O
-Linked Chains of Mucin 277
Gal residues (1,37,38). Most of the enzymes synthesizing blood group ABO, Lewis,
and other antigenic determinants act on O- and N-glycans as well as glycolipids. By
contrast, sialyltransferases often prefer one type of glycoconjugate (39). Two
sialyltransferase families, α3- and α6-sialyltransferase, act preferably on mucin-
type O-glycans.
α3-Sialyltransferase acts on Gal residues of cores 1 and 2 (Fig. 1, path l) (24,40,41).
The enzyme is developmentally regulated in thymocytes (42) and increased in leuke-
mia cells (43) and several cancer models (3,30). The α3-sialyltransferase has been
localized to medial and trans-Golgi compartments (44). The sialylation reaction cata-
lyzed by this enzyme has an important role in keeping O-glycan chains short and
sialylated. Since the enzyme acts relatively early in the O-glycan extension pathways
(Fig. 1, path l), it has the ability to compete with branching and elongation reactions.
Once core 1 is α3-sialylated, it is no longer a substrate for extension reactions
although it can still be converted to the disialylated core 1 by α6-sialyltransferase
(Fig. 1, path m).
The α6-sialyltransferase (Fig. 1, path j) that acts on GalNAc-R to form the
sialyl-Tn antigen, sialylα2-6GalNAc-Th/Ser (45), requires glycoproteins as substrate
and cannot act on GalNAc-benzyl or nitrophenyl substrates (46,47). However,
another type of α6-sialyltransferase (α6-sialyltransferase III) does not have a pep-
tide requirement, but is specific for the α3-sialylated core 1 structure (48). The
disialylated core structure can probably be synthesized by α6-sialyltransferase III
and other α6-sialyltransferases (Fig. 1, path m). Modifications of the sialic acid
residues of mucins include O-acetylation, catalyzed by specific O-acetyltransferases
acting in the Golgi (49).
The common sulfate ester linkages in mucins are SO
4
-6-GlcNAc and SO
4
-3-Gal.
Several types of sulfotransferases have been described that act on the 6-position of
GlcNAc (50) or the 3-position of Gal of core 1 (Fig. 1, path k) and N-acetyllactosamine
structures (51,52). Sulfated oligosaccharides appear to play an important role in cell
adhesion through binding to selectins and in the control of bacterial binding (53,54).
Sulfation also functions in directing the biosynthetic pathways of complex O-glycans
by blocking certain reactions. For example, sulfation of core 1 prevents the branching
reaction to form core 2 (51).
The enzymes catalyzing the reactions depicted in Fig. 1 assemble mucin-type
O-linked carbohydrate chains and are listed in Table 1, together with their substrates,
enzyme products, and the high performance liquid chromatography (HPLC) condi-
tions of product separation. Probably none of these enzymes are specific for mucins,
but also act on other glycoproteins that carry O-glycans, and can act on various glyco-
peptides with O-linkages. In vitro, many of these enzymes utilize synthetic compounds
as substrates in which the peptide chain is replaced by a hydrophobic group. The
substrate should be clean, specific, and easy to isolate in order to determine the
enzyme activity and specificity accurately. For a few enzymes, purified mucins with
defined glycosylation are available as substrates. However, mucins are usually too
heterogeneous in their carbohydrate structures, and therefore the use of synthetic com-
pounds with defined structure is preferred. In addition, it is much easier to determine
278 Brockhausen
the product structure of synthetic substrates as a proof of the assayed activity. When a
compound is a potential substrate for several glycosyltransferases present in the
enzyme preparation, or several reactions occur in sequence, the various products
have to be separated and identified. This can usually be achieved by HPLC. With the
exception of β1,6-GlcNAc-transferases, UDP-sugar binding enzymes require the pres-
ence of divalent metal ion for optimal activity. Thus, measuring a β6-GlcNAc-trans-
ferase activity in the presence of EDTA will eliminate the activity of other
GlcNAc-transferases potentially acting on the same substrate. Enzymes utilizing
CMP-sialic acid may be stimulated by metal ions but usually can act in their absence.
