ANALYTICALBIOCHEMISTRY

186,127-134

(1990)

A Highly Sensitive Fluorometric Assay for Sialyltransferase Activity Using CMP-9-fluoresceinyl-NeuAc as Donor H. J. Gross, Institut

Received

fiir

U. Sticher,

Biochemie

August

and R. Brossmer

II, Universitat

Heidelberg,

Im Neuenheimer

Feld 328, 6900 Heidelberg,

Federal Republic

of

Germany

2,1989

This paper presents a very sensitive fluorometric assay for sialyltransferase activity based on the transfer of 5-acetamido-S-deoxy-S-fluoresceinylthioureidoneuraminic acid onto distinct glycoproteins, thus allowing determination of acceptor specificities. Acceptor protein-bound fluorescence was quantified after gel filtration which separated fluorescent sialoglycoprotein from the fluorescence-labeled CMP-glycoside donor. Kinetic constants obtained for five different purified sialyltransferases indicated that CMP-S-fluoresceinyl-NeuAc was a suitable donor substrate for each enzyme, affording low K, values and V,,, values comparable in magnitude (15-100%) to that obtained with the parent CMP-NeuAc. Sensitivity was enhanced 200- to lOOO-fold compared to the radiometric sialyltransferase assay as it is used routinely. The method was applied to determination of the kinetic properties of purified rat liver cY2,6+ialyltransferase with four separate glycoprotein acceptors differing in glycan structure, employing very small amounts of donor, acceptor, and enzyme, and to the study of sialyltransferase activity of the human promyelocytic cell line HL-60 toward three different acceptors. o isso Academic PWS, he.

Sialyltransferases are important enzymes of glycoprotein and glycolipid biosynthesis catalyzing the incorporation of sialic acids into terminal positions of glycoconjugate glycans (l-4). There is growing interest in these enzymes in biochemistry and clinical chemistry. They are, for example, useful tools for studies on receptor determinants in vitro (5-8). Since the quantitative and qualitative sialylation patterns of tumor cells seem to be significantly different from those of nontransformed cells (g-11), determination of sialyltransferase activity 0003-2697/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form

Inc.

reserved.

in biological samples as well as kinetic data on the acceptor specificity of the different enzymes in normal and transformed cells provides important information. Up to now, radiolabeled CMP-NeuAc’ was routinely required in studies on sialyltransferase activity. To separate the radiolabeled sialoprotein acceptor from the donor CMP-glycoside, the assays required either precipitation, ion-exchange chromatography, or adsorption of the glycoprotein product formed, and subsequent liquid scintillation photometry (12-14). The procedure is laborious especially because it involves handling of radioactive compounds. Thus there is a demand for a sialyltransferase assay that is simple to perform and at the same time very sensitive. This paper describes such a method employing a fluorescent CMP-glycoside as donor substrate and a liquid chromatography system. The new assay requires only very small amounts of both donor and acceptor substrate. MATERIALS

AND

METHODS

Materials. All chemicals used were of analytical grade and purchased from Merck (Darmstadt) or Serva (Heidelberg). Crystalline N-acetylneuraminic acid was prepared in this laboratory (15). Cytidine 5’-monophosphate (CMP) was obtained from Boehringer (Mannr Abbreviations used: FPLC, fast protein liquid chromatography; NeuAc, 5-acetamido-3,5-dideoxy-fi-D-neuraminic acid; 9 - fluoresceinyl - NeuAc, 5 - acetamido - 9 - fluoresceinylthioureido - NeuAc, [5-acetamido-9-(3-fluoresceinylthioureido)-3,5,9-t~~xy-~-D-glyce~D - galacto - 2 - nonulosonic acid]; CMP - NeuAc, cytidine - 5 -mono phospho - N- acetylneuraminic acid, CMP - 9 - fluoresceinyl_ NeuAc, cytidine - 5 - monophospho - 9 - fluoresceinyl - NeuAc; PMSF, phenyl3GalNAcP1, 4(NeuAco12,3)methylsulfonyl fluoride; Gnnl, GalPl, Gal@,4Glc-ceramide; ST, sialyltransferase; BSA, bovine serum albumin; Enzymes: Gal/31, 4GlcNAc o2,6-sialyltransferase (EC 2.4.99.1); Galfll, 4(3)GlcNAc o2,3sialyltransferase (EC 2.4.99.5); Gal@, 3GalNAc a2,3-sialyltransferase (EC 2.4.99.4); GalNAc cY2,6-sialyltransferase (EC 2.4.99.3).

