Iozzo Renato V. Proteoglycan Protocols

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86 Shworak

that the pellet formation site is oriented to the front. The GAG pellets are extremely small

and very difficult to see. At best, they might only be detected as a slightly cream-colored

hazy line when viewed from a tangential aspect. If extremely high incorporation is achieved,

the

S generates sufficient secondary Bremsstrahlung photons to detect the pellet with a

hand-held sodium iodide tube. Carefully aspirate off ethanol to generate a nearly “dry”

pellet, (see Note 20).

5. Dissolve GAG. Place 10 µL of water onto the pellet of each tube and sit for 10 min. Add

50 µL of water to the first tube, vortex briefly and centrifuge, then transfer the contents to the

second tube, vortex, and spin. Repeat until the forth tube contains the entire 90 µL. Repeat

this process with a 49-µL water wash. Remove 0.5 µL and determine radioactivity by

liquid-scintillation counting. Typically, ~70% of the DEAE

S is recovered.

6. Chondroitinase treatment. The chondroitin component is eliminated by GAG lyase

digestion in the reaction shown in Table 4. Incubate the reaction at 37°C for 1 h, add an

additional 4 µL of chondroitinase ABC and incubate an additional hour.

7. Purify HS. The reaction proteins are next removed by phenol extraction followed by

ethanol precipitation to purify the

S-labeled HS away from chondroitin degradation prod-

ucts: Add 1.2 mL of PCI to the reaction, cap, and vortex at maximum for 30 s. Separate the

phases by centrifugation at 10,000g, room temperature, for 2 min. Transfer the aqueous

phase to a 2-mL screw-cap tube containing 4 µL of 5 M NaCl. To enhance recovery, add

50 µL of water to the initial reaction tube, vortex, spin, then recover and pool with the

first aqueous phase (~200 µL total volume). Add 1 mL of absolute ethanol, centrifuga-

tion at 10,000g, room temperature, for 30 min and aspirate, as described in Note 20. Add

200 µL of 0.0005% Triton X-100, let sit at room temperature for 10 min, then resuspend

by two quick vortex/spin cycles. Remove 0.5 µL and determine radioactivity by liquid-

scintillation counting. Snap-freeze sample on liquid nitrogen and store at –80C°.

4. Notes

1. The described recipe maximizes L cell production of HS. Others might find it more

convenient to use a preformulated medium such as sulfate-free BME (Life Technologies,

# 31300) or MEM (Life Technologies, # 22300). The medium is CO

equilibrated to

maximize reproducibility. Equilibrate the medium prior to the addition of

S so as to

minimize the possibility of contamination.

2. High levels of

S can be safely manipulated when diligent respect is paid to the three

basic principles governing radioactive lab work: (1) minimize personal exposure time

and (2) use appropriate shielding. In particular, always consider the secondary γ-radia-

Table 4

Chondroitinase ABC Reaction

Stock solution Final concentration Required volume

Entire

S-labeled GAG sample 139 µL

5 M NaCl 30 mM 0.9 µL

1 M ammonium acetate (pH 7.0) 25 mM 3.75 µL

10 mg/mL BSA 67 µg/mL 1 µL

2 mg/mL Glycogen 13 µg/mL 1 µL

5 U/mL Chondroitinase ABC 20 mU/reaction 4 µL

Total volume: 150 µL

Preparation of High–Specific-Activity Heparan Sulfate 87

tion emissions, as this is an underappreciated point for high-level

S work. High-level

stocks of

S should always be stored in lead containers. (3) Monitor for contamination.

Individuals who use levels greater than 5 mCi at once should wear a radiation badge and

should undergo a urine test shortly after use, to rule out personal contamination. The

institution’s radiation protection office should be consulted in advance regarding the poli-

cies and procedures for use and disposal of such materials.

3. Certain lots of CHAPS (3-[(3-chloamidopropl)-dimethylammonio]-1-propanesulfonic

acid) contain an impurity that causes a gradual precipitant formation, which clogs col-

umns. To test for the contaminant, store the 18% CHAPS stock at 4°C for 1 wk. If a

precipitate has formed, then the supplier must send a replacement from a different lot.

