanin has

tentatively been identified as the cytolytic agent. This is the first

example of which we are aware of an immune function for mol- luscan hemocyanin.

This research was supported by Grant MCB 97-26771 from the National Science Foundation.

Hemolysis appears to result from the insertion of the hemo-

lytic protein into the plasma membrane of the target red cell to establish hydrophilic pores that mediate osmotic lysis of the cell. We draw this conclusion because the macromolecular osmolites dextran-4 (Mr 4-6 kDa) and dextran-8 (Mr 8-12 kDa) blocked

hemolysis completely. In this situation, the dextrans appear to act as osmolites that are too large to enter the red cell through the

presumed hydrophilic channels and thus act to balance the osmotic

pressure gradient across the red cell membrane and thus to protect the cell against osmotic damage (6). The molecular size of dex- tran-4 is approximately 1.7 nm (7), indicating that the effective

pore size is smaller than this value. We suggest that Busycon has a cytolytic system in the plasma

that functions to destroy invading pathogens. Hemocyanin has

tentatively been identified as the cytolytic agent. This is the first

example of which we are aware of an immune function for mol- luscan hemocyanin.

This research was supported by Grant MCB 97-26771 from the National Science Foundation.

Protein (pg/ml) Protein (pg/ml) Figure 1. Hemolysis of horse red cells by the proteins precipitated by

4% PEG-8000. Hemolysis was conducted in 0.15 M NaCl, 0.14 M dex- trose, 10 mM CaCI2, 10 mM Tris, pH 7.3. The hemolytic activity of the

plasma was determined with triplicate samples using horse red blood cells obtained from Becton Dickinson and Company, Cockeysville, MD. The reaction mixtures contained 3 X 107 washed red cells in a final volume of 800 JIl. The samples were incubated at 22-23?C for 4 h, and the reaction was terminated by adding 2 ml of ice-cold phosphate-buffered saline

containing 5 mM ethylenediaminetetraacetic acid, followed by centrifuga- tion to remove the red cells. The extent of hemolysis was determined by monitoring released hemoglobin in the supernatant by the optical absor- bance at 412 nm and was compared to full hemolysis produced by hypo- tonic lysis of the red cells. The filled circles report hemolysis in the

presence of 10 mM Ca+2; the open circle reports hemolysis in a Ca+2-free buffer system.

Figure 1. Hemolysis of horse red cells by the proteins precipitated by 4% PEG-8000. Hemolysis was conducted in 0.15 M NaCl, 0.14 M dex- trose, 10 mM CaCI2, 10 mM Tris, pH 7.3. The hemolytic activity of the

plasma was determined with triplicate samples using horse red blood cells obtained from Becton Dickinson and Company, Cockeysville, MD. The reaction mixtures contained 3 X 107 washed red cells in a final volume of 800 JIl. The samples were incubated at 22-23?C for 4 h, and the reaction was terminated by adding 2 ml of ice-cold phosphate-buffered saline

containing 5 mM ethylenediaminetetraacetic acid, followed by centrifuga- tion to remove the red cells. The extent of hemolysis was determined by monitoring released hemoglobin in the supernatant by the optical absor- bance at 412 nm and was compared to full hemolysis produced by hypo- tonic lysis of the red cells. The filled circles report hemolysis in the

presence of 10 mM Ca+2; the open circle reports hemolysis in a Ca+2-free buffer system.

Literature Cited

1. Law, S. D., and K. B. M. Reid. 1988. Complement, IRL Press, Oxford.

2. Armstrong, P. B., S. Swarnakar, S. Srimal, S. Misquith, E. A. Hahn, R. T. Aimes, and J. P. Quigley. 1996. J. Biol. Chem. 271: 14717- 14721.

3. Herskovits, T. T., S. E. Carberry, and G. B. Villanueva. 1985. Biochim. Biophys. Acta 828: 278-289.

4. Herskovits, T. T., A. E. Guzman, and M. G. Hamilton. 1989.

Comp. Biochem. Physiol. 92B: 181-187. 5. Waxman, L. 1975. J. Biol. Chem. 250: 3796-3806. 6. Hatakeyama, T., H. Nagatomo, and N. Yamasaki. 1995. J. Biol.

Chem. 270: 3560-3564. 7. Scherrer, R., and P. Gerhardt. 1971. J. Bacteriol. 107: 718-735.

Literature Cited

1. Law, S. D., and K. B. M. Reid. 1988. Complement, IRL Press, Oxford.

2. Armstrong, P. B., S. Swarnakar, S. Srimal, S. Misquith, E. A. Hahn, R. T. Aimes, and J. P. Quigley. 1996. J. Biol. Chem. 271: 14717- 14721.

3. Herskovits, T. T., S. E. Carberry, and G. B. Villanueva. 1985. Biochim. Biophys. Acta 828: 278-289.

4. Herskovits, T. T., A. E. Guzman, and M. G. Hamilton. 1989.

Comp. Biochem. Physiol. 92B: 181-187. 5. Waxman, L. 1975. J. Biol. Chem. 250: 3796-3806. 6. Hatakeyama, T., H. Nagatomo, and N. Yamasaki. 1995. J. Biol.

Chem. 270: 3560-3564. 7. Scherrer, R., and P. Gerhardt. 1971. J. Bacteriol. 107: 718-735.

Reference: Biol. Bull. 197: 277-279. (October 1999)

Hyaluronic Acid: A Component of the Aggregation Factor Secreted by the Marine Sponge, Microciona prolifera

William J. Kuhnsl, Max M. Burger2, and Eva Turley' (Marine Biological Laboratory, Woods Hole, Massachusetts, 02543)

Reference: Biol. Bull. 197: 277-279. (October 1999)

Hyaluronic Acid: A Component of the Aggregation Factor Secreted by the Marine Sponge, Microciona prolifera

William J. Kuhnsl, Max M. Burger2, and Eva Turley' (Marine Biological Laboratory, Woods Hole, Massachusetts, 02543)

The aggregation factor (MAF) of Microciona sponge is a se- creted proteoglycan with a molecular weight of about 20 X 106 daltons. As observed by electron microscopy (EM), MAF has a sunburst type structure comprising a number of linear, primarily

Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada, M5G 1X8.

2 Friedrich Miescher Institute, Box 2543, CH 4002, Basel, Switzerland.

Correspondence should be addressed to Dr. Kuhns.

The aggregation factor (MAF) of Microciona sponge is a se- creted proteoglycan with a molecular weight of about 20 X 106 daltons. As observed by electron microscopy (EM), MAF has a sunburst type structure comprising a number of linear, primarily

Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada, M5G 1X8.

2 Friedrich Miescher Institute, Box 2543, CH 4002, Basel, Switzerland.

Correspondence should be addressed to Dr. Kuhns.

carbohydrate appendages extending from a rounded protein core (1). The integrity of the whole molecule is maintained by calcium, which unites the low molecular weight subunits of MAF to each other via salt linkages, and to cells by a linkage that is not yet defined. In the absence of calcium, MAF disintegrates; concomi-

tantly, EM reveals partially denuded cores ("bracelets"), and cell- cell binding is lost (2). A form of MAF-cell binding that is

calcium-independent is presumed to exist (3) but has not been characterized. We hypothesized that polymers of the disaccharide hyaluronic acid (HA) might play a role in cell attachment, because

carbohydrate appendages extending from a rounded protein core (1). The integrity of the whole molecule is maintained by calcium, which unites the low molecular weight subunits of MAF to each other via salt linkages, and to cells by a linkage that is not yet defined. In the absence of calcium, MAF disintegrates; concomi-

tantly, EM reveals partially denuded cores ("bracelets"), and cell- cell binding is lost (2). A form of MAF-cell binding that is

calcium-independent is presumed to exist (3) but has not been characterized. We hypothesized that polymers of the disaccharide hyaluronic acid (HA) might play a role in cell attachment, because

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REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

its constituent monosaccharides (glucuronic acid and N-acetylglu- cosamine) are known to be present in MAF glycans (4). Among our aims was to test our hypothesis that MAF contains an HA polymer and to show that this polymer constitutes a central link in the binding of MAF to an HA-binding protein (HABP).

Various HABPs have been identified in vertebrates; moreover, the consensus amino acid sequence at the HA binding site can be expressed by the formula (B(X6_7)B), where B is arginine (R) or lysine (K), and X is any non-acidic amino acid, except that one or more must be a basic residue (5). An HA-binding peptide sequence has recently been identified within the amino acid sequence of the MAF core protein, which was translated from the cDNA (6). The sequence of the novel sponge binding site, designated MAF- HABP, is RRYRNRVR; it clearly fits the requirements of the consensus (5). MAF-HABP has been synthesized at the University of Toronto Biotechnology Center and has been probed, in a West- ern blot, with a biotin-labeled hyaluronic acid (BHA), as described by us in an earlier report (7).

A second non-MAF HABP has also been demonstrated by immunohistochemistry on Microciona cell surfaces. The antibody probe was prepared from RHAMM protein, i.e., the receptor for hyaluronic acid (HA) mediated motility. This protein occurs on vertebrate cell membranes and possesses amino acid sequences that, again, conform to the consensus formula given above (5). Double labeling studies indicated that immunoreactivity to FITC- labeled anti-RHAMM could be blocked by the prior exposure of cells to BHA; conversely, a reaction with BHA was blocked by previous exposure to anti-RHAMM (5). We concluded that MAF binding to a cell could occur, if a RHAMM on the cell surface were linked to the MAF-HABP via an HA polymer, and if this polymer were a component of the MAF macromolecule.

In the experiments to be described, we provide further evidence in support of this hypothesis. First we show that treatment of MAF with hyaluronidase (HAase), an HA depolymerizing enzyme, ab- rogates MAF binding to an HABP (experiment 1). Second, we establish that when MAF is premixed with synthetic MAF-HABP,

2pI 1 2 3

Premix + = water

, i*"I

,

W'ft - .

+= HAase. , i

Figure 1. Dot blots illustrating the effects of hyaluronidase (HAase) and synthetic MAF-HABP on the binding of purified Microciona aggrega- tion factor (MAF) to biotin-labeled, hyaluronic acid binding protein (B- HABP). Experiment I (Lanes 1 and 2): Upper blots, MAF premixed with water, incubated, and spotted, the spots treated with B-HABP, and the color developed (methods in text); Lower blots, MAFpremixed with HAase (method as above). Experiment 2 (Lane 3, Upper blot): MAF premixed with synthetic MAF-HABP (method as above). Lane 3, Lower blot: color control.

Figure 2. A semi-schematic diagram drawn upon a portion of an electron micrograph adapted from (1). MAF is visualized by us as a three-dimensional pattern (grey circles and lines) etched within a stack of very thin grey carpets. A portion of the pattern on one layer of the stack is outlined in dark black; it represents a nest of MAF macromolecules attached to five Microciona cells (only fragments of cell membranes are shown as curved hatched structures). The core protein of the MAF are circular structures, and the thin lines extending away from the cores are the primarily carbohydrate arms. The arms appear to bind to other MAF cores as well as to cell membranes. Cores also seem able to bind to one another, but there may be overlapping that cannot be visualized in one dimension. Arms that appear to be short, or to have a free end, may be attached to an underlying portion of MAF. Similarly, portions of cores that appear to lack arms may have arms that extend vertically to MAF in a different horizontal plane. A single MAF-HABP (see text) is thought to occur on each protein core; it is attached to HA (thick dark line) which binds at its distal end to a cell surface HABP (short dark lines at right angles to the HA). RHAMM is an example of a cell surface HABP, but other molecules with similar HA binding motifs may exist in sponges.

binding occurs, and the MAF is subsequently incapable of binding to a labeled HABP probe (experiment 2).

Adult Microciona sponge was processed, in accordance with previous reports, to yield the crude adhesive proteoglycan (MAF) (8, 9). Purified MAF was isolated by cesium gradient centrifuga- tion, and the concentrated product was desalted. Viscosity was reduced by extensive dialysis in de-ionized water containing 1 mM EDTA. The final compound was lyophilized and reconstituted at 1 mg/ml as needed. Synthetic MAF-HABP was used in these studies at a concentration of 1 mg/ml.

For experiment 1, aliquots of MAF were premixed with an equal volume of a solution of hyaluronidase (HAase). The latter solution was prepared by adding 50 units of HAase, together with protease inhibitors (25 /xg/ml) in acetate buffer at pH 5.0, to make 50 Al. Mixtures in a total volume of 10 udl, as well as dilution controls lacking HAase, were incubated at 4?C overnight. For experiment 2, MAF was premixed with synthetic MAF-HABP,

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COMPARATIVE BIOCHEMISTRY COMPARATIVE BIOCHEMISTRY

both products being combined in equal amounts and incubated as above.

Dot blots were prepared for both experiments by spotting 2 ,u1 of one of the two premixtures on nitrocellulose. Non-specific reactive sites were blocked with 1% bovine serum albumin in

phosphate buffered saline. The spots were then treated with 2 ,ug of biotin-labeled hyaluronic acid binding protein (B-HABP) (Seikagaku No. 400763-1) for one hour, and then washed. This was followed by streptavidin-conjugated peroxidase and color

development with 3'3 diaminobenzidine and hydrogen peroxide. If

binding occurred, a brown coloration would appear at sites where substrates were applied to the nitrocellulose.

In experiment 1, two preparations of MAF gave a strong brown color at the sites of application of B-HABP; but MAF that was incubated with HAase showed no coloration. This result shows that the depolymerization of HA prevents the binding of MAF to B-HABP. In experiment 2, the site that contained MAF pre-treated with synthetic MAF-HABP remained almost colorless after the

application of biotin-labeled HABP. This finding indicated that HA in MAF had combined with the synthetic binding site and was unable to bind to the HABP probe. Taken together, the results of the two experiments demonstrate that the MAF macromolecule contains HA, presumably in polymeric form (Fig. 1).

These results extend our earlier findings that had demonstrated the

binding of biotin-labeled HA to the synthetic MAF-HABP (7). The existence of an HA binding amino acid sequence within the MAF core peptide suggests that its coupling to HA is a likely event. Our belief that HA-HABP coupling derives from a single site on the MAF core is supported by recent studies of Femandez-Busquets (manu- script in preparation). The occurrence of RHAMM, or other HA

both products being combined in equal amounts and incubated as above.

Dot blots were prepared for both experiments by spotting 2 ,u1 of one of the two premixtures on nitrocellulose. Non-specific reactive sites were blocked with 1% bovine serum albumin in

phosphate buffered saline. The spots were then treated with 2 ,ug of biotin-labeled hyaluronic acid binding protein (B-HABP) (Seikagaku No. 400763-1) for one hour, and then washed. This was followed by streptavidin-conjugated peroxidase and color

development with 3'3 diaminobenzidine and hydrogen peroxide. If

binding occurred, a brown coloration would appear at sites where substrates were applied to the nitrocellulose.

In experiment 1, two preparations of MAF gave a strong brown color at the sites of application of B-HABP; but MAF that was incubated with HAase showed no coloration. This result shows that the depolymerization of HA prevents the binding of MAF to B-HABP. In experiment 2, the site that contained MAF pre-treated with synthetic MAF-HABP remained almost colorless after the

application of biotin-labeled HABP. This finding indicated that HA in MAF had combined with the synthetic binding site and was unable to bind to the HABP probe. Taken together, the results of the two experiments demonstrate that the MAF macromolecule contains HA, presumably in polymeric form (Fig. 1).

These results extend our earlier findings that had demonstrated the

binding of biotin-labeled HA to the synthetic MAF-HABP (7). The existence of an HA binding amino acid sequence within the MAF core peptide suggests that its coupling to HA is a likely event. Our belief that HA-HABP coupling derives from a single site on the MAF core is supported by recent studies of Femandez-Busquets (manu- script in preparation). The occurrence of RHAMM, or other HA

receptors on Microciona cell populations may thus provide a means of MAF-cell binding that is calcium independent and distinct from the low molecular weight anionic carbohydrate epitopes previously de- scribed (10, 11). A signalling role for HA and its receptors has also been proposed (7); such linkages may enable Microciona cells to cross-communicate (Fig. 2).

Literature Cited

1. Henkart, P., S. Humphreys, and T. Humphreys. 1973. Biochem- istry 12: 3045-3050.

2. McLaurin, J., T. Franklin, W. Kuhns, and P. Fraser. 1999. Amy- loid (In press).

3. Jumblatt, J., V. Schlup, and M. Burger. 1980. Biochemistry 19: 1038-1042.

4. Misevic, G., and M. Burger. 1986. J. Biol. Chem. 261: 2853-2859. 5. Yang, B., L. Zhang, and E. Turley. 1993. J. Biol. Chem. 268:

8617-8623. 6. Fernandez-Busquets, X., R. Kammerer, and M. Burger. 1996.

J. Biol. Chem. 271: 23558-23565. 7. Kuhns, W., X. Fernandez-Busquets, M. Burger, M. Ho, and E.

Turley. 1998. Biol. Bull. 195: 216-218. 8. Misevic, G., J. Finne, and M. Burger. 1987. J. Biol. Chem. 262:

5870-5877. 9. Misevic, G., and M. Burger. 1993. J. Biol. Chem. 268: 4922-4929.

10. Spillmann, D., K. Hard, J. Thomas-Oates, J. Vliegenthart, G. Misevic, M. Burger, and J. Finne. 1993. J. Biol. Chem. 268: 13378-13387.

11. Spillmann, D., J. Thomas-Oates, A. van Kuik, J. Vliegenthart, G. Misevic, M. Burger, and J. Finne. 1995. J. Biol. Chem. 270: 5089-5097.

receptors on Microciona cell populations may thus provide a means of MAF-cell binding that is calcium independent and distinct from the low molecular weight anionic carbohydrate epitopes previously de- scribed (10, 11). A signalling role for HA and its receptors has also been proposed (7); such linkages may enable Microciona cells to cross-communicate (Fig. 2).

Literature Cited

1. Henkart, P., S. Humphreys, and T. Humphreys. 1973. Biochem- istry 12: 3045-3050.

2. McLaurin, J., T. Franklin, W. Kuhns, and P. Fraser. 1999. Amy- loid (In press).

3. Jumblatt, J., V. Schlup, and M. Burger. 1980. Biochemistry 19: 1038-1042.

4. Misevic, G., and M. Burger. 1986. J. Biol. Chem. 261: 2853-2859. 5. Yang, B., L. Zhang, and E. Turley. 1993. J. Biol. Chem. 268:

8617-8623. 6. Fernandez-Busquets, X., R. Kammerer, and M. Burger. 1996.

J. Biol. Chem. 271: 23558-23565. 7. Kuhns, W., X. Fernandez-Busquets, M. Burger, M. Ho, and E.

Turley. 1998. Biol. Bull. 195: 216-218. 8. Misevic, G., J. Finne, and M. Burger. 1987. J. Biol. Chem. 262:

5870-5877. 9. Misevic, G., and M. Burger. 1993. J. Biol. Chem. 268: 4922-4929.

10. Spillmann, D., K. Hard, J. Thomas-Oates, J. Vliegenthart, G. Misevic, M. Burger, and J. Finne. 1993. J. Biol. Chem. 268: 13378-13387.

11. Spillmann, D., J. Thomas-Oates, A. van Kuik, J. Vliegenthart, G. Misevic, M. Burger, and J. Finne. 1995. J. Biol. Chem. 270: 5089-5097.

Reference: Biol. Bull. 197: 279-281. (October 1999)

Biosynthesis of Tyrosine O-Sulfate by Cell Proteoglycan from the Marine Sponge, Microciona prolifera Octavian Popescu1, Rey Interior4, Gradimir Misevic2, Max M. Burger3, and William J. Kuhns4

(Marine Biological Laboratory, Woods Hole, Massachusetts, 02543)

Reference: Biol. Bull. 197: 279-281. (October 1999)

Biosynthesis of Tyrosine O-Sulfate by Cell Proteoglycan from the Marine Sponge, Microciona prolifera Octavian Popescu1, Rey Interior4, Gradimir Misevic2, Max M. Burger3, and William J. Kuhns4

(Marine Biological Laboratory, Woods Hole, Massachusetts, 02543)

Tyrosine sulfation is a post-translational modification of pro- tein that takes place in the trans-Golgi system (1). The inclusion of such a modified tyrosine in their native sequence confers

strong bioactivity to a variety of ligands and peptides involved in motility, secretion, cell binding, and the promotion of com-

plement action and blood coagulation (2-7). Tyrosine sulfa- tion is catalyzed by a specific tyrosylprotein sulfotransferase

(TPST); the enzyme has a particularly strong affinity for peptide substrates with a consensus sequence that features acidic amino acid residues adjacent, or at least close to, tyrosine (8). Se-

1 Institute for Biological Research, Cluj/Napoca, Romania (O.P.). 2 Biocenter, University of Basel, Switzerland. 3Friedrich Miescher Institute, Box 2543, CH4002, Basel, Switzerland. 4 Hospital for Sick Children, 555 University Avenue, Toronto, Ontario,

Canada, M5G 1X8.

Tyrosine sulfation is a post-translational modification of pro- tein that takes place in the trans-Golgi system (1). The inclusion of such a modified tyrosine in their native sequence confers

strong bioactivity to a variety of ligands and peptides involved in motility, secretion, cell binding, and the promotion of com-

plement action and blood coagulation (2-7). Tyrosine sulfa- tion is catalyzed by a specific tyrosylprotein sulfotransferase

(TPST); the enzyme has a particularly strong affinity for peptide substrates with a consensus sequence that features acidic amino acid residues adjacent, or at least close to, tyrosine (8). Se-

1 Institute for Biological Research, Cluj/Napoca, Romania (O.P.). 2 Biocenter, University of Basel, Switzerland. 3Friedrich Miescher Institute, Box 2543, CH4002, Basel, Switzerland. 4 Hospital for Sick Children, 555 University Avenue, Toronto, Ontario,

Canada, M5G 1X8.

quences with these criteria can be identified in translated por- tions of the cDNA encoding the protein core of the Microciona adhesive proteoglycan (MAF) (9). A related earlier finding of ours established that restriction of environmental sulfate in Microciona cell suspensions is accompanied by a diminution of secreted MAF and a loss of cell motility (10). These findings motivated a search for a functional correlate. We now describe, for the first time, the biosynthesis of tyrosine sulfate by cell

preparations from the marine sponge, Microciona prolifera. Cell suspensions (107/ml) were prepared in sulfate-free arti-

ficial seawater (ASW) from whole sponge fragments and ro- tated for 24 h at 16?C. The cells were then pulsed with 50 ,tCi of sulfur-35 (DuPontNEN NEX-041) and 50 ,uCi tyrosine-ring 3,5 3H (NEN) for 8 h. The pellets were washed 3 times: lysates were prepared in 20 mM Tris (pH 7.4) containing 1% Triton- X-100 and protease inhibitors (25 /ug/ml). Several aliquots (20

quences with these criteria can be identified in translated por- tions of the cDNA encoding the protein core of the Microciona adhesive proteoglycan (MAF) (9). A related earlier finding of ours established that restriction of environmental sulfate in Microciona cell suspensions is accompanied by a diminution of secreted MAF and a loss of cell motility (10). These findings motivated a search for a functional correlate. We now describe, for the first time, the biosynthesis of tyrosine sulfate by cell

preparations from the marine sponge, Microciona prolifera. Cell suspensions (107/ml) were prepared in sulfate-free arti-

ficial seawater (ASW) from whole sponge fragments and ro- tated for 24 h at 16?C. The cells were then pulsed with 50 ,tCi of sulfur-35 (DuPontNEN NEX-041) and 50 ,uCi tyrosine-ring 3,5 3H (NEN) for 8 h. The pellets were washed 3 times: lysates were prepared in 20 mM Tris (pH 7.4) containing 1% Triton- X-100 and protease inhibitors (25 /ug/ml). Several aliquots (20

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