127

128

GROSS,

STICHER,

heim); cytidine 5’-triphosphate from Biomol (Ilvesheim); and bovine serum albumin, Triton CF-54, and Triton X-100 from Sigma (Munich). Both acetonitrile grade E and grade S were from Zinsser (Frankfurt). CMP-NeuAc (free CMP contamination below 4%) and CMP-9-fluoresceinyl-NeuAc (free CMP contamination below 5%) were prepared enzymatically as described previously (16,17). CMP-[3H]NeuAc (26.2 Ci/mmol) was purchased from New England Nuclear. al-Acid glycoprotein (18) and Zn-ala-glycoprotein (19) were gifts of Dr. Karl Schmid (Boston), and antifreeze glycoprotein [fractions 3-5 from serum of Pagothenia borchgrevinki (20)] was donated by Dr. Robert E. Feeney (Davis). Fetuin was obtained from Serva (Heidelberg) and further purified on Sepharose 6B. Antithrombin III was kindly donated by Behring Werke (Marburg) and GM1 from Dr. Pallmann KG (Munich). Galfll,4GlcNAc a2,6-ST (0.9 U/ml, 1.7 U/mg) was purified from rat liver as described (21), and Galpl,4GlcNAc (Y~,~-ST (443 mu/ml, 1.5 U/mg) from human liver (Sticher and Brossmer, unpublished results). Rat liver Galpl,4(3)GlcNAc a2,3-ST (0.104 U/ml) and both Gal&3GalNAc a2,3-ST (58 mu/ml) and GalNAc a2,6ST (4.4 U/ml) from porcine submaxillary glands were supplied by Dr. James C. Paulson (Los Angeles) (22-24). Protein determination. Protein content was determined by the Bio-Rad protein assay (Bio-Rad, Munich) using bovine serum albumin as standard. Desialylation of sialoglycoproteins. After treatment with Vibrio cholerae sialidase (25) asialo-al-acid glycoprotein, asialofetuin, asialo-antithrombin III, or asialoZn-a2-glycoprotein each contained about 0.2% bound NeuAc. Galactose acceptor sites. Sites were expressed in terms of galactose content of asialo-al-acid glycoprotein, antifreeze glycoprotein, asialofetuin, fetuin, asialo-antithrombin III, asialo-Zn-trz-glycoprotein, or GM1 as described previously (16). Fluorometric standard assay. The standard reaction mixture (30 ~1) for rat liver Galpl,4GlcNAc a2,6-ST and Galpl,4(3)GlcNAc a2,3-ST contained 62.5 mM sodium cacodylate buffer, pH 6.0, 1 mg/ml BSA, 0.5% Triton CF-54, 1.5 mg/ml asialo-cui-acid glycoprotein (690 PM galactose acceptor sites), and 3-100 pM CMP-g-fluoresceinyl-NeuAc. The concentration of asialo-al-acid glycoprotein in terms of galactose acceptor sites was about fourfold higher than the K, value with both rat liver ST (26). The standard reaction mixture for Galpl,4GlcNAc a2,6-ST from human liver was identical to that for rat liver, except that pH value was 6.5 and Triton X-100 (0.1%) was used instead of Triton CF-54. The standard reaction mixture (30 ~1) for porcine Gal&3GalNAc a2,3-ST and GalNAc a2,6-ST contained 62.5 mM sodium cacodylate, pH 6.5, 1 mg/ml

AND

BROSSMER

BSA, 0.1% Triton X-100, 1.05 mg/ml antifreeze glycoprotein (1500 pM galactose acceptor sites), or 10 mg/ml asialofetuin (1875 pM total galactose acceptor sites), or 10 mg/ml fetuin (1875 pM total galactose acceptor sites), or 1.15 mg/ml GM1 (1500 pM total galactose acceptor sites), and 3-200 pM CMP-9-fluoresceinyl-NeuAc. The concentration of antifreeze glycoprotein in terms of galactose sites was 1.5-fold the K, of porcine GalNAc (u2,6-ST and 3-fold that of porcine (Y~,~-ST (24,27). The concentration of the 0-glycosidically bound galactose acceptor sites of asialofetuin or fetuin was calculated to be 25% of the total galactose sites (28,29). In accordance with the published structure of antifreeze glycoprotein, an equimolar of galactose/N-acetylgalactosamine ratio was assumed (30). Reaction was initiated in each case by addition of the respective sialyltransferase and, after incubation at 37°C terminated by addition of 4 ~1 0.1 M CTP. Corresponding controls were performed with enzyme in the presence of 4 ~10.1 M CTP. The assay mixture was kept in the dark. Fluorometric separation system. Two to ten microliters of the assay mixture was applied to the liquid chromatography system consisting of a FPLC pump (Pharmacia), Rheodyne injection valve (20-~1 sample loop), Hitachi F-1000 fluorescence spectrophotometer (excitation at 490 nm, emission at 520 nm), and a Latek one-channel recorder. Separation of fluorescently labeled glycoprotein from donor substrate was performed on a column (0.4 X 12.5 cm) of Sephadex G-50 fine operated isocratically with 0.1 M Tris/HCl, pH 8.5, at 0.5 ml/ min. The amount of fluorescent NeuAc incorporated was calculated with respect to external standards of 9fluoresceinyl-NeuAc and CMP-9-fluoresceinyl-NeuAc (each 0.1 nmol). Ganglioside GILll sialylated with g-fluoresceinyl-NeuAc was separated from the fluorogenic CMP-glycoside using the Sephadex G-50 fine column described above operated with 0.1 M Tris/HCl pH 8.5 containing 100 mM NaCl and 0.3% Triton X-100. Special fluorometric assays. Kinetic data on rat liver a2,6-ST for different glycoprotein acceptors were obtained with an assay reduced to 10 /*l total reaction volume containing the concentrations mentioned above. Inhibition of rat liver a2,6-ST by the fluoresceinated glycoprotein product was determined as follows. The assay (30 ~1) was performed in duplicate as above (30 pM CMP-9-fluoresceinyl-NeuAc) in the absence and in the presence of asialo-cu,-acid glycoprotein presialylated with either 9-fluoresceinyl-NeuAc or NeuAc (1.7 pg containing 225 nmol9-fluoresceinyl-NeuAc/mg or 200 nmol NeuAc/mg); initial rates were determined after 20 min at 37°C as described above. HPLC sialyltransferase assay. The reaction mixture (80 ~1) was composed as described for the fluorometric assay. The reaction was terminated by addition of 1.3 ml

SENSITIVE

FLUOROMETRIC

ice-cold 1% phosphotungstic acid in 0.5 N HCl. Assay tubes were placed on ice for 20 min and then centrifuged at 9500g for 20 s (16). The sediment was washed once with phosphotungstic acid (16), and extracted with a mixture (1 ml) of cold ethanol/O.1 M Tris, pH 7.5 (9/l, v/v), left for 30 min at 4”C, and centrifuged for 2 min at 9500g. The extraction was repeated once and the final sediment containing the glycoprotein dissolved in 20 ~1 1 N NaOH. A glycoprotein recovery of 85% was calculated by the method outlined previously (16,31). Corresponding controls were lacking sialyltransferase. The glycoprotein was dissolved in 1 N NaOH (20 pl), acidified with 25 ~1 1 N HCL (50 ~1 total, final pH l), and hydrolyzed for 1 h at 80°C (14); an external g-fluoresceinyl-NeuAc standard (0.4 mM) was treated under identical conditions. About 85% of the fluorescent NeuAc transferred was released from the glycoprotein by this acid hydrolysis. Then 10 ~1 1 N NaOH was added, and aliquots (5-20 ~1) were analyzed by analytical HPLC for the amount of fluorescent NeuAc liberated (see below). The extent of degradation of 9-fluoresceinyl-NeuAc liberated during acid hydrolysis was 15%. Analytical HPLC was performed as described previously (16,17,32). Radiometric sialytransferase assay. The reaction mixture (80 ~1) was composed as described for the fluorometric assay, except that CMP-9-fluoresceinyl-NeuAc was replaced by CMP-[3H]NeuAc (7000 cpm/nmol, 30500 PM). The assay was performed as described previously (21). Sialyltransferase activity in HL-60 culture cells. Cells were washed three times with phosphate-buffered saline and lysed for 45 min at 4°C with a mixture of 0.1 M Tris/ HCl, 100 mM NaCl, 1.5 mM MgCl,, 0.1 mM PMSF, 10 pg/ml Aprotinin, and 0.5% Triton X-100 (33). The suspension was then centrifuged for 15 min at 1750 X g (33). Aliquots of the final supernatant (2-5 ~1) were analyzed for sialyltransferase activity applying the fluorometric assay (see above, 30 min, 20 PM CMP-9-fluoresceinyl-NeuAc) without BSA. Either asialo-al-acid glycoprotein (1.5 mg/ml), antifreeze glycoprotein (1.5 mg/ml), or asialofetuin (3.5 mg/ml) was employed as acceptor substrate. An assay without added glycoprotein served to determine transferase activity toward endogenous acceptors. Corresponding controls were performed in the presence of 12 mM CTP. RESULTS Sialyltransferase

Assay

Fluorometric sialyltransferase measurements were made by application of a new assay, which is based on quantification of protein-bound fluorescence after gel filtration. Glycoprotein sialylated with 9-AuoresceinylNeuAc separated well from the corresponding CMP-gly-

SIALYLTRANSFERASE

129

ASSAY

B

A

h ,

1

;

;

1

I

I

,

I

4

I

I

I

I

5

6

7

8

9

10

11

12

13

)

(min) FIG. 1. Gel filtration on Sephadex G-50 fine of a sialyltransferase assay with CMP-9-fluoresceinyl-NeuAc as donor substrate. (A) asialo-al-acid glycoprotein labeled with 9-fiuoresceinyl-NeuAc, (B) CMP-9-fluoresceinyl-NeuAc.

coside, up to a sample volume of 20 ~1 (Fig. 1). Recovery of fluorescently labeled glycoprotein after chromatography on Sephadex G-50 fine was at least 95%. The fluorescence yield was linear with a concentration of g-fluoresceinyl-NeuAc up to at least 0.1 nmol. Protected against light, the fluorescence yield of transferred g-fluoresceinyl-NeuAc was stable for at least 5 h either at room temperature or at 4°C. The transfer at 3 PM CMP-9-fluoresceinyl-NeuAc was linear with time up to 30 min employing 0.015 mU rat liver a2,6-ST (45% donor substrate consumption) (Fig. 2); using the same amount of enzyme, the transfer at 20 and 50 FM was linear only up to about 25% donor substrate consumption, respectively (Fig. 2). This may be explained by product inhibition, as asialo-al-acid glycoprotein resialylated with 9-fluoresceinyl-NeuAc proved to be an efficient inhibitor of rat liver c~2,6ST; transfer was inhibited by 60% in the presence of 1.7 pg fluorescent glycoprotein (13 PM protein-bound g-fluoresceinylNeuAc) . Furthermore, incorporation of 9-fluoresceinyl-NeuAc at 50 /*M CMP-glycoside was linear with the amount of enzyme up to 0.021 mU rat liver a2,6-ST, representing 20% donor substrate consumption. To confirm the incorporation values obtained fluorometrically, the time course for transfer of g-fluoresce-

130

GROSS,

STICHER,

AND

BROSSMER

(min) FIG. 2. Time course for the incorporation CMP-9-fluoresceinyl-NeuAc. Fluorometric under Materials and Methods.

of 9-fluoresceinyl-NeuAc assay was performed

with

inyl-NeuAc was also calculated by analytical HPLC after release of the bound fluorescent NeuAc analog by acid hydrolysis (HPLC sialyltransferase assay). The values obtained by HPLC did not differ significantly (less than 15%) from that obtained fluorometrically. Kinetic

Data for CMP-9-flzmresceinyl-NeuAc

Kinetic constants of five purified sialyltransferases for CMP-9-fluoresceinyl-NeuAc were determined by applying the fluorometric method (Table 1). The acceptor substrate for each rat liver ST and for human liver ST was asialo-al-acid glycoprotein bearing the N-linked terminal glycan sequence Gal/R,LiGlcNAc (26); the acceptor for each porcine submaxillary gland ST was antifreeze glycoprotein bearing the O-linked disaccharide Galfl1,3GalNAc (24,27); asialofetuin and fetuin, which contain both of these glycan structures, served as alternative acceptors for the porcine enzymes. Additionally, GM,, was employed as acceptor for porcine Gal/31,3GalNAc c~2,3-ST; separation of the ganglioside labeled with 9-fluoresceinyl-NeuAc from the fluorescent CMP-glycoside was also achieved by gel filtration, which was performed as outlined previously with a buffer containing 0.3% Triton X-100 (34). With each sialyltransferase, the fluorescent CMPNeuAc analog yielded lower K, values compared with those determined for the parent CMP-NeuAc (Table 1). V,,, values differed significantly depending on the en-

into asialo-a,-acid 0.015 mU Galfll,

glycoprotein at 3 pM (X), 4GlcNAc a2,6-sialyltransferase

20 PM (O), and 50 pM (0) (rat liver) as described

zyme (15-100% compared to V,,, of CMP-NeuAc, Table 1). V,,, values of porcine GalNAc (Y~,~-ST and Gal&3GalNAc a2,3-ST increased markedly when asialofetuin or fetuin was employed instead of antifreeze glycoprotein as aceptor substrate (Table 1). Kinetic data of rat liver a2,6-ST examined by the HPLC sialyltransferase assay (Km = 10 PM, relative V,,, = 1.0) were not significantly different from the values obtained fluorometrically (K, = 7 PM, relative V,,, = 1.0, Table 1). Kinetic constants of each ST for parent CMP-NeuAc were determined by radiometric assay. K,,, and V,,,,, values (Table 1) obtained were in accordance with those described previously (21,24,26,27,31). Kinetic

Data of Acceptor

Substrates

Table 2 shows K,,, and V,,, values for four different asialoglycoproteins with Galpl,4GlcNAc (Y~,~-ST, employing CMP-9-fluoresceinyl-NeuAc as donor substrate. The reaction mixture could be reduced from 30 to 10 ~1 total assay volume, yielding identical data. Kinetic constants for each acceptor glycoprotein obtained radiometrically with CMP-[3H]NeuAc were not significantly different (Table 2). However, applying the fluorometric assay with 10 ~1 total assay volume, consumption of glycoprotein was 15%, and that of rat liver a2,6-ST was about 0.1% compared to the radiometric assay.

SENSITIVE

FLUOROMETRIC

SIALYLTRANSFERASE TABLE

Comparison

of Apparent

Kinetic

Constants

Sialyltransferase Galpl,4GlcNAc (rat liver) Gal/31,4GlcNAc (human liver) Galfll,4(3)GlcNAc (rat liver) Galpl,3GalNAc (porcine)

o12,6ST

CMP-S-fluoresceinyl-NeuAc CMP-[3H]NeuAc CMP-S-fluoresceinyl-NeuAc CMP-[“H]NeuAc CMP-S-tluoresceinyl-NeuAc CMP-[3H]NeuAc CMP-S-Auoresceinyl-NeuAc

cu2,6-ST o12,3-ST (u2,3-ST

CMP-[3H]NeuAc CMP-S-fluoresceinyl-NeuAc

GalNAc cu2,6-ST (porcine)

1

of Five Purified Sialyltransferases and CMP-NeuAc

Donor

CMPj3H]NeuAc

131

ASSAY

for CMP-9-fluoresceinyl-NeuAc

Relative

Acceptor

KITI (PM)

AORM AORM AORM AORM AORM AORM AFG AF G AFM;: AFG AF F AFG

7 45 2 15 a 65 2.5 4 3 6 30 35 25 500

1.0 1.0 0.8 1.0 0.25 1.0 0.15 0.5 0.15 1.0 0.2 0.85 0.75 1.0

V maxa

Relative Vmax /Km

(l/mM) 143 22 400 67 31 15 60 125 50 166 7 24 30 2

Note. Assays were performed in duplicate as described under Materials and Methods using at least five concentrations of CMP-S-fluoresceinyl-NeuAc or CMP-[3HJNeuAc near the respective Km value. Assays were performed within linear limits of time and amount of enzyme. Kinetic parameters were obtained from Hanes plots (47). Acceptors: rat liver ST and human liver ST, asialo-a,-acid glycoprotein (AORM); uorcine ST. antifreeze elvconrotein (AFG). asialofetuin (AF). fetuin (F), or Gr,,i. ’ Relative to the respective V,,,,, obtained with CMP-NeuAc (1.0). ~ -”

-

al-Acid glycoprotein and fetuin contain a mixture of di-, tri- and tetraantennary complex-type glycans and only triantennary glycans, respectively (28,29,35). In contrast, antithrombin III and Zn-cYa-glycoprotein are composed entirely of biantennary glycans (19,36). Referring to the K,,, values (Table 2), rat liver a2,6-ST preferred the biantennary structure, whereas, in compari-

TABLE

2

Apparent Kinetic Parameters of Galpl,4GlcNAc alyltransferase (Rat Liver) for Different Glycoprotein tors with CMP-9-fluoresceinyl-NeuAc as Donor

n2,6-SiAccep-

Relative Acceptor

K, (PM)

glycoprotein

Asialo-a,-acid glycoprotein Asialofetuin Asialo-antithrombin III Asialo-Zn-a,-glycoprotein

350 250 180 135

(3409 (235b) (160’) (1409

V maxa (relative 100 130 115 95

(100b) (1159 (1156) (80b)

%)

VmIKm (l/mM) 2.8 5.2 6.4 7.0

Note. Fluorometric assay (10 ~1 assay volume) was performed in duplicate as described under Materials and Methods using five concentrations of the acceptor substrate near the respective K,,, values. CMPS-Auoresceinyl-NeuAc was fixed at the saturating concentration of 30 pM. Kinetic data were obtained from Hanes plots (47). a Relative to the value obtained with asialo-o,-acid glycoprotein (100% = 4.0 pmol/min). b Values obtained by the radiometric assay with CMP-[3H]NeuAc as donor are given for comparison.

son, affinity for asialo-al-acid glycoprotein, usually applied as acceptor for this enzyme, was only 50%. Sensitivity of the Fluorometric Assay Sensitivity was compared to that of the radiometric assay, which employs 3H-labeled CMP-NeuAc as donor substrate and which is routinely used for quantifying sialyltransferase activity (19). The labeling extent (7500 cpm/nmol) was of the same order of magnitude as commonly used for radiometric sialyltransferase assays (14,22,37-43). The fluorometric detection limit for activity of rat liver (u2,6-ST and porcine ~u2,3-ST using standard assay conditions amounted to 0.001 and 0.005 pU, respectively (enzymatic fluorescence transfer to unspecific fluorescence adsorption 1.5/l); this is about lOOO-fold and 200fold, respectively, lower than the detection limit of the radiometric assay (1.0 pU, representing 150 cpm). Sialyltransferase Activity Line HL-60

of Human Promyelocytic Cell

The Triton extract (5 ~1) from about 4 X lo4 cells (12 pg protein) served to determine the sialyltransferase activity toward different acceptor glycoproteins, employing asialofetuin for total activity, asialo-n,-acid glycoprotein for GalP1,4GlcNAc-specific ST, and antifreeze glycoprotein for GalP1,3GalNAc-specific ST. Relative to the protein content of the cell extract, the specific activ-

132

GROSS,

STICHER,

AND

BROSSMER

NH I 2 HO

dH FIG. 3.

Structure

OH

of CMP-9-fluoresceinyl-NeuAc.

ities obtained with asialofetuin, asialo-al-acid glycoprotein, and antifreeze glycoprotein were 13, 11, and 3 pU/ mg, respectively; the endogenous activity amounted to 1.6 pU/mg. Enzymatic transfer was two- to sixfold higher, depending on the acceptor, than the respective nonspecific adsorption determined in the presence of the ST inhibitor CTP. DISCUSSION

Previously, we reported on the conversion of 9-fluoresceinyl-NeuAc to the activated CMP-glycoside and transfer of the fluorescent analog onto glycoproteins by rat liver and porcine submaxillary gland a2,6-sialyltransferase (17). The novel fluorometric sialyltransferase assay that we describe in this paper is based on the enzymatic transfer of 9-fluoresceinyl-NeuAc onto glycoprotein acceptors and subsequent monitoring of the protein-bound fluorescence after gel filtration. This assay exceeds in sensitivity the common radiometric methods in use for the detection of very low enzyme activities. As the donor substrate carries the fluorescent label, the method allows determination of the acceptor specificity of sialyltransferases employing glycoproteins with defined glycan structures. To ascertain the general applicability of CMP-g-fluoresceinyl-NeuAc (Fig. 3) as synthetic substrate, kinetic data for five purified sialyltransferases were determined (Tables 1 and 2). Though the enzymes differed in their specificity for the acceptor glycan and for the glycosidic linkage formed, a higher affinity for the fluorescent CMP-NeuAc analog relative to parent CMP-NeuAc was observed in each case (Table 1). The reaction rates of the sialyltransferases studied were of the same order of magnitude for both donors (Table 1). GalNAc a2,6-sialyltransferase gave a low rate of incorporation of the fluorescent NeuAc analog into antifreeze glycoprotein (20%, Table l), which has been already described (17). With asialofetuin or fetuin as acceptor, V,,, rates measured with CMP-9-fluoresceinyl-NeuAc increased by about fourfold (Table 1) in accordance with results ob-

tained previously with other CMP-NeuAc analogs modified at C-9 (31). Radiometric procedures to determine sialyltransferase activity are well established. However, to achieve a sensitivity comparable to that of the fluorometric assay at saturating concentrations of CMP-glycoside, the radiametric assay requires a high specific labeling. Such saturating reaction conditions (fourfold K,,, value) for the enzymes shown in Table 1 (24-2000 pM CMP-[3H]NeuAc) would require lo-800 &i/assay of the expensive CMP-[3H]NeuAc. Sensitivity of the fluorometric assay is about 200- to lOOO-fold higher than that of the standard radiometric assay, a result which allows the detection of low activities and the performance of kinetic studies with sialyltransferases that are available only in small amounts. The fluorometric assay also avoids the special precautions necessary in the handling of radioactive compounds. Moreover, procedures for separation of the radiolabeled glycoprotein, such as ion-exchange chromatography, acid precipitation, and adsorption to membrane filters, are not needed. The fluorometric assay described offers several further advantages. Saturating concentrations of the fluorescent donor substrate are achieved at 2- to 17-fold lower values than with CMP-NeuAc (Table 1). This minimizes unspecific adsorption of fluorescent material to the protein acceptor, which is an inherent disadvantage of many Auorometric assays. Routinely, the assay volume is 30 ~1, and can be reduced further to 10 ~1without loss of precision and sensitivity. Compared to the standard radiometric assay, both the favorable K,,, value for the fluorescent donor and the small reaction volume reduce the consumption of donor substrate to about 2% and that of acceptor substrate to about 15%. The latter is especially attractive when scarce biologically active glycoproteins are to be studied. Recently, a fluorogenic acceptor derived from lactose was employed to determine sialyltransferase activity (42). However, studies on the acceptor specificity, which is the most important property of an unknown sialyl-

SENSITIVE

SIALYLTRANSFERASE

FLUOROMETRIC

transferase, require compounds with different glycan structures. Thus the fluorometric assay presented here shows definite advantages with respect to identification and characterization of sialyltransferases occurring in a biological sample. Furthermore all sialyltransferases studied so far showed only low affinity for lactose as acceptor (24,26,27,45,46). Therefore a high concentration of the fluorogenic lactose derivative is required for a sufficient transfer. With the novel assay, very small amounts of a purified sialyltransferase are needed to study the properties of glycoproteins, differing in glycan structure, as acceptor substrates. Such information is especially important for in vitro sialylation studies of soluble or membranebound glycoconjugates with purified enzymes. Thus, the assay was applied to determine the kinetic data of purified rat liver cu2,6-sialyltransferase for four glycoprotein acceptors. It is noteworthy that the results can be obtained with less than 0.1% of the quantity of enzyme that is needed for the standard radiometric assay (Table 2). Gangliosides could also be used as acceptor in the fluorometric assay; this was demonstrated using GM1 and porcine a2,3-sialyltransferase (Table 1). The results acquired with the human promyelocytic cell line HL-60 show that sialyltransferase activity can be determined reliably even with a small cell number. It should be noted that the number of cells employed in this study is not at the lower limit. The results obtained with asialo-a,-acid and antifreeze glycoprotein demonstrate the existence of at least two sialyltransferases in these cells differing in acceptor specificity. The new method should be useful as an alternative to the commonly applied radiometric assays. It is highly sensitive; is economical with respect to consumption of donor, acceptor, and enzyme; is suitable for quantitation of sialyltransferases in small amounts of biological samples; and allows differentiation between defined glycan acceptor specificities.

4. Durrie,

R., and Rosenberg,

5. Markwell, Sci. USA

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A highly sensitive fluorometric assay for sialyltransferase activity using CMP-9-fluoresceinyl-NeuAc as donor.

This paper presents a very sensitive fluorometric assay for sialyltransferase activity based on the transfer of 5-acetamido-9-deoxy-9-fluoresceinylthi...
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