4. Solubility of guanidine

·HCl is highly dependent on purity. High quality material is readily

soluble at 4 M, whereas less expensive grades are insoluble.

5. This treatment of the column matrix blocks “nonspecific” sites that will irreversibly bind

GAG. Consequently, quantitative recover of extremely small amounts of radiolabeled

proteoglycans (<1 fmol or <200 pg of a 200-kDa molecule) can be obtained.

6. Under these conditions the enzyme can withstand multiple freeze–thaw cycles. PBS must

be used; if water is used the activity is destroyed by the freezing process.

7. Wear two sets of latex gloves to minimize risk of radioactive contamination. It is advisable to have

a separate pipet gun dedicated for radioactive work, as this equipment is easily contaminated.

8. This recipe makes sufficient medium to cover a monolayer in a flask that has an actual

growth surface area of <75 cm

. Some flasks have larger surfaces, which can lead to drying

out of the monolayer. In this case, the use 5 mL base medium, 0.5 mL fetal bovine serum,

5.75 µL of 0.1 M Na

, and 200 mCi of Na

9. The labeling mixture has a total sulfate concentration of ~200 µM. This is the minimum

concentration to prevent undersulfation of GAGs in L cells (12); thus,

S-labeled HS is

of normal structure and maximal specific activity. The formulation has only three sulfate

sources, fetal bovine serum, cold sodium sulfate, and carrier-free Na

which respec-

tively provide about 70, 30, and 100 µM. Antibiotics are purposefully omitted, as these

can contribute substantial amounts of sulfate, which will reduce net incorporation. Expect

~2 × 10

cpm of

S-labeled HS. If reduced radioactivity is required, decrease the input

and compensate with cold carrier to maintain total sulfate at 200 µM.

10. Certain cell types (e.g., CHO) efficiently salvage sulfate from amino acid degradation and

do not generate undersulfated GAG, even when grown in sulfate-free media (13,14). For

such cells, efficient incorporation can be obtained with lower external sulfate concentration.

CHO cells can be labeled using 4 mL of Ham’s F12 medium (contains 6 µM sulfate)

supplemented with either 10% fetal bovine serum and 100 mCi Na

, or dialyzed

fetal bovine serum and 8 mCi Na

(both conditions employ a specific activity of

~270,000 Ci/mol, so comparable incorporations are obtained).

11. It is important that the incubator shelf be level so that the cell monolayer does not dry out.

The flask is sealed to prevent off-gassing of volatile

S-labeled metabolites, which certain

cell types can generate. If sealing is insufficient, then such compounds may be captured by

placing the flask on a bed of activated charcoal during the incubation period. After the

incubation, monitor the incubator to ensure that contamination has not occurred.

12. Multiple rinses are necessary to remove “free”

S, which can copurify with GAG and

confound later steps. The majority of the input radioactivity is contained within the cul-

ture medium and the first two washes. Certain institutions require that high-level radio-

active waste be kept in a minimal volume. For this reason, it may be necessary to collect

this material separate from other contaminated waste fluids.

88 Shworak

13. This procedure was originally optimized to recover quantitatively small levels of labeled

proteoglycans from L cells, but can be modified for proteoglycans from other sources.

The loading and initial washing pH facilitate removal of acidic proteins, whereas the urea

disrupts protein complexes and prevents hydrophobic interactions with the column matrix.

These very stringent conditions may not allow for efficient binding of free GAG chains,

or proteoglycans with GAG chains that are short, undersulfated, or derive from GAG

biosynthetic mutants. To optimize recoveries for problem samples, reduce the NaCl con-

centration in both UAW and the preparative load buffer (equivalent proportional

reductions), then monitor total

S recovery and its corresponding susceptibility to diges-

tion with GAG lyases. An optimal NaCl level maximizes the recovery of lyase-sensitive

material while minimizing non-lyase-sensitive material.

14. Elution is performed by the application of multiple small aliquots so as to reduce the flow

rate of the eluate. This maximizes sample recovery and minimizes sample volume.

15.Watch the cap to ensure that the O-ring becomes compressed during closing. A perfect seal

is required to prevent leakage of sample during vortexing.

16.Classically, β-elimination is performed under basic conditions with high concentrations of

borohydride. The base cleaves the glycosidic bond to a substituent that is in a β-orientation

relative to an electron-withdrawing group. For proteoglycans, cleavage occurs between the

linkage region xylose and the corresponding serine residue of the core protein. This works

best with intact proteins rather than proteolytically degraded material, since cleavage cannot

occur if the carboxyl group of the serine is free. High borohydride is included in general

polysaccharide analysis to prevent the “peeling reaction” — a sequential cyclic process

whereby the terminal sugar undergoes oxidative modification followed by β-elimination.

High borohydride is not required for proteoglycans, since the linkage xylose does not

undergo peeling. Do not use high levels of borohydride, as violent bubbling occurs upon

neutralization, which will cause extensive radioactive contamination.

17. To maximize recovery, hold the tube vertical and remove the aqueous phase by drawing with

a P-1000 pipetter from the gradually descending meniscus. When the meniscus approaches

the interphase, stop and transfer the harvest to a clean tube. Next, tip the tube on a ~45° angle

to break the surface tension between the aqueous and organic phases (slight tapping of the tube

may be required). The aqueous phase will detach from the lower tube wall and form a ball on

the uppermost surface. Place a P-200 tip inside the ball and withdraw solution until the tip gets

close to the protein interphase. Pool the recovered material.

18. Efficient (i.e., > 90% recovery) precipitation of GAG requires a minimum of 4.5 volumes of

ethanol, as well as carrier glycogen and a minimum of 100 mM NaCl. In high ethanol,

sodium has low solubility. The described protocol dilutes the sample so that the salt remains

in solution during the precipitation of GAG.

19. To prevent pellet loss, it is best not to let the tubes sit for any length of time. It is preferable

to turn the centrifuge off just short of 30 min, so that the samples can be removed the instant

the centrifuge stops.

20. Slow aspiration is necessary to prevent pellet loss. This is accomplished with a 25-gage

needle connected to the vacuum line. For right handed people, tubes are held in the left hand

with the pellet positioned to the right side (providing a side-on view of the pellet). Initially,

hold tubes vertically and maintain the needle bezel at the level of the meniscus and parallel to

the tube wall. Always point the needle opening away from the pellet. Aspiration speed is

controlled by slowly lowering the needle, and by slightly pressing the bezel against the

tube wall. A slow speed ensures complete fluid removal without leaving behind residual

drops. As the meniscus approaches the pellet region, start tilting the tube to the right so

Preparation of High–Specific-Activity Heparan Sulfate 89

that the pellet is under fluid for as long as possible. Ultimately, the tube will be nearly

horizontal with the pellet at the lowest point and the bezel will be a pressed against the

opposite tube wall directly above the pellet.

References

1. Bame, K. J. and Esko, J. D. (1989) Undersulfated heparan sulfate in a chinese hamster

ovary cell mutant defective in heparan sulfate N-sulfotransferase. J. Biol. Chem. 264,

8059–8065.

2. Bame, K. J., Lidholt, K., Lindahl, U., and Esko, J. D. (1991) Biosynthesis of heparan

sulfate. Coordination of polymer-modification reactions in a chinese hamster ovary cell

mutant defective in N-sulfotransferase. J. Biol. Chem. 266, 10,287–10,293.

3. Shworak, N. W., Shirakawa, M., Colliec-Jouault, S., Liu, J., Mulligan, R. C., Birinyi, L. K.,

and Rosenberg, R. D. (1994) Pathway-specific regulation of the synthesis of anticoagulantly

active heparan sulfate. J. Biol. Chem. 269, 24,941–24,952.

4. Ozeran, J. D., Westley, J., and Schwartz, N. B. (1996) Kinetics of PAPS translocase:

evidence for an antiport mechanism. Biochemistry 35, 3685–3694.

5. Klaassen, C. D. and Boles, J. W. (1997) Sulfation and sulfotransferases 5: the importance of

3'- phosphoadenosine 5'-phosphosulfate (PAPS) in the regulation of sulfation. Faseb J.

11, 404–418.

6. Sjoberg, I. and Malmstrom, A. (1982) Biosynthesis of dermatan sulphate in cultured fibro-

blasts. Characterization of newly synthesized glycans from cells and microsomes. Eur. J.

Biochem. 128, 29–34

7. Kimura, J. H., Caputo, C. B., and Hascall, V. C. (1981) The effect of cycloheximide on

synthesis of proteoglycans by cultured chondrocytes from the Swarm rat chondrosarcoma.

J. Biol. Chem. 256, 4368–4376

8. Shworak, N. W., Fritze, L. M. S., Liu, J., Butler, L. D., and Rosenberg, R. D. (1996)

Cell-free synthesis of anticoagulant heparan sulfate reveals a limiting activity which modi-

fies a nonlimiting precursor pool. J. Biol. Chem. 271, 27,063–27,071.

9. Liu, J., Shworak, N. W., Fritze, L. M. S., Edelberg, J. M., and Rosenberg, R. D. (1996)

Purification of heparan sulfate

D-glucosaminyl 3-O-sulfotransferase. J. Biol. Chem. 271,

27,072–27,082.

10. Liu, J., Shworak, N. W., Sinay, P., Schwartz, J. J., Zhang, L., Fritze, L. M., and Rosenberg,

R. D. (1999) Expression of heparan sulfate

D-glucosaminyl 3-O-sulfotransferase isoforms

reveals novel substrate specificities. J. Biol. Chem. 274, 5185–5192.

11. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory

Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, MA.

12. Humphries, D. E., Silbert, C. K., and Silbert, J. E. (1986) Glycosaminoglycan production by

bovine aortic endothelial cells cultured in sulfate-depleted medium. J. Biol. Chem. 261,

9122–9127.

13. Keller, J. M. and Keller, K. M. (1987) Amino acid sulfur as a source of sulfate for

sulfated proteoglycans produced by Swiss mouse 3T3 cells. Biochim. Biophys. Acta

926, 139–144.

14. Esko, J. D., Elgavish, A., Prasthofer, T., Taylor, W. H., and Weinke, J. L. (1986) Sulfate

transport-deficient mutants of Chinese hamster ovary cells. Sulfation of glycosaminoglycans

dependent on cysteine. J. Biol. Chem. 261, 15,725–15,733.

Detection of Sulfotransferase Isoforms 91

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

Selective Detection of Sulfotransferase Isoforms

by the Ligand Affinity-Conversion Approach

Nicholas W. Shworak

1. Introduction

1.1. Perspective

The profound functional diversity of heparan sulfate (HS) proteoglycans is largely

engendered by the regulated structural complexity of the glycosaminoglycan (GAG)

component (reviewed in ref. 1). Ultimately, the status of the heparan sulfate biosyn-

thetic machinery dictates fine structure that encodes binding sites for protein ligands.

In part, regulation of fine structure is made possible through the existence of multiple

isoforms for most of the HS sulfotransferases (2,3). Examination of the heparan sul-

fate 3-O-sultotransferase (3OST) family shows that each isotype exhibits a unique

sequence specificity (4). All 3OST enzymes place a sulfate group in the 3-O-position

of glucosamine residues within heparan sulfate; however, each isoform recognizes

and modifies a distinct sequence context. Consequently, only 3OST1 generates the

antithrombin-binding site, whereas 3OST3 makes binding sites for gD, an envelope pro-

tein of herpes virus (4,5). The existence of a multitude of sulfotransferases necessitates a

facile approach for the specific detection of individual isoforms present in crude extracts.