Unless they are released and secreted, or are produced as soluble recombinant
enzymes, glycosyltransferases are membrane-bound enzymes, and their activities are
stimulated by detergents.
A convenient way of identifying and quantifying glycosyltransferase products is by
the use of nucleotide-sugar donors that contain
14
C or
3
H-labeled radioactive sugar.
Similarly, the sulfate moiety of PAPS can be labeled with
35
S. Calculations of
sulfotransferase activities must take into account the relatively short half-life of
35
S (about 87 d).
2. Materials
2.1. Preparation of Enzymes
1. 0.25 M Sucrose.
2. 0.9% NaCl.
3. Potter-Elvehjem hand homogenizer.
4. Low-speed centrifuge (10,000g).
5. Ultracentrifuge (100,000g).
6. Small pieces of tissue, or cells.
2.2. Preparation of Substrates and Standard Compounds
1. Commercially available oligosaccharides: GlcNAc, GalNAcα-benzyl, Galβ1-3
GalNAcα-benzyl, GlcNAcβ1-3 GalNAcα-p-nitrophenyl [pnp], Galβ1-4 GlcNAc,
GlcNAcβ1-3 Galβ-methyl (Sigma, St. Louis MO); Galβ1-3 GalNAcα-pnp (Toronto
Research Chemicals, Toronto, Canada).
2. Thr-peptides, synthesized by Hans Paulsen, University of Hamburg, Germany
(15,55).
3. Frozen sheep submaxillary glands (Pel-Freez, Rogers, AR) to isolate ovine submaxillary
mucin (OSM), 0.1 N H
2
SO
4
, bovine testicular β-galactosidase (Boehringer, Laval,
Canada), 0.1 M Na-citrate buffer, Sephadex G25 column.
4. Components of enzyme assays to prepare product standards enzymatically GlcNAcβ1-3
GalNAcα-benzyl, GlcNAcβ1-6 (GlcNAcβ1-3) GalNAcα-pnp, GlcNAcβ1-3 Galβ1-4
GlcNAc, GlcNAcβ1-6 (GlcNAcβ1-3 Galβ1-3) GalNAcα-benzyl, GlcNAcβ1-6
(GlcNAcβ1-3) Galβ-methyl, sialylα2-3 Galβ1-3 GalNAcα-pnp, SO
4
-3 Galβ1-3
GalNAcα-benzyl, SO
4
-3 Galβ1-4 GlcNAc.
5. Enzymatically prepared substrates: GlcNAcβ1-6 (Galβ1-3) GalNAcα-benzyl, sialylα2-3
Galβ1-3 GalNAcα-pnp
O
-Linked Chains of Mucin 279
6. Nuclear magnetic resonance (NMR) and mass spectrometers, reagents for methylation
analysis.
2.3. Separation and Identification
of Glycosyltransferase and Sulfotransferase Products
1. HPLC apparatus.
2. HPLC columns C18, NH
2
(amine), PAC (cyano-amine).
3. Acetonitrile/water mixtures.
4. Dionex system for high-performance anion-exchange chromatography (HPAEC).
5. Bio-Gel P4 or P2 column (80 × 1.6 cm) (Bio-Rad, Hercules, CA).
6. Ion-exchange columns (AG1 × 8, 100–200 mesh, Bio-Rad).
7. High-voltage electrophoresis apparatus, 1% Na-tetraborate, Whatman No. 1 paper.
8. C18 Sep-Pak columns, methanol.
9. 0.05 M KOH/1 M NaBH
4
for β-elimination.
10. Scintillation fluid, scintillation counter.
2.4. Polypeptide
α
-GalNAc-Transferase Assays
1. 5% Triton X-100.
2. 0.5 M N-morpholino ethanesufonate (MES) buffer, pH 7.
3. 0.05 M Adenosine 5'-monophosphate (AMP) to inhibit pyrophosphatases.
4. 0.5 M MnCl
2
.
5. 10 mM UDP-GalNAc (2000 dpm/nmol) donor substrate.
6. 5 mM Acceptor substrate solution: Thr-containing peptide.
7. Enzyme homogenate or solution.
2.5.
β
3- and
β
6-GlcNAc-Transferase Assays
1. 5% Triton X-100.
2. 0.5 M MnCl
2
(for β3-GlcNAc-transferases only).
3. 0.5 M MES buffer, pH 7.
4. 0.05 M AMP.
5. 0.5 M GlcNAc to inhibit N-acetylglucosaminidases.
6. 50 mM γ-galactonolactone (if substrate with terminal Gal is used) to inhibit
galactosidases.
7. 10 mM UDP-GlcNAc (2000 dpm/nmol).
8. 5 mM Acceptor substrate solution: GalNAcα-benzyl, Galβ1-3 GalNAcα-benzyl, Galβ1-4
GlcNAc, GlcNAcβ1-3Galβ-methyl, or GlcNAcβ1-6 (Galβ1-3) GalNAcα-benzyl.
9. Enzyme homogenate or solution.
2.6. Core 1
β
3-Gal- and
β
4-Gal-Transferase Assays
1. 5% Triton X-100.
2. 0.5 M MnCl
2
.
3. 0.5 M MES buffer, pH 7.
4. 0.05 M AMP.
5. 0.05 M γ-galactonolactone.
6. 10 mM UDP-Gal (2000 dpm/nmol).
280 Brockhausen
7. 5 mM Acceptor substrate solution: GalNAcα-benzyl or GlcNAc.
8. Enzyme homogenate or solution.
2.7.
α
3- and
α
6-Sialyltransferase Assays
1. 5% Triton X-100.
2. 0.5 M Tri-HCl buffer, pH 7.
3. 0.05 M AMP.
4. 10 mM CMP-sialic acid (2000 dpm/nmol).
5. 5 mM Acceptor substrate solution: DS-OSM. with 3 mM GalNAc concentration, Galβ1-3
GalNAcα-pnp, or sialylα2-3 Galβ1-3 GalNAcα-pnp.
6. Enzyme homogenate or solution.
7. High-voltage electrophoresis apparatus.
8. 20 mM EDTA/2 % Na-tetraborate.
9. 1% Na-tetraborate.
10. Whatman No. 1 paper.
11. HPLC apparatus.
2.8. Sulfotransferase Assays
1. 5% Triton X-100.
2. 0.1 M magnesium-acetate.
3. 0.1 M NaF to inhibit sulfatases.
4. 0.5 M Tris-HCl buffer, pH 7
5. 0.05 M adenosine triphosphate (ATP).
6. 0.1 M 2,3-Mercaptopropanol to inhibit PAPS degradation.
7. 0.3 mM PAPS (2000 dpm/nmol).
8. 5 mM Acceptor substrate solution: Galβ1-3GalNAcα-benzyl, Galβ1-4 GlcNAc, or
GlcNAcβ1-3Galβ-methyl.
9. Enzyme-homogenate or solution.
10. High-voltage electrophoresis apparatus.
11. 20 mM EDTA/2% Na-tetraborate.
12. 1% Na-tetraborate.
13. Whatman No. 1 paper.
14. HPLC apparatus.
15. Dionex system for HPAEC.
3. Methods
3.1. Preparation of Enzymes
Ideally, enzymes are present in the highly purified state, and soluble in the assay
mixture. A number of enzymes have been purified. However, these procedures
depend on the specific enzyme and tissue and may take several months or years.
Therefore, purification protocols are not described here. Purified enzymes may be
stable at 4°C for months but are usually more stable at lower temperatures. With
tissue homogenates or microsomes, however, this is rarely the case. The enzyme
preparations inevitably contain interfering substances and degradative enzymes. For
example pyrophosphatases and phosphatases that degrade nucleotide sugar donors,
O
-Linked Chains of Mucin 281
glycosidases that degrade substrates and products, and proteases that degrade
the peptide moiety of substrates and products may be present, and deactivate the
enzyme to be assayed. These unwanted reactions can be suppressed with specific
inhibitors.
1. To prepare crude homogenate, hand homogenize tissue in 10 times the volume of 0.25 M
sucrose. For most studies of crude enzymes, this preparation is sufficient. The homoge-
nate can be stored at –20°C for a few months, or at –70°C for several years. If sufficient
material is available, a more enriched enzyme fraction can be prepared as microsomes.
Microsomes may be prepared from homogenates by first removing a low-speed pellet by
centrifugation at 10,000g, followed by the precipitation of microsomes from the superna-
tant at 100,000g. The microsomal pellet is hand homogenized in 10 times the volume of
0.25 M sucrose.
2. Enzymes from cultured cells are prepared similarly. Harvest cells from the culture plate,
and wash three times with 0.9% NaCl by gently stirring and centrifuging cells. After
washing, hand homogenize cells in 0.25 M sucrose (1 mL/10
8
cells) and store as
described in step 1.
3.2. Preparation of Substrates and Standard Compounds
Substrates may be purchased, prepared by chemical synthesis or combined chemi-
cal-enzymatic synthesis, or prepared by enzymatic synthesis or degradation from
natural glycoproteins.
1. GalNAc-OSM is prepared from purified sheep submaxillary mucin, ovine submaxil-
lary mucin (OSM) (29). Treat OSM with 0.1 N H
2
SO
4
for 1 h at 80°C to remove sialic
acid and fucose. For high purity, follow by digestion with bovine testicular β-galac-
tosidase (56), which removes the small amount of β1-3–linked Gal residues present
in OSM (30).
2. Substrate and product compounds that are not commercially available are synthesized
with a known source of the desired enzyme under the conditions described for the stan-
dard transferase assay.
3. Low molecular weight compounds are isolated by gel filtration on Bio-Gel P4 or P2 col-
umns, followed by HPLC (Table 1). The purity and linkages of all compounds should be
verified by mass spectrometry (MS) and
1
H-NMR. The concentrations of individual sug-
ars can be determined after acid hydrolysis (1 h at 80°C with 33% trifluoroacetic acid for
sialic acid–containing compounds, 1 h at 100°C with 6 N HCl for neutral sugars) by
HPAEC (Dionex system) as described in Subheading 3.4.5.
3.3. Separation and Identification
of Glycosyltransferase and Sulfotransferase Products
To demonstrate that an enzyme activity is synthesizing a certain sugar linkage, the
product has to be isolated and its structure determined. This is especially important
when a new enzyme activity is to be assayed or when a novel variant of a known
activity is expected.
1. Produce large amounts of glycosyltransferase product in a standard assay, possibly after
incubation for 8–24 h, and pass through an AG1 × 8 column to remove nucleotide sugar
282 Brockhausen
and negatively charged molecules. For sulfotransferase products, run high-voltage elec-
trophoresis after the standard assay.
2. Purify low molecular weight compounds by HPLC or using the Dionex system, as de-
scribed in Subheading 3.4.5. Low molecular weight compounds separated by high-volt-
age electrophoresis, can be eluted off the paper with water, by placing the paper into
syringes and centrifuging at low speed. Borate is removed by repeated flash evaporation
with methanol.
3. Purify mucin substrates by passing incubation mixtures through AG1 × 8 columns,
followed by gel filtration on Bio-Gel P4 or P2. O-glycans are released from mucins
by β-elimination (0.05 N KOH/1 M Na BH
4
at 45°C for 16 h). After neutralization,
purify reduced O-glycan-alditols by gel filtration on Bio-Gel P4 or P2 columns, and
by HPLC.
4. Carry out structural analysis of all low molecular compounds and oligosaccharide-
alditols by NMR, MS (fast atom bombardment, electrospray, or matrix-assisted laser
desorption ionization), and methylation analysis. If small amounts of product are
available, chromatographic methods, including HPLC and the Dionex system, with
the use of standard compounds, and sequential glycosidase digestion are useful
(29,57–60).
3.4. Glycosyltransferase Assays (
see
Notes 1 and 2)
3.4.1. Ion-Exchange Assay
The ion exchange assay is simple, quick, and inexpensive, and can be applied to all
transferase assays using neutral acceptor substrates.
1. After the incubation, stop the reaction with 100 mL of ice-cold water. Apply mixture
to a column (a Pasteur pipet) of 0.4 mL of AG1 × 8, which removes excess radioac-
tive nucleotide sugar. Wash the column three times with 0.6 mL of water and collect
the eluate.
2. Add 5 mL of scintillation fluid and estimate radioactivity with a scintillation counter.
Since the radioactivity in the eluate includes free radioactive sugar (originating from
nucleotide sugar breakdown) and radioactive products from various endogenous sub-
strates, the radioactivity of assays lacking exogenous substrates has to be substracted
from the disintegrations per minute obtained. The specific enzyme activity is calculated
as nanomoles/(hour
.
milligrams of protein).
3. Regenerate AG1 × 8 columns with 5 M NaCl, followed by thorough washing with water.
3.4.2. C18 Column Assay
The C18 column (Sep-Pak) assay can be applied when substrates contain a hydro-
phobic group. This method is often not reliable when charged (sialylated or sulfated)
products are formed, unless very large hydrophobic groups are present in the enzyme
product. A methyl aglycone group does not provide sufficient hydrophobicity to bind
to Sep-Pak C18 columns.
1. After the incubation, apply the mixture onto a Sep-Pak C18 column, previously washed
in water. Wash columns with 5 mL of water to elute excess nucleotide sugar and free
radioactive sugar.
O
-Linked Chains of Mucin 283
2. Elute radioactive product with 5 mL of methanol and count in 5 mL of scintillation fluid.
3. Regenerate Sep-Pak columns with 10 mL methanol followed by 20 mL of water.
3.4.3. HPLC Assay
All assays using low molecular weight substrates can be carried out by HPLC.
Depending on the structure of the enzyme product, various conditions are used
(Table 1). This method usually allows the separation of enzyme product from all other
components of the assay; it also can separate multiple products. The structure of the
product can be identified by HPLC if standard compounds are available. HPLC can be
carried out after the incubation mixtures have been passed through AG1 × 8 or
Sep-Pak columns.
1. Adjust the HPLC conditions, using substrate and product standards so that product elutes
in 20–40 min. The flow rate for analytical columns is usually 1 mL/min but can be
reduced for better resolution. Acetonitrile/water mixtures are usually successful. When
compounds have charged groups (sialic acid and sulfate), water is replaced by buffer at
low pH, e.g., 15 mM KH
2
PO
4
, pH 5.4. For glycopeptides, water may be replaced by 0.1%
trifluoroacetic acid.
2. Inject an aliquot of the assay mixture into the HPLC, collect fractions, measure absor-
bance at about 200 nm and radioactivity of fractions, and compare elution times to
those of standard compounds. This will assess the identity and quantity of the enzyme
products.
3.4.4. High-Voltage Electrophoresis
Mucin products and sialylated and sulfated products can be separated on paper by
high-voltage electrophoresis.
1. Stop enzyme reactions with 10 µL of 20 mM EDTA/2% borate.
2. After the incubation, apply samples and radioactive standards on paper, separated by
2.5 cm. Wet paper with 1% borate buffer, let the buffer evenly soak in, and place paper so
that samples are just above the surface of the borate buffer in the electrophoresis tank.
Run electrophoresis at 1500 V for about 1 h. The current is about 150 mA.
3. Dry paper, cut into 2-cm strips, add 7 mL of scintillation fluid, and measure radioactivity
with a scintillation counter.
3.4.5. Dionex System
The Dionex system HPAEC allows the quantification of small amounts of free
monosaccharides, or oligosaccharides, for which standards are available. It can also
achieve excellent separation of mono- and oligosaccharides and, in particular, sialy-
lated and sulfated compounds that would otherwise be difficult to obtain by HPLC.
1. For free sugars, use 15 mM NaOH as the mobile phase; for sialylated and sulfated
compounds, use 15 mM NaOH/0.1 M Na-acetate, or higher concentrations of
Na-acetate.
2. Sugars can be detected amperometrically. Once an elution time has been established,
collect a fraction of eluting samples and detect radioactivity by scintillation counting.
284Brockhausen
Table 1
Mucin Glycosyltransferases, Their Substrates and Products, and HPLC Conditions for Product Isolation
HPLC
Path Enzyme Substrate Product column % AN
a Polypeptide α-GalNAc-T Thr-peptide GalNAc-Thr-peptide C18 0–20
b Core 1 β3-Gal-T 1. GalNAcα-Bn Galβ
3GalNAcα-Bn C18 10
2. GalNAcα-OSM Galβ3GalNAcα-OSM — —
c Core 3 β3-GlcNAc-T 1. GalNAcα-Bn GlcNAcβ3GalNAcα-Bn C18 10
2. GalNAcα-OSM GlcNAcβ3GalNAcα-OSM — —
d Core 2 β6-GlcNAc-T Galβ3GalNAcα-pnp GlcNAcβ6(Galβ3)GalNAcα
-pnp C18 8
e Core 4 β
6-GlcNAc-T GlcNAcβ3GalNAcα-pnp GlcNAcβ6(GlcNAcβ3)GalNAcα-pnp C18 8
f β
4Gal-T GlcNAc Galβ4GlcNAc NH2 85
gi β3-GlcNAc-T Galβ4GlcNAc GlcNAcβ3Galβ4GlcNAc NH2 82
h Elongation β3-GlcNAc-T GlcNAcβ6(Galβ3)- GlcNAcβ6(GlcNAcβ3Galβ3)- C18 8
GalNAcα-Bn GalNAcα-Bn PAC 82
iI β6-GlcNAc-T GlcNAcβ3Galβ-CH
3
GlcNAcβ6(GlcNAcβ3)Galβ-CH
3
NH2 85
j α6-sialyl-T GalNAcα-OSM Sialylα6GalNAcα-OSM — —
284
O
-Linked Chains of Mucin285
285
k Gal 3-sulfo-T Galβ3GalNAcα-Bn SO
4
-3-Galβ3GalNAcα-Bn NH2 80
a
l α3-sialyl-T Galβ3GalNAcα-pnp Sialylα3Galβ3GalNAcα-pnp NH2 80
a
m α6-sialyl-T III Sialylα3Galβ3GalNAcα-pnp Sialylα3Galβ3 (sialylα2-6) GalNAcα-pnp NH2 80
a
a
AN, acetonitrile; Bn, benzyl; Gal, galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; OSM, ovine submaxillary
mucin; pnp, paranitrophenyl; T, transferase; C18, reversed phase C18 column; NH2, amine column; PAC, mixed cyano-amine column.
Compounds
containing large peptides (OSM) and charged groups (sialic acid, sulfate) are separated by high-voltage electrophoresis. Sulfat
ed compounds also
separate well in the Dionex system. Low molecular weight compounds are separated by HPLC, and eluted in acetonitrile/water mixtures. The paths
(a–l) catalyzed by transferases refer to those indicated in Fig. 1.
b
HPLC separation is carried out at 80% acetonitrile and 20% 15 mM KH
2
PO
4
or KHCO
3
, pH 5.4.
286 Brockhausen
If more than 10 µL of sample are collected, neutralize the solution before adding
scintillation fluid.
3.5. Assays for Specific Enzymes
Nucleotide donor substrates should be radioactively labeled with
14
C or
3
H (sugar)
for sensitivity and unequivocal identification of enzyme product.
3.5.1. Assay for Polypeptide GalNAc-transferase (path a) (Note 3)
1. Add the following to the incubation mixture to a total volume of 40 µL: 8 µL of 5 mM
peptide substrate, dried in the assay tube, 4 µL of 50 mM AMP, 1 µL of 0.5 M MnCl
2
,
10 mL of 0.5 M MES, pH 7.0, 1 µL of 5% Triton X-100, 4 µL of 10 mM UDP-
*
GalNAc,
10 µL of enzyme source.
2. Incubate 1 h at 37°C, and then proceed as described for ion-exchange or HPLC assay.
3.5.2. Assay for Core 3
β
6-GlcNAc-Transferase (path c)
1. Add the following to the incubation mixture to a total volume of 40 µL: 16 µL of 5 mM
GalNAcα-benzyl substrate, dried in the assay tube, 4 µL of 50 mM AMP, 1 µL of 0.5 M
MnCl
2
, 10 µL of 0.5 M GlcNAc, 10 µL of 0.5 M MES, pH 7.0,1 µL of 5% Triton X-100,
4 µL of 10 mM UDP-
*
GlcNAc, 10 µL enzyme source.
2. Incubate for 1 h at 37°C, and then proceed as described for ion-exchange assay, C18
column, or HPLC assay.
3.5.3. Assay for Core 2
β
6-GlcNAc-Transferase (path d)
1. Add the following to the incubation mixture to a total volume of 40 µL: 16 µL of 5 mM
Galβ1-3GalNAcα-benzyl or Galβ1-3 GalNAcα-pnp substrate, and 10 µL of 0.5 M
GlcNAc, dried in the assay tube, 4 µL of 50 mM AMP, 10 µL of 0.5 M MES, pH 7.0,1 µL
of 5% Triton X-100, 8 µL of 50 mM γ-galactonolactone, 4 µL of 10 mM UDP-
*
GlcNAc,
10 µL of enzyme source.
2. Incubate for 1 h at 37°C, and then proceed as described for ion-exchange, C18 column, or
HPLC assay.
3.5.4. Assay for Core 4
β
6-GlcNAc-Transferase (path e)
1. Add the following to the incubation mixture to a total volume of 40 µL: 16 µL of 5 mM
GlcNAcβ1-3 GalNAcα-pnp substrate, dried in the assay tube, 4 µL of 50 mM AMP,
10 µL of 0.5 M GlcNAc, 10 µL of 0.5 M MES, pH 7.0, 1 µL of 5% Triton X-100, 4 µL of
10 mM UDP-
*
GlcNAc, 10 µL of enzyme source.
2. Incubate for 1 h at 37°C, and then proceed as described for ion-exchange, C18 column, or
HPLC assay.
3.5.5. Assay for i
β
3-GlcNAc-Transferase (path g)
1. Add the following to the incubation mixture to a total volume of 40 µL: 16 µL of 5 mM
Galβ1-4GlcNAc substrate, dried in the assay tube, 4 µL of 50 mM AMP, 1 µL of 0.5 M
MnCl
2
, 10 µL of 0.5 M GlcNAc, 10 µL of 0.5 M MES, pH 7.0, 1 µL of 5% Triton X-100,
4 µL of 10 mM UDP-
*
GlcNAc, 10 µL of enzyme source.
2. Incubate for 1 h at 37°C, and then proceed as described for ion-exchange or HPLC assay.
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