Andrew P Spicer

1. Introduction

Hyaluronan (HA) is synthesized at the plasma membrane as a free linear polymer of composition [01^4GlcAp1^3GlcNAc]n (1-3). All models suggest that polymerization occurs at the inner face of the plasma membrane while the polymer is coordi-nately translocated or extruded across the membrane to the extracellular face of the cell [for review, see (2-5)]. In mammals (6), and all vertebrates (Spicer, unpublished data), HA is synthesized by any one of three HA synthases (HAS). The three HAS proteins are encoded by three related yet distinct genes (6,7). All HAS proteins are predicted integral plasma membrane proteins with N-terminal and C-terminal transmembrane domains separating a large cytoplasmic domain [for review, see (4)]. Any one HAS protein is capable of catalyzing the de novo synthesis of HA (8), suggesting that each is capable of specifically binding both UDP-sugar substrates and creating the alternate 01^3 and the 4 glycosidic bonds. Indeed, the related prokaryotic HA synthase, spHAS, from the Gram-positive bacterium, Streptococcus pyogenes, has been purified to apparent homogeneity and is capable of synthesizing high molecular mass HA chains in vitro, when provided with a source of UDP-GlcA, UDP-GlcNAc, and Mg2+ ions (9).

The vertebrate HAS proteins have not yet been successfully purified in a soluble and active form. Thus, all current approaches to the detection of HA synthase activity are performed on intact cells or on membrane preparations derived from cells transfected with a particular HA synthase expression vector or from cells that express endogenous HA synthase activity (see Note 1). In this chapter, three relatively simple procedures for the detection of HA synthase activity will be described.

2. Materials

2.1. General

All three protocols described here use cultured mammalian cell lines. These methods could be equally well applied to the investigation of cell lines derived from other vertebrates and potentially invertebrates. All cells are cultured on tissue culture plastic at 37°C in a humidified atmosphere of 5% CO2. Most cell lines are cultured in medium containing 10% fetal bovine serum plus 5 mM l-glutamine and antibiotics (penicillin and streptomycin). Phospate-buffered saline (PBS), calcium- and magnesium-free, is used throughout for washing cell monolayers.

2.2. Particle Exclusion Assay

1. Fixed horse or sheep erythrocytes (Sigma), reconstituted to a final cell density of 5 x 108 cells/mL in PBS, supplemented with 1 mg/mL bovine serum albumin (BSA). Erythrocytes should be washed and pelleted at least twice in this buffer before use, to remove traces of sodium azide. These erythrocyte suspensions can be stored for long periods at 4°C, and need simply to be mixed well before each use to avoid clumps. Pipetting several times with a plugged Pasteur pipet is most effective.

2. Streptomyces hyaluronate lyase (10) (Sigma or Calbiochem) reconstituted to 400 turbidity-reducing units (TRU) per mL in 20 mM sodium acetate, pH 5.0, and stored as frozen aliquots (20 TRU/ 50-^L aliquot) at -80°C.

3. Inverted phase-contrast microscope with digital camera or other recording device.

2.3. In Vitro HA Synthase Assay

1. Sterile (autoclaved) hypotonic lysis buffer (LB): 10 mM KCl, 1.5 mM MgCl2, and 10 mM Tris-HCl, pH 7.4, stored at 4°C.

2. Protease inhibitors: aprotinin, leupeptin, and phenyl methyl sulfonyl fluoride (PMSF) (Calbiochem); dissolved and used at the manufacturer's recommended concentrations.

3. Prechilled disposable cell scrapers or rubber policemen and 1-mL pipets.

4. Sterile screw-capped 2-mL microcentrifuge tubes.

5. Small (2-mL reservoir) Dounce homogenizer with type B pestle.

6. Refrigerated microcentrifuge, centrifuge, or microcentrifuge/centrifuge located in cold room.

7. Micro-BCA assay kit (Pierce) and flat-bottomed enzyme-linked immunosorbent assay (ELISA) plates.

9. UDP-[14C]-glucuronic acid (>180 mCi/mmol; usually 250-350 mCi/mmol), NEN-Dupont, ICN, or Amersham. Can also use UDP-[3H]-GlcNAc (NEN-Dupont, 20-45 Ci/mmol). Aliquot UDP-[14C]-GlcA into 0.25-^Ci aliquots (sufficient for one reaction) and store at -20°C.

10. 10x HA synthase buffer consisting of 50 mM dithiothreitol, 150 mM MgCl2, and 250 mM HEPES, pH 7.1 (sterile-filtered and stored at 4°C).

11. UDP-GlcNAc (stock solution of 100 mM aliquotted and stored at -20°C) and UDP-GlcA (stock solution of 5 mM aliquotted and stored at -20°C). Dissolved in hypotonic lysis buffer.

12. Streptomyces hyaluronate lyase (reconstituted as described under Subheading 2.2.2.).

14. Whatman 3MM chromatography paper (big sheets), cut widthwise into strips 2 cm wide. Before cutting into strips, draw lines across the entire length of the sheet as follows: solid line 2.5 cm from the top; dashed line 6 cm from the top; solid line 8 cm from the top; solid line 10 cm from the top. Cut into individual strips, then fold strips forward at first line, then backwards at second line. A large number of strips can be made in advance and stored in a suitable dry location.

15. Chromatography solvent consisting of a 130/70 mix of absolute ethanol/ 1 M ammonium acetate, pH 5.5. It is better to make the solvent fresh before each use by simply mixing ethanol with 1 M ammonium acetate, pH 5.5.

16. Chromatography chamber.

17. Scintillation vials, liquid scintillation fluid, and liquid scintillation counter. Alternatively, an imaging device capable of detecting 14C could be used to scan each strip and determine the number of counts at the origin.

2.4. Metabolic Labeling with [3H]-Acetate or [3H]-Glucosamine

1. Cell line growing in appropriate medium at appropriate density.

2. [3H]-glucosamine (20-45 Ci/mmol, NEN-Dupont or Amersham), or [3H]-acetate (2-5 Ci/ mmol, NEN-Dupont or Amersham).

3. 0.25% trypsin, 1 mM EDTA solution (Life Technologies).

4. 1.3% (w/vol) potassium acetate/95% ethanol solution.

5. Protease Type XIV (Sigma), resuspended in 100 mM Tris-HCl, pH 8.0.

7. Streptomyces hyaluronate lyase, reconstituted and aliquotted as described under Subheading 2.2.2.

8. 12% (w/vol) cetyl pyridinium chloride (CEPC) (Sigma) solution (made up in deionized water).

10. Methanol.

11. Liquid scintillation counter, liquid scintillant, and scintillation vials.

3. Methods

3.1. General

Any of the three methods described here can be used to detect HA synthesis. The first, the particle exclusion assay, can be used as a rapid assessment of putative HA synthase activity in, e.g., transfected cells, or as a rapid assessment of possible endogenous HA synthase activity in established or primary cell cultures (see Note 1). The second in vitro assay is more quantitative in nature, providing a direct measure of the HA biosynthetic capacity of a given cell population (see Note 2). The third assay, metabolic labeling of HA, provides a measure of the HA being synthesized within a cell culture over a given period (see Note 3).

3.2. Particle Exclusion Assay

This very simple and now classical assay permits the visualization of the HA-dependent pericellular matrix (11) (see Note 4).

1. Cells are plated on tissue culture plastic at a suitable cell density to achieve 30-40% confluence the next day. Duplicate wells are established for each experiment.

2. Cells can either be assayed directly (if an endogenous HA synthase activity is being studied) or transfected with an expression vector carrying a putative HA synthase.

3. 48-72 h post-transfection or at least 24 h postplating (endogenous HA synthases), fixed red blood cells are added to cell cultures. Amounts added are as follows: 24-well plate, 1 x 107/well; 6-well plate, 5 x 107/well. Swirl dishes gently to distribute the cell suspension evenly.

4. Culture dishes are returned to the incubator for an additional 15 min to allow erythrocytes to settle on the monolayer.

5. Dishes are removed from the incubator (plates may need to settle on the microscope stage for a few minutes prior to viewing) and examined under phase contrast using the 10x and 20x objectives to scan individual wells for evidence of cells with pericellular coats. Positive cells are viewed using the 40x objective (see Note 5) and the image is captured for computer analysis (see Note 6) or displayed on a screen such that the outline of the cell and pericellular matrix can be traced. Tracing onto transparency paper works well for this approach.

6. For quantitation of relative pericellular matrix size, at least 20 cells are imaged per well. The relative pericellular coat size is expressed as the area of the pericellular matrix plus the area of the cell, divided by the area of the cell. Hence, a cell without a pericellular matrix will have a relative value of 1.0. In contrast, cells that are actively synthesizing HA may have ratios of 2.0 or more.

7. After imaging or tracing, 10 TRU of Streptomyces hyaluronate lyase (for 6-well plate) are added directly to one well of each pair (see Note 7). The second well receives an equivalent volume (25 ^L) of 20 mM sodium acetate pH 5.0. Dishes are returned to the incubator and incubated for at least 1 h more at 37°C.

8. After 1 h, dishes are swirled to redistribute the erythrocyte layer, and steps 4-6 are repeated.

3.3. In Vitro HA Synthase Assay

1. Cell cultures are established on 15-cm tissue culture plates. Generally, between 2 and 4 plates are necessary to obtain sufficient material for in vitro assays (see Notes 8 and 9).

2. Individual 15-mL conical tubes are filled (10-15 mL) with either PBS (2 per plate) or hypotonic lysis buffer (LB) (3 per plate) and placed on ice. These prechilled solutions will be used for the washes outlined below. Tubes can be reused each time. It is not necessary to cap tubes.

3. Prelabel 2-mL screw-capped microcentrifuge tubes and place on ice. Three tubes should be labeled for each plate. Label as follows: "nuclei," "membranes," and "lysate."

4. Cultures of subconfluent, proliferating cells (for endogenous HA synthase measurement) or transfected cells (72 h posttransfection is best) are removed from the tissue culture incubator and placed on ice in a large tray (see Note 10). Six plates can be processed at one time.

5. Culture medium is aspirated from each plate and plates are washed twice with cold, prealiquotted PBS. For each wash, buffer is poured gently onto each plate, the plate is swirled gently, then buffer is removed by aspiration.

6. Each plate is washed twice with cold hypotonic lysis buffer. After the second wash, LB is added to each plate and the plates are incubated on ice for 10 min to allow cells to swell.

7. After the 10 min incubation, each plate is treated separately in turn. Buffer is aspirated from the plate and the plate is tilted such that any remaining buffer drains to one side and can be removed by aspiration.

8. 1 mL of LB, supplemented with the protease inhibitors aprotinin and leupeptin (LB+), and prepared just prior to use, is added to the plate. Using a prechilled sterile cell scraper, the monolayer is scraped from the plate into the 1 mL of LB+ (see Note 11).

Table 1

In Vitro HA Synthase Assay

Component

Volume (or amount)

Cell membranes (add last) LB buffer

10X HA synthase buffer UDP-GlcNAc (100 mM) UDP-GlcA (5 mM) Aprotinin (0.2 mg/mL in PBS) Leupeptin (5 mg/mL in water) UDP-[14C]GlcA

25-100 ^g (not to exceed 73.5-75.5 ^L volume) to a final volume of 100 ^La 10 ^L

1 ^L (or 0 ^L for specificity control reaction) 1 ^L 1 ^L 1 ^L

aCalculate amount of LB required to bring final volume of each reaction to 100 |L before assembling reactions. Assemble each reaction according to the order in the table, adding the cell membranes last.

9. The suspension is transferred into the mortar of a prechilled, 2-mL Dounce homogenizer. 10 ^L of PMSF is added to the suspension. Cells are disrupted by 5-10 twisting strokes using a B-type pestle and the homogenate is transferred to the prechilled tube labeled, "nuclei" (see Note 12). Leave tube on ice until all plates have been processed.

10. Pellet nuclei and organelles by spinning all tubes at 4000g for 5 min in a refrigerated microcentrifuge.

11. Transfer supernatants to prechilled, labeled tubes marked "membranes," and spin at high speed (approximately 20,000g) for 15 min in a refrigerated microcentrifuge to pellet cell membranes.

12. Transfer supernatants to prechilled, labeled tubes marked "lysate."

13. Carefully resuspend individual membrane pellets in 50 ^L of LB+. Pool resuspended membranes from equivalent samples.

14. Remove 5-10 ^L of each sample (after pooling) to a separate tube for determination of protein content using, for instance, a Micro-BCA assay. If the remainder of the sample will not be immediately used, store at -80°C until required (see Note 13).

15. Determine protein content using a Micro-BCA assay. Five microliters of each sample should be brought up to 205 ^L by addition of 200 ^L of LB plus 1% SDS (w/v) (see Note 14). The individual samples can then be split into duplicate wells of an ELISA plate (100 ^L/well).

16. Assemble in vitro synthase assays as outlined in Table 1. Typically, between 25 and 100 ^g of total membrane protein are used per assay (see Notes 15-17).

17. Incubate assays for 1-2 h (1 h is standard) at 37°C in a heat block or water bath. Establish duplicate or triplicate reactions wherever possible.

18. After incubation, stop the reactions by heating at 100°C for 3-5 min. Pulse-spin to collect condensate.

19. Thaw Streptomyces hyaluronate lyase (Hase) aliquots (one per reaction) at room temperature and label tubes to correspond to each reaction under study. Add 50 ^L for each reaction to appropriately labeled HA lyase tubes. These tubes will represent the +Hase tubes. To the remaining 50 ^L of each reaction, add 50 ^L of 20 mM sodium acetate, pH 5.0. These tubes will serve as the mock treated group (-Hase). Incubate all tubes for a minimum of 3 h at 60°C.

20. Add 20% sodium dodecyl sulfate (SDS) to each reaction to a final concentration of 1% (w/v) and heat reactions for 5 min at 100°C.

21. Spot each reaction onto the origin (bounded by the lines at 8 and 10 cm from the top) of individual prelabeled paper chromatography strips, taking care to avoid spreading beyond the origin. This can most easily be achieved by applying each reaction as two separate additions, allowing drying between each application.

22. Assemble paper chromatography chamber and elute overnight with 130/70 (absolute etha-nol/1 M ammonium acetate, pH 5.5) by descending paper chromatography (see Notes 18 and 19).

23. Cut out origins with scissors and transfer to individual scintillation vials containing 2 mL of deionized water. Ensure that paper is immersed in the water by mixing well.

24. Add liquid scintillation cocktail to each tube, mix well, and count using liquid scintillation counting (LSC) (see Note 20).

25. Assuming that dpm are approximately equivalent to cpm, the HA synthase activity of each sample can be determined from the LSC results and expressed as pmol/mg membrane protein/h. Calculations should take into account the specific activity of the labeled sugar and the relative molar amounts of unlabeled and labeled UDP-sugar. Compare results from +Hase (generally between 60% and 100% of the HA-dependent counts should be removed by Hase treatment) and -Hase, and reactions performed in the presence (+UDP-GlcNAc) and absence (-UDP-GlcNAc) of UDP-GlcNAc.

3.4. Metabolic Labeling with [3H]-Glucosamine or [3H]-Acetate

1. Aspirate medium and wash cell cultures briefly with PBS.

2. Replace cell culture medium with medium containing [3H]-glucosamine (20 ^Ci/mL of medium) or [3H]-acetate (100 ^Ci/mL of medium). Return cultures to the incubator and incubate for the desired amount of time for the study in question (see Note 21).

3. Collect medium, containing free radiolabeled glucosamine or acetate and radiolabeled mac-romolecules (including HA), and transfer to a 15-mL conical tube (see Note 22). Wash twice with PBS (2 mL for a single well of a 6-well plate) and add these wash solutions to the previously collected medium. This final solution represents the released or "cell-free HA."

4. Cell-surface HA can also be collected by trypsinization. This removes most of the remaining cell-surface localized HA. Trypsinize cells per the requirements for the cell line under study. Neutralize with complete medium containing serum, and pellet cells by centrifugation. Remove the supernatant to a fresh tube. This will represent the cell surface HA. If desired, this solution can be pooled with the cell-free HA, if total extracellular HA is being monitored. Alternatively, this solution can be treated separately as, "cell-surface HA."

5. Cell number should be determined using, for instance, a hemocytometer or Coulter counter.

6. Three volumes of 1.3% potassium acetate/95% ethanol are added to each solution and the samples are mixed and placed at -20°C overnight.

7. Total macromolecules are collected by centrifugation at 2500g for 15 min at 4°C.

8. Precipitates are diluted in 500 ^L of 0.5% (w/v) protease XIV (Sigma) solution (in 100 mM Tris-HCl, pH 8.0) and incubated on a rocking platform overnight at 37°C (see Note 23).

9. Three volumes of 1.3% potassium acetate/95% ethanol are added to each sample. Samples are mixed well and placed at -20°C overnight.

10. Macromolecules are collected by centrifugation at 2500g for 15 min at 4°C.

11. Precipitates are resuspended in 1 mL of 20 mM sodium acetate, pH 6.0.

12. Five TRU (12.5 ^L) of Streptomyces hyaluronate lyase, or 12.5 ^L of 20 mM sodium acetate, pH 5.0, are added to duplicate 200-^L aliquots of each sample, and tubes are incubated overnight at 60°C (see Note 24).

13. 50-^L (0.25 volume) of 12% CEPC solution are added to each tube. Tubes are mixed well by vortexing and incubated at 37 °C for 1 h.

14. Samples are centrifuged for 15 min at 10,000g at 4°C.

15. Pellets are washed with 500 ^L of 0.05% CEPC, 50 mMNaCl, and spun again as described in step 13.

16. Final pellets can be resuspended in 150 ^L of methanol and added directly to scintillation vials containing liquid scintillant, and analyzed by liquid scintillation counting (see Note 25). Comparison of Hase-treated and mock-treated will give a measure of the total HA-dependent counts in each sample. HA-dependent counts should be related to the cell density in the respective cultures.

17. Alternatively, if an indication of the relative length of labeled HA chains is desired, final pellets can be resuspended in a buffer suitable for size-exclusion chromatography.

4. Notes

1. Many established and primary cell lines synthesize significant amounts of HA through endogenous HA synthase activities. These include most embryonic fibroblasts, such as 3T3 and 3T6 cells, the mouse oligodendroglioma cell line (G26-24), human lung fibroblasts (WI38), primary keratinocytes and dermal fibroblasts, and various chondrocyte cultures. Most of these cell lines (for instance, 3T6, G26-24, WI38, and dermal fibroblasts) express predominantly Has2 (Spicer, unpublished data). There are several important cell lines however, that synthesize reduced levels or no detectable HA. These include the SV40-trans-formed African green monkey kidney cell lines, COS-1 and COS-7 (no detectable activity), human embryonic kidney (HEK293) cell line (no detectable activity), and various Chinese hamster ovary lines (low levels). The latter group of cell lines has proven extremely useful for functional screening of putative HA synthases through transfection experiments (6,8,16).

2. Endogenous HA synthase activities can be markedly affected by cell density (12). In general, HA synthase activity is highest in subconfluent, proliferating cultures and lowest in confluent, growth-arrested cultures (12). This may in part reflect regulation of HA synthase messenger RNA levels (13). Thus, the cell density is an important consideration in the overall experimental design. Any variations in cell density from one experiment to the next should, therefore, be noted.

3. Measurements of metabolically labeled HA can also be affected by receptor-mediated uptake and degradation (through hyaluronidase action). Hence, measurement of meta-bolically labeled HA may reflect the steady-state HA levels resulting from both synthesis and degradation. In cell cultures that are not actively endocytosing and degrading HA, extracellular (in the cell culture medium) HA levels may climb steadily and reach a steady-state level at confluence. In contrast, in those cell cultures that are actively endocytosing and degrading HA, extracellular HA levels may start to decline at confluence as the balance between synthesis and degradation is likely to shift in favor of uptake and degradation.

4. Some cell lines can assemble a HA-dependent pericellular matrix from exogenously supplied HA. This is dependent on the presence of proteoglycans and upon the expression of a cell surface HA receptor such as CD44 (14).

5. When viewing HA-dependent pericellular matrices in cell cultures grown in small wells, the cell density can vary across the well. In particular, cell densities may be much higher at the periphery of the well. Thus, it is important to consider local fluctuations in cell density when scanning a well.

6. Computer programs such as NIH-Image (http://rsb.info.nih.gov/nih-image/index.htmL) can be used very effectively to determine the relative areas of the HA-dependent pericellular matrix and the cell, either from directly captured images, or from scanned images of traced cells. Using NIH-Image, for instance, the area corresponding to the cell only is filled and the area calculated. Next, the pericellular matrix plus the cell are filled and the total area calculated. Data can be imported into a spreadsheet program such as Microsoft Excel for rapid calculation of ratios.

7. It is not necessary to remove the erythrocytes from each well during treatment with Streptomyces hyaluronate lyase.

8. The number of plates that will be necessary for a given experiment will be determined in part by the characteristics of the cell or cells under study. Transfected COS cells can be processed at 72 h posttransfection, at which time most cultures will be confluent. Under these conditions, 3 plates will usually yield between 100 and 200 ^g of crude cell membranes.

9. The method detailed here describes the relatively small-scale preparation of membranes for in vitro HA synthase assay. If larger amounts of membranes are required, a larger Dounce homogenizer may be used along with 15-mL conical tubes and centrifuge tubes suitable for use in a larger centrifuge, rather than screw-capped microcentrifuge tubes. In these instances, cell suspensions from multiple 15-cm plates, or from roller bottles, can be homogenized at the same time.

10. It is vitally important to ensure that plates sit flat on the ice, such that there are no "dry" spots on the plate. Do not leave the plates empty for any length of time between washes.

11. When scraping the monolayer into the LB+, do not scrape back and forth. Instead, work systematically across the plate, from left to right.

12. It may be necessary to determine the optimal number of strokes required to break open cells leaving nuclei intact. This can be achieved through transferring a drop of the homogenate to a microscope slide, cover slipping, and observing under low (4x objective) and high (20x objective) magnification. Accept a lower efficiency of cell breakage rather than risk damage to nuclei. Chromatin released into the homogenate can induce aggregation of membrane vesicles and dramatically reduce yields.

13. Resuspended membrane preparations retain significant enzyme activity even if freeze-thawed up to 3-4 times. However, for maximum enzyme activity and reproducibility, membrane preparations should be aliquotted into 25- to 50-^L aliquots (depending on protein content) and stored at -80°C.

14. Addition of SDS is important in order to solubilize the cell membranes prior to the BCA assay. Likewise, it is important to serially dilute any standards (such as BSA), which may be used for the generation of a standard curve, in the same buffer (LB plus 1% SDS).

15. The synthase buffer described here contains Mg2+ as the cation. The vertebrate HA synthases and the Streptococcal enzyme prefer Mg2+. However, if this procedure is used to assay putative invertebrate or prokaryotic HAS enzymes, alternate cations, such as Mn2+, might be considered before a membrane preparation or cell lysate is considered negative for HA synthase activity. Similarly, alternative reaction conditions, such as pH, temperature and substrate concentration, should be considered.

16. It is easiest to assemble reactions in 2-mL screw-capped tubes, as several rounds of heating and boiling are required for the whole procedure.

17. Assemble at least one specificity-control reaction in which UDP-GlcNAc (or the unlabeled sugar) is omitted from the reaction. HA synthesis requires both UDP-GlcA and

UDP-GlcNAc. Hence, any labeled product generated in the absence of UDP-GlcNAc is, by definition, not HA.

18. Place chromatography strips into the chromatography chamber as follows: the first fold (2.5 cm from the top of the strip) is placed under the glass rod; the second fold (6 cm from the top of the strip) takes the strip over the rail and allows it hang in a vertical manner. Once all strips have been placed into the chamber, solvent is added to the top of the reservoir.

19. If multiple chromatography strips are eluted at the same time, ensure that adjacent strips do not touch at their origins.

20. Set up the scintillation counter such that counts achieve 95% confidence, or proceed for 10 min, whichever comes first.

21. Generally, cells grown in individual wells of a 6-well tissue-culture plate will yield sufficient labeled HA over a 24- to 48-h period.

22. Approximately 70% or more of the radiolabeled HA synthesized by a given cell line will be found in the medium. Most of the remaining HA will be associated with the cell surface. This may occur through several mechanisms, including continued interaction with the HA synthase, interaction with specific cell-surface HA receptors, or through incorporation into the pericellular matrix through homophilic interaction with other HA chains, or through heterophilic interaction with specific HA-binding proteins. If desired, the intracellular HA fraction, a small percentage of the total HA, can be collected through lysis of the cell pellet using, for instance, treatment with a PBS solution supplemented with 0.5-1% (w/v) Triton X-100 or NP-40. The recent report demonstrating intracellular HA and its dynamic distribution during the cell cycle (15) suggests that intracellular HA may play previously unimagined roles in cell biology.

23. Protease XIV treatment will digest any protein molecules that may carry covalently linked, radiolabeled carbohydrate chains.

24. As molecules other than HA will be metabolically labeled by culture in the presence of [3H]-glucosamine or [3H]-acetate, it is important to determine the amount of radiolabeled HA in each sample. This can be determined by treatment of part of each sample with Streptomyces hyaluronate lyase.

25. Liquid scintillation counting will provide an accurate measure of the amount of HA produced by a given cell line. Size-exclusion chromatography, for instance as described (16), can provide a measure of the amount of HA and the relative molecular mass of the chains.

References

1. Weissman, B. and Meyer, K. (1954) The structure of hyalobiuronic acid and of hyaluronic acid from umbilical cord. J. Am. Chem. Soc. 76, 1753-1757.

2. Philipson, L. H. and Schwartz, N. B. (1984) Subcellular localization of hyaluronan synthetase in oligodendroglioma cells. J. Biol. Chem. 259, 5017-1023.

3. Prehm, P. (1984) Hyaluronate is synthesized at plasma membranes. Biochem. J. 220, 597-600.

4. Weigel, P. H., Hascall, V. C., and Tammi, M. (1997) Hyaluronan synthases. J. Biol. Chem. 272, 13,997-14,000.

5. Weigel, P. H. (1998) Bacterial hyaluronan synthases. Hyaluronan Today Internet Glycoforum, http://www.glycoforum.gr.jp/science/hyaluronan/HA06/HA06E.html.

6. Spicer, A. P., and McDonald, J. A. (1998) Characterization and molecular evolution of a vertebrate hyaluronan synthase (HAS) gene family. J. Biol. Chem. 273, 1923-1932.

7. Spicer, A. P., Seldin, M. F., Olsen, A. S., Brown, N., Wells, D. E., Doggett, N. A., Itano, N., Kimata, K., Inazawa, J., and McDonald, J. A. (1997) Chromosomal localization of the human and mouse hyaluronan synthase (HAS) genes. Genomics 41, 493-497.

8. Itano, N., Sawai, T., Yoshida, M., Lenas, P., Yamada, Y., Imagawa, M., Shinomura, T., Hamaguchi, M., Yoshida, Y., Ohnuki, Y., Miyauchi, S., Spicer, A. P., McDonald, J. A., and Kimata, K. (1999) Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties. J. Biol. Chem. 274, 25,085-25,092.

9. DeAngelis, P. L. and Weigel, P. H. (1994) Immunochemical confirmation of the primary structure of streptococcal hyaluronan synthase and synthesis of high molecular weight product by the recombinant enzyme. Biochemistry 33, 9033-9039.

10. Ohya, T. and Kaneko, Y. (1970) Novel hyaluronidase from Streptomyces. Biochim. Biophys. Acta 198, 607-609.

11. Clarris, B. J. and Fraser, J. R. (1968) On the pericellular zone of some mammalian cells in vitro. Exp. Cell Res. 49, 181-193.

12. Matuoka, K., Namba, M., and Mitsui, Y. (1987) Hyaluronate synthetase inhibition by normal and transformed human fibroblasts during growth reduction. J. Cell Biol. 1104, 1105-1115.

13. Watanabe, K. and Yamaguchi Y. (1996) Molecular identification of a putative human hyaluronan synthase. J. Biol. Chem. 271, 22,945-22,948.

14. Knudson, W., Bartnik, E., and Knudson, C. B. (1993) Assembly of pericellular matrices by COS-7 cells transfected with CD44 lymphocyte-homing receptor genes. Proc. Natl. Acad. Sci. (USA) 90, 4003-4007.

15. Evanko, S. P. and Wight, T. N. (1999) Intracellular localization of hyaluronan in proliferating cells. J. Histochem. Cytochem. 47, 1331-1342.

16. Brinck, J. and Heldin, P. (1999) Expression of recombinant hyaluronan synthase (HAS) isoforms in CHO cells reduces cell migration and cell surface CD44. Exp. Cell Res. 252, 342-351.

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