This chapter describes establishing a ligand affinity-conversion assay, based on a

procedure that has been used to measure selectively the activity of 3OST1 (6). Exten-

sive suggestions are included to assist others in the application of this approach to

other glycosaminoglycan biosynthetic enzymes. Thus, the methods should be viewed

as modifiable elements of a general approach to measure the sequence specific activ-

ity of sulfotransferases that generate binding sites for protein ligands.

1.2. Principle, Advantages, and Limitations of the Ligand

Affinity-Conversion Approach

The ligand affinity-conversion approach is based on two tenets that permit a

two-step assay. (1) An appropriately selected HS source will contain biosynthetic pre-

92 Shworak

cursors for the binding site of a protein ligand. Precursors are “partially” synthesized

structures that require modification by a single sulfotransferase to generate a func-

tional binding site. Fulfillment of this criterion enables the cell-free synthesis of ligand-

binding sites. Thus, cell extracts containing the sulfotransferase of interest are incubated

with

S-labeled HS and the sulfate donor adenosine 3'-phosphate 5'-phosphosulfate

(PAPS). The sequence specific action of the enzyme converts precursors into func-

tional binding sites. (2) The affinity of the ligand for its completed binding site is

substantially higher than for the corresponding precursor structure(s). Such a ligand is

used in the second assay step to capture the newly generated, high-affinity portion of

S-labeled HS. Thus, sulfotransferase activity is indirectly quantified through the produc-

tion of HS that contains high-affinity ligand-binding sites. The measured enzyme activity

is expressed in arbitrary units of affinity conversion.

The pros and cons of this technique are determined by the reaction conditions,

which differ from typical HS sulfotransferase assays. Classically, activity is deter-

mined directly by quantifying the transfer of radioactivity from the labeled sulfate

donor (PAP

S) onto various GAG acceptors (7). This approach is quite facile for

measuring the abundant N-sulfotransferases (8) but detection of rare enzymes in

crude cell extracts requires extensive modifications (9). Subcellular fractionation is

frequently required to overcome the high levels of background incorporation, which

result from endogenous sulfotransferases and sulfate acceptors contained within

whole cell extracts. The affinity-conversion approach eliminates high backgrounds

by using

S-labeled HS in conjunction with unlabeled PAPS. This arrangement

allows for the selective detection of a specific sulfotransferase activity within even

crude cell extracts (6).

The

S-labeled HS substrate is generated by metabolically labeling cells with

, diluted to any desired specific activity. Indeed, cells can even be labeled

with undiluted Na

to generate a substrate of ~1.2 × 10

Ci/mol (6). Consequently,

cell-free reactions can contain as little as 350 attomol (10

-18

mol) of substrate and can

detect as little as 0.35 attomol of product (6). In contrast, sensitivity of the classic

assay is restricted by the specific activity of PAP

S. Micromolar concentrations are

require for enzyme activity, which limits practical specific activity to ~400 Ci/mol

(8,10) and minimum detection to ~50 pmol of product. Thus, the affinity-conversion

approach also provides for tremendous sensitivity.

It is important to note that assay specificity can be limited by the structural heteroge-

neity of HS, which allows HS to function as a substrate for multiple sulfotransferases.

(The affinity-conversion assay minimizes this problem by exploiting a protein ligand to

focus detection on only the reaction(s) that generate the ligand’s binding site.) Neverthe-

less, it is possible that a ligand-binding site can be generated by distinct enzymes that

modify different precursor structures. Thus, a single HS sample may contain multiple

precursor structures and so could detect the multiple activities. Clearly, care must be

taken in characterizing extracts from novel sources and in identifying the particular HS

sample that is most selective for monitoring the enzyme of interest. In this regard, cell

mutants that are defective in the production of specific ligand binding sites (11,12) may

serve as idea sources for an exquisitely selective HS substrate.

Detection of Sulfotransferase Isoforms 93

2. Materials

Refer to Chapter 9, Subheading 2.1., for required general materials.

2.1. Generation of Crude Cell Extract

1. A cell line that contains the enzymatic activity of interest.

2. PBS and trypsin/EDTA. See Chapter 9, Subheading 2.2., item 3.

3. Lysis/sample buffer (100 mL): 0.25 M sucrose, 1% Triton X-100, make from stocks of 1

M sucrose (autoclaved) and 20% Triton X-100. Store at 4°C.

4. Bradford reagent (Bio-Rad).

2.2. Cell-Free Synthesis of Ligand-Binding Sites

1. 1 M MES, pH 7.0 (100 mL). The sodium salt of 2-[N-morpholino]ethanesulfonic acid

(MES) is obtained from Sigma. Solutions are stored in a dark bottle at 4°C.

2. 20% Triton X-100; 10 mg/mL BSA and 20-mg/mL chondroitin sulfate. See Chapter 9,

Table 2 and Subheading 2.3., item 5.

3. 2 M MnCl

(15 mL). Filter sterilize and store at room temperature.

4. 1 M MgCl

(15 mL). Filter sterilize and store at room temperature.

5. 10-mg/mL protamine chloride (Sigma) (10 mL). Aliquots (1 mL) are stored at –20°C.

6. 25 mM PAPS (0.5 mL). 3'Phosphoadenosine 5' phosphosulfate (PAPS) (Sigma). Dis-

solve in ethanol/water (50/50 v/v), make 25-µL aliquots, snap-freeze on liquid nitrogen,

and store at –80°C, see Note 1.

S-labeled heparan sulfate. See Note 2.

8. Cell extract. From the method of Subheading 3.1.

9. Quantitation standard of sulfotransferase of interest, see Note 3.

10. Standard buffer. The buffer for quantitation standards, see Note 4.

11. Sample buffer. The cell extract lysis buffer, see Subheading 2.1., item 3.

12. 96-well PCR tube plates with sealing tape (Stratagene #410088 and #410152) are ideal for

analysis of large numbers of samples.

13. A PCR cycler (optional). Convenient for incubations of large sample sets. Machines with

heated lids are preferred to prevent sample evaporation and lid condensation.

14. 167 mM NaCl, 13.3-µg/mL glycogen (10 mL). Store at room temperature.

15. A centrifuge capable of holding 96-well plates.

16. A multichannel pipetter (for analysis of large sample sets).

2.3. Quantitation of Protein-Heparan Sulfate Complexes

by Solid-Phase Capture

1. 96-Well dot-blot apparatus (Schleicher and Schuell).

2. Whatman 3 mm paper.

3. Nitrocellulose membrane (BA85, Schleicher and Schuell).

4. 6 × binding buffer (15 mL). 60 mM Tris-HCl, pH 8.0, 3 M NaCl, 1.2-mg/mL BSA, store

at 4°C. (see Note 5).

5. Wash buffer (100 mL). 10 mM Tris-HCl, pH 8.0, 0.5 M NaCl. (see Note 5).

6. A one-hole punch. Obtain from any stationary store.

3. Methods

Upon verification of two criteria, this assay can be tailored to detect the activity of

any HS sulfotransferase that creates a high-affinity binding site for a protein ligand.

First, the protein ligand of interest must preferentially bind either to a subpopulation

94 Shworak

of HS or to HS from only a subset of cell types. Second, two specific cell lines must be

identified—an expressive line that produces high levels of the ligand-binding site

and a nonexpressive line that produces little or no ligand-binding site. If necessary,

one might wish to mutagenize the former to create the latter (11,12). The nonexpressive

line is the source of the

S-labeled HS substrate, whereas the expressive cell line

supplies the crude extract with the sulfotransferase activity of interest (see Subhead-

ing 3.1.). These reagents are required for establishing conditions appropriate for

sulfotransferase activity (see Subheading 3.2.). The ligand of interest is used to frac-

tionate in-vitro-synthesized reaction products and thereby determine the percentage

S-HS chains containing newly generated binding sites (see Subheading 3.3.).

This last step requires the greatest degree of customization, and the reader is advised

to consider alternate approaches to measure protein·GAG interactions that are

described extensively in Part III. Once established, the assay can be used to moni-

tor sulfotransferase activity of crude cell or tissue extracts, or of derived purified

fractions.

3.1. Generation of Crude Cell Extract

1. Grow tissue culture cells in a 25-cm

flask to the desired growth state.

2. Wash monolayers twice with 2 mL of PBS (at room temperature), as described in Chapter

9, Subheading 3.1., step 5.

3. Remove cells by trypsinization. Add 1 mL trypsin and incubate at 37°C until cells round

up, (see Note 6). Add 3 mL of tissue culture medium containing 10% fetal bovine serum

and dislodge cells from the flask by tituration.

4. Wash cells. Transfer the suspension to a 15-mL conical tube containing 10 mL of PBS

(room temperature). Mix, then pellet cells by centrifugation at 500g for 5 min. Remove

as much supernatant as possible and resuspend the pellet in 1 mL of PBS. Transfer the

suspension to a 1.5-mL microfuge tube and pellet cells at 1000g for 5 min. Aspirate off

medium. Optional. If desired, pellets can be snap-frozen on liquid nitrogen and stored

at –80°C.

5. Lyse cells. Place pellets on ice and add 0.5 mL of 0.25 M sucrose with 1% Triton X-100

(ice chilled). Cells are disrupted by vortexing on high for at least 30 s. Sit on ice for 10

min, then vortex for an additional 30 s (see Note 7).

6. Pellet nuclei. Centrifuge 10,000g for 10 min, then transfer the crude lysate (supernatant)

to a clean tube.

7. Determine protein content by Bradford procedure (13), (see Note 8).

8. Snap-freeze samples on liquid nitrogen and store at –80°C.

3.2. Cell-Free Synthesis of Ligand-Binding Site

1. Reaction design. Reactions for experimental samples and for quantitation standards

should all contain the same components (see Table 1). The standard curve should have at

least six different enzyme concentrations as well as two zero enzyme (background con-

trol) points. The curve range should span the anticipated sample activities. All sample

reactions should contain equal amounts of cell extract protein, (see Note 9).

2. Reaction setup. Reactions are assembled on ice using individual 0.6-mL tubes for a small

number of samples, or a 96-well plate (with adhesive plastic lid) for a large sample set. First

pipet the required amounts of standard buffer and sample buffer (see Subheading 2.2

items 10 and 11). Then add the standards and individual samples.

Detection of Sulfotransferase Isoforms 95

3. Reaction mix. A 10% excess of reaction mix is generated, on ice, by combining the Table

2 ingredients in the listed order. (see Notes 10 and 11 regarding optimization for certain

sulfotransferases and sources of crude extract). After adding the mix to each reaction, tubes

are sealed, mixed by inversion (so as not to make bubbles), briefly centrifuged, then placed

in a 37°C incubator for 3 h to overnight, (see Note 12). If 96-well plates are employed, it

is convenient to incubate the reaction in a PCR cycler.

4. Removal of protein.

a. Small sample sets. To each tube, add 150 µL of 167 mM NaCl, 13.3 µg/mL glycogen

and 300 µL of PCI. Vortex each tube at maximum for 20 s, then separate the phases

by centrifugation at 10,000g, at room temperature for 2 min (see Chapter 9, Subhead-

ing 4, Notes 17 and 19). Remove as much aqueous (top) phase as possible (but do not

take interphase material) and transfer to a 1.5-mL screw-cap tube containing 1 mL of

absolute ethanol. Centrifugation at 10,000g, at room temperature, for 30 min and aspi-

rate, as described in Chapter 9, Subheading 4, Note 20. The “wet” pellet is directly

resuspended in ligand binding buffer (Subheading 3.3., step 1a). (see Note 13).

b. Large sample sets. Heat the plate in a PCR cycler at 95°C for 15 min to denature

proteins, then cool the plate to room temperature. Mix the samples by inverting the

plate and then centrifuge at maximum speed for 10 min to pellet the protein. Using a

multichannel pipetter, transfer 40 µL of supernatant to a clean tube plate.

3.3. Quantitation of Protein-Heparan Sulfate Complexes

by Solid Phase Capture

This procedure exploits the observation that nitrocellulose does not bind free HS but

does bind the protein component of a protein·HS complex. Consequently, filtration through

nitrocellulose can be used to specifically capture protein·HS complexes for quantitation.

1. Form protein·HS complexes.

a. Small sample set. Make a 1 X binding buffer (33 mL per sample provides a 10%

excess) by combining appropriate amounts of 6 X binding buffer and water with pro-

Table 1

Design of Assay Reactions

Standards Samples

Standard 0–10 µL0 µL

Standard Buffer 10–0 µL 10 µL

Subtotal: 10 µL 10 µL

Sample 0 µLX µL

Sample Buffer 10 µL 10–X µL

Subtotal: 10 µL 10 µL

Reaction Mix 30 µL 30 µL

Total: 50 µL 50 µL

96 Shworak

tein ligand (for determining appropriate ligand concentration see Note 14). Add

30 µL per tube, let sit 10 min, then lightly vortex samples (do not make bubbles) and

incubate at room temperature for at least 20 min.

b. Large sample set. A 5 X binding buffer is generated and 10 µL are added per sample.

Plates are capped with fresh sealing tape, mixed by inversion, briefly spun to pellet

liquid, and incubated at room temperature for at least 20 min.

2. Assemble dot blot apparatus. Cut two pieces of Whatman paper and one piece of nitrocel-

lulose membrane, sized for the apparatus (~11 cm × 8 cm), (see Note 15). On the filter

support plate, position both pieces of Whatman paper, on top of this place the nitrocellu-

lose membrane, and finally add and clamp down the sample manifold. Put 100 µL of

wash buffer in all of the wells, and let sit for 10 min to saturate the membrane. Apply a

gentle vacuum so as to just remove residual liquid from wells. Add 100 µL of wash buffer

to all wells that are to receive sample, and to all adjacent wells. Proceed to step 3. In the

meantime, capillary action will absorb much of the applied fluid.

3. Determine input radioactivity. Samples are centrifuged at maximum for 5 min to pellet

insoluble material. For each sample, remove 2.5 µL (small sample set) or 4 µL (large set),

mix with 3 mL of scintillant, and place in a liquid scintillation counter.

4. Capture complexes. All sample wells should still be wet (see Note 16). If any are dry, add

50 µL of wash buffer. Transfer 25 µL (small set) or 40 µL (large set) of each sample to it’s

respective manifold well. When applying, pump the pipet up and down once to mix the

sample with the preexisting well buffer. Apply a gentle vacuum. Once all wells are dry,

maintain the vacuum and rinse each sample well with 200 µL of wash buffer. Once dry,

remove vacuum, disassemble the apparatus, and place the membrane in a fume hood to

expedite air drying.

5. Quantitation of bound radioactivity. The manifold’s O-rings should leave an impression

in the membrane that reveals the location of each samples “dot.” A one hole punch is used

to excise each dot, which is placed in a 7 mL scintillation vial containing 3 mL of

scintillant. Radioactivity levels are then determined by liquid scintillation counting.

6. Calculate background-corrected percentage bound. For each reaction, first calculate the

percentage of

S-HS that is converted to ligand-bound material:

% bound = cpm bound (step 5) ÷ input cpm (step 3 cpm × 10) × 100%

Table 2

Mix Composition for a Single Reaction

Concentration

Stock Working Volume per reaction

O ~15.5 µL

MES, pH 7 1 M 50 mM 2.5 µL

Triton X-100 20% 1% 2.5 µL

MnCl

2 M 10 mM 0.25 µL

MgCl

1 M 5 mM 0.25 µL

Chondroitin sulfate 20 mg/mL 0.4 mg/mL 1 µL

BSA 10 mg/mL 1.2 mg/mL 6 µL

PAPS 25 mM 0.5 mM 1 µL

S-HS ~10

cpm/µL10

cpm ~1 µL

Total: 30 µL