Characterization of human plasma sialyltransferase using a novel fluorometric assay

Clinica Chimica Acta, 197 (1991) 237-248 0 1991 Elsevier Science Publishers B.V. 0009-8981/91/$03.50 ADONIS 0009898191001002

237

CCA 04964

Characterization of human plasma sialyltransferase using a novel fluorometric assay H.J. Gross

and R. Brossmer

institut fir Biochemie II der Universitiit Heidelberg (Received

23 February

1990; revision

Key words: Sialyltransferase;

Human

received 5 December

plasma; Fluorometric Acceptor specificity

(Germany)

1990; accepted

assay;

13 December

1990)

CMP-9-fluoresceinyl-NeuAc;

Summary

We have characterized human plasma sialyltransferase using a new fluorometric assay based on incorporation of a fluorescent NeuAc analogue into different acceptor glycoconjugates. This enables an exact characterization of acceptor specificity and kinetic properties. The data obtained indicate the presence of at least two distinct plasma sialyltransferases: one specific for N-linked complex type glycan acceptors, and the other for GalNAc-residues on O-linked glycan acceptors. The first enzyme turned out to have very similar properties to a purified human liver sialyltransferase, supporting an earlier hypothesis that liver is the main enzyme source. The new fluorometric assay presented here may be suitable for answering the question as to whether plasma sialyltransferase is a useful diagnostic parameter in pathology.

Abbreviations: HPLC, high performance liquid chromatography; NeuAc, 5-acetamido-3,5-dideoxy-/3-Dneuraminic acid; 9-Fluoresceinyl-NeuAc, 5-acetamido-9-(3-fluoresceinylthioureido)-3,5,9-trideoxy-~-Dglycero-D-galacto-2-nonulosonic acid; CMP-NeuAc, cytidine-5’-monophospho-N-acetylneuraminic acid; CMP-9-fluoresceinyl-NeuAc, cytidine-5’-monophospho-9-fluoresceinyl-NeuAc; GMl, Galpl,3GalNAcpl,4(NeuAca2,3)Gal~l,4Glc-ceramide; ST, sialyltransferase; DTE, dithioerythritol. Enzymes; Galpl,4GlcNAc a2,6-sialyltransferase (EC 2.4.99.1); Galbl,3GalNAc a2,3_sialyltransferase (EC 2.4.99.4); GalNAc a2,6+ialyltransferase (EC 2.4.99.3). Correspondence to: Hans Jttrgen Gross, Institut fur Biochemie II, Universitlt Heidelberg, 69 Heidelberg. lm Neuenheimer Feld 328, Germany.

238

Introduction Expression, activity and specificity of sialyltransferases, which catalyze terminal sialylation in the biosynthesis of glycoconjugate glycan structures [1,2], are of growing interest in biochemistry and clinical chemistry. For example, the quantitative and qualitative sialoglycoconjugate pattern of tumor cell surfaces seems to be significantly different from that of non-transformed cells [3-51, which may be a result of changes in the activity and specificity of the sialyltransferases expressed ([6-lo]; for review, see [ll]). In addition, increased sialyltransferase activity levels have been reported in sera of patients during inflammation and tumor growth [ 1 l-131. Studies presented previously indicated a correlation between an enhanced level of plasma sialyltransferase and clinical parameters of some types of cancer such as tumor mass, stage of disease and metastasis [11,13]. In contrast, others have claimed that an increased level of serum sialyltransferase is not a general phenomenon connected with malignant transformation. Therefore, the determination of sialyltransferase activity in different body fluids may provide important biochemical and diagnostic information. We now present a novel assay for the determination of sialyltransferase activity in human blood based on a newly developed fluorometric method described recently [14]. The procedure avoids handling of radiolabelled compounds and time consuming procedures to separate the glycoprotein product formed, and thus it is suitable for routine determination. With a sensitivity superior to common radiometric procedures [14], the assay permits measurement of the low activity levels of healthy probands towards distinct acceptor glycans, and has proved useful to uncover the existence of at least two plasma sialyltransferases, acting on glycoproteins, but differing in specificity and kinetic properties. Materials and methods Materials All chemicals used were of analytical grade and purchased from Merck (Darmstadt) or Serva (Heidelberg). Cytidine-5’-trisphosphate (CTP) was from Biomol (Ilvesheim), bovine serum albumin and Triton X-100 from Sigma (Munchen). CMP-NeuAc (CMP contamination below 4%) and CMP-9-fluoresceinyl-NeuAc (CMP contamination below 5%) were prepared enzymatically as described previously [15,16]. CMP-(3H)NeuAc (26.2 Ci/mmol) was purchased from New England Nuclear (Boston). a,-Acid-glycoprotein [17] was a generous gift of Dr. Karl S&mid (Boston) and antifreeze glycoprotein (Fractions 3-5, from serum of Pagothenia borchgrevinki [18]) was kindly donated by Dr. Robert E. Feeney (Davis). Fetuin was obtained from Serva (Heidelberg) and further purified on Sepharose 6B. Ganglioside G,, was obtained from Pallmann KG (Miinchen). Galpl,3GalNAc a2,3- and GalNAc (Y~,~-ST purified from porcine submaxillary glands as described previously [19,20] were obtained from Dr. J.C. Paulson (Los Angeles). GalPl,4GlcNAc (Y~,~-ST from

239

rat- and human liver were isolated in this laboratory according to [21,22], also a Gal/31,3GalNAc (Y~,~-STwas purified 6 OOO-foldfrom porcine liver (unpubl. results). Methods Protein determination

Protein content was determined by the Bio-Rad protein assay (Bio-Rad, Miinchen) using bovine serum albumin as standard. Desialylation of sialoglycoproteins After treatment with Vibrio cholerae sialidase (EC 3.2.1.18) [23] asialoq-acid

glycoprotein, or asialofetuin contained each about 0.2% bound NeuAc. Degalactosylation of antifreeze glycoprotein

Antifreeze glycoprotein or galactoglycoprotein (3 mg) were treated with P-galactosidase (EC 3.2.1.23) purified from bovine testis (0.5 U, 3.0 U/mg) for 48 h at 37 o C. Galactosidase was separated by affinity chromatography on thiogalactosideSepharose 4B, and the agalacto-glycoprotein desalted on Sephadex G-100. The final agalacto-glycoprotein was tested as acceptor for Galj31,3GalNAc ~u2,3-and GalNAc c~2,6-ST purified from porcine submaxillary glands; incorporation of NeuAc into agalacto-antifreeze glycoprotein or agalacto-galactoglycoprotein with the first enzyme was below 2%, with the latter enzyme 45%, relative to the transfer value obtained with the parent glycoprotein under similar conditions. Galactose acceptor sites

Sites were expressed in terms of galactose content of asialo-q-acid glycoprotein, antifreeze glycoprotein, galactoglycoprotein, asialofetuin, and G r,,n as described previously [16]. Fluorometric sialyltransferase assay

Human blood was obtained by venipuncture (3 mmol/l sodium EDTA as anticoagulant), and erythrocytes were separated by centrifugation at 800 x g for 10 min (4’ C). The resulting supematant (EDTA-plasma) was used for the determination of sialyltransferase activity. Standard assay [14]

The standard reaction mixture (30 ~1) for determination of serum sialyltransferase contained 62.5 mmol/l sodium cacodylate buffer, pH 6.8, and 25 pmol/l or 40 pmol/l CMP-9-fluoresceinyl-NeuAc. The following acceptor concentrations were used: 1.6 g/l asialo-q-acid glycoprotein (720 pmol/l galactose acceptor sites), or 1.7 g/l antifreeze glycoprotein (2 300 pmol/l galactose/N-acetylgalactosamine acceptor sites), or 4 g/l asialofetuin (830 pmol/l total galactose acceptor sites), or 4 g/l fetuin (830 pmol/l total galactose acceptor sites), or 1.5 g/l agalacto-antifreeze glycoprotein (2200 pmol/l Nacetylgalactosamine acceptor sites), or 2.0 g/l G,, (2100 ~mol/l galactose accep-

240

tor sites). An assay without exogenously added glycoprotein served to determine the activity towards endogenous acceptors. According to the published carbohydrate structure of antifreeze glycoprotein an equimolar ratio of galactose/N-acetylgalactosamine was assumed [24]. The reaction routinely was initiated by addition of 5 ~1 of plasma (about 300 pg protein) and terminated after appropriate times of incubation at 37 ’ C by addition of 4 ~1 0.1 mol/l CTP. Corresponding controls were carried out in the presence of 12 mmol/l CTP. Assay tubes were protected from light. Kinetic assay Kinetic data for CMP-9-fluoresceinyl-NeuAc were determined using duplicate fluorometric assays as above with asialo-a,-acid glycoprotein (720 pmol/l galactose sites) and antifreeze glycoprotein (2 300 pmol/l galactose sites) at 5 concentrations of donor substrate near the respective K, value. Kinetic data of acceptor glycoproteins were obtained using duplicate fluorometric assays as above with CMP-9-fluoresceinyl-NeuAc (30 pmol/l) at 5 concentrations of acceptor substrate, in terms of galactose sites, near the respective K, value. Fluorometric separation system The liquid chromatography system was composed as described previously [14], a Hitachi D-2000 Chromatointegrator was employed instead of the one-channel recorder. 2-10 ~1 of the reaction mixture were analysed by chromatography on a column of Sephadex G 50 fine exactly as described previously [14]. Results Plasma sialyltransferase assay Fluorometric plasma sialyltransferase measurements were performed applying the new highly sensitive assay for this enzyme class described previously, which is based on the transfer of 9-fluoresceinyl-NeuAc onto glycoprotein acceptors and quantification of protein-bound fluorescence after gel filtration [14]. No fluorescence quenching of protein-bound 9-fluoresceinyl-NeuAc was caused by plasma up to 5 ~1, by Triton X-100 up to 1% or divalent cations up to 10 mmol/l. Protected against light, fluorescence yield was stable during storage at 4’ C for at least 3 days. The initial transfer rate of plasma ST for each acceptor glycoprotein was linear with respect to time for at least 2.5 h (Fig. 1). The same result was obtained in presence of 0.1% Triton X-100. Product formation was linear with the amount of plasma up to 600 pg and 300 pg plasma protein with asialo-cri-acid glycoprotein and antifreeze glycoprotein, respectively. In contrast, in presence of 0.1% Triton X-100, product formation with asialo-a,-acid glycoprotein was linear only up to 100 pg plasma protein. Thus, the plasma sialyltransferase assay was performed routinely without detergent.

241

35-

30-

25rT 0

E LP.

20-

15-

. 30

60

90

120

150

180

Fig. 1. Time course for the transfer of 9-fluoresceinyl-NeuAc by plasma ST onto aisalofetuin (0 -.)> X ), antifreeze glycoprotein (0 0), and endogenous acceptor asialo-q-acid glycoprotein (X Standard fluorometric assay was performed in duplicate at 25 pmol/l CMP-9(A -A). fluoresceinyl-NeuAc as described in ‘Materials and Methods’.

Effect of divalent cations, detergent and anticoagulants enhance the activity of plasma The divalent cation, Mn2’, did not significantly ST, employing either asialo-q-acid glycoprotein or asialofetuin as acceptor, whereas 5 mmol/l Mg2+ increased the activity by about 10% with both acceptors (Table I). Whereas Zn” did not affect enzyme activity, HgZf and Cu2+ inhibited the plasma enzyme, each to a different extent. The complexing agent EDTA was used as anticoagulant because it did not significantly influence plasma ST activity up to 10 mmol/l. In contrast, presence of sodium citrate or heparin in the standard incubation mixture reduced plasma ST activity (Table I). The latter anticoagulant markedly decreased the activity (60-85%) at 12.5 U/ml, a concentration commonly used to prevent blood coagulation (Table I). Addition of the nonionic detergent Triton X-100 to the standard reaction mixture (5 ~1 plasma) highly inhibited ST activity (Table I); applying only 1 ~1 of plasma, inhibition by the detergent was significantly lower (Table I). Specificity

of plasma sialyltransferase

The fluorometric determination of plasma ST activity was performed with different acceptors (Fig. 1, Table II): Asialofetuin served to determine the total activity (75% Gal sites on N-linked and 25% on O-linked glycans (for structure, see [25,26])). Asialo-q-acid glycoprotein and antifreeze glycoprotein were used to differentiate between activity towards the acceptor structure Gal/31,4GlcNAc on N-linked glycans and Galj31,3GalNAc on O-linked glycans, respectively [24,27]. In order to define exactly the acceptor specificity of the plasma enzyme towards

242

TABLE

I

influence of detergent, activity of plasma ST

divalent

Additives

cations,

sodium

EDTA,

Activity

standard assay + 0.1% T&n X-100 + 0.5% Triton X-100 + 1.0% Triton X-100 + 1.25 mmol/l MgCl 2 + 5 mmol/l M&l, + 1.25 mmol/l MnCl 2 + 5 mmol/l MnCl 2 + 0.01 mmol/l HgCl z + 0.1 mmol/l cuc12 + 1 .o mmol/l CUCI 2 + 1 .O mmol/l ZnCl 2 + 2.5 mmol/l NaEDTA + 5 mmol/l NaEDTA + 10 rrmtol/l NaEDTA + 12.5 IE/ml Heparin + 20 mmol/l Na citrate + 1 mmol/l DTE + 0.05 mol/l NaCl + 0.1 mol/l NaCl + 0.5 mol/l NaCl

DTE, sodium

chloride

and anticoagulants

on the

(W)

AF

AORM

100 19 (60) a 15 (65) a 10 (50) a 96 115 96 105 _

100 16 (100) = 13 (105) a 14 (115) a _ 110 105 104

_ _ _ _

78 71 99 92 95 90 39 _

loo _ 14 64 73 69 59 13

67 100 85 33

Standard fluorometric assay was performed in duplicate at 25 pmol/l CMP-9-fluoresceinyl-NeuAc for 30 min employing asialofetuin (AF) or asialo-q-acid glycoprotein (AORM) as described in ‘Materials and Methods’. a Assay performed with only 60 ng of plasma protein ( = 1 ~1).

TABLE Plasma plasma Acceptor

II ST activity determined of four probands

with several specific acceptor

glycoproteins

Relative

substrate

Asialo-fetuin Asialo-q-acid glycoprotein Antifreeze glycoprotein Agalacto-antifreeze glycoprotein Agalacto-galactoglycoprotein G En?ogenous

calculated

(830 ymol/l Gal) (720 pmol/l Gal) (2 300 pmol/l Gal/GalNAc) (2 200 c mol/l GalNAc) (2 300 pmol/l GalNAc) (2 100 pmol/l Gal/GalNAc)

acceptors

Standard fluorometric assay was performed at 40 pmol/l described in ‘Materials and Methods’. The concentration acceptor sites of each acceptor are given for comparison.

as mean value from

activity

(%)

100 75 30 25 90 2.5 2

CMP-9-fluoresceinyl-NeuAc for 45 min as of total galactose or N-acetylgalactosamine

243

O-linked glycans, activity was additionally determined with agalacto-antifreeze glycoprotein and agalacto-galactoglycoprotein, which both contain only GalNAc-residues O-linked to serine or threonine [28]. The activity measured with antifreeze glycoprotein (30%, Table II) was almost identical to that obtained with the corresponding agalacto-glycoprotein (25% Table II), identifying this enzyme as a GalNAc-ST acting on O-glycosidically linked glycans. This conclusion was supported by the high activity measured with agalacto-galactoglycoprotein. Activity determined with ganglioside G,,, which contains the terminal glycan sequence GalPl,3GalNAc [29], reflected only the level towards the endogenous acceptor (Table II). Corresponding controls for unspecific fluorescence adsorption were performed in presence of 12 mmol/l CTP which was sufficient to completely inhibit ST activity [30]. Kinetic properties of plasma sialyltransferase

Kinetic data obtained for CMP-9-fluoresceinyl-NeuAc and for acceptor glycoproteins are shown in Table III. The K, value for the fluorescent donor CMP-glycoside obtained with asialo-cu,-acid glycoprotein (4-6 pmol/l) and antifreeze glycoprotein (30-45 pmol/l) was markedly different (Table III) indicating two distinct

TABLE III Apparent K, value of plasma ST for CMP-9-fluoresceinyl-NeuAc, antifreeze glycoprotein Plasma ST (range determined from 4 probands): K, for CMP-9-~uores~~yl-Ne~c K,

for AORM

Rat liver Galj31,4GlcNAc a2,6-ST: K, for CMP-9-fluoresceinyl-NeuAc XI, for AORM Human liver Galpl,rlGlcNAc a2,6-ST: K, for CMP-9-fluoresceinyl-NeuAc K, for AORM Porcine submaxillary gland GalNAc a2,6-ST: K, for CMP-9-fluoresceinyl-NeuAc

asialo-q-acid

glycoprotein

and

4-6 pmol/l (acceptor AORM) 30-45 pmol/l (acceptor AFG) 360-450 pmol/l a (donor CMP-9-fluoresc.-NeuAc) ir~mol/l (acceptor AORM) 340 pmol/l a {donor CMP-9-fluoresc.-NeuAc) 2 gmol/l (acceptor AORM) 380 gmol/l a (donor CMP-9-fluoresc.-Net&) 35 pmol/l (acceptor AFG)

Fluorometric assay for kinetic measurements was performed as described in ‘Materials and Methods’. Incubation was performed for 30 min with a&lo-q-acid glycoprotein (AORM), and for 45 min with antifreeze glycoprotein (AFG). Kinetic data were obtained from Hanes plots [34) employing plasma of four healthy probands. Kinetic data of purified rat liver, human liver ST and porcine submaxillary gland ST are given for comparison 1141. a Given as concentration of galactose sites.

244

ST specificities in human plasma. Considering these kinetic measured with antifreeze glycoprotein in Table II represents Sialyltransferase

parameters, the activity only the I’,,,,/2 value.

activity levels

Sialyltransferase activity obtained with asialo-a,-acid glycoprotein and antifreeze glycoprotein obtained with several plasma samples from five healthy probands ranged from 25 to 40 pU/ml, and 10 to 15 $J/ml, respectively (standard assay conditions). A significant activity could be obtained with only 1 ~1 plasma during 20 min incubation time. An activity of 0.0025 /.LJ represented the lower detection limit at 20 min incubation time (fluorescence intensity of sample/blank = 2.0). Activity was stable during storage of the plasma at 4” C for 3 weeks, and was preserved by deep freezing for at least several weeks. Radiometric

assay

The plasma ST activity was determined additionally by a radiometric assay procedure [22] using asialo-a,-acid glycoproteiriXand antifreeze glycoprotein: Activity determined fluorometrically was about 10% lower ‘in each case than the values obtained radiometrically (data not shown). However, the radiometric assay required a 7-fold higher amount of plasma and a 2.5 to 4-fold longer incubation time in order to yield a significant incorporation of (3H)NeuAc. This comparison demonstrates that, despite of the use of a NeuAc analogue with a bulky fluorescent substituent at position C-9, plasma ST reached similar transfer values with parent NeuAc and the fluorescent derivative. Discussion In general, function and origin of sialyltransferase activity in human blood as well as an increase observed during some pathological conditions such as inflammation and malignant transformation remains speculative [ll]. Some authors have assumed the liver to be the main organ for secretion of plasma sialyltransferase [ll]. In this context, a previous report showed very similar properties of porcine liver and porcine serum sialyltransferase [31], but another described significant kinetic differences between the human liver and serum enzyme [ll]. The reason suggested for elevation of glycosyltransferase activities in general in the plasma of cancer patients was, predominantly, a degradation of transformed cells or a shedding from their surface [ll]. Alternatively, erythrocytes were identified as possible main source for human serum sialyltransferase [32]. Frequently studies on serum sialyltransferase used subsaturating radiometric assays to measure the low activities, as such assays allow a higher specific labelling. Hence, fluorometric methods combine high sensitivity with the possibility of saturating donor concentrations. Moreover, these methods avoid handling of radiolabelled compounds. Recently, a fluorescent acceptor disaccharide derived from lactose was synthe-

245

sized and found to be suitable for determining a serum sialyltransferase activity by a fluorometric HPLC-assay [33]. In contrast with the assay presented in this paper, the use of a distinct fluorogenic acceptor only allowed detection of enzyme activity towards the sequence Gal/31,4Glc. The present sialyltransferase assay, which is based on the transfer of a fluorescent NeuAc analogue, 9-fluoresceinyl-NeuAc, onto different acceptor glycoconjugates, permits an easy detection of the low sialyltransferase activity in human plasma; the application of a fluorescent donor substrate allows a differentiation between the activity towards distinct glycan acceptor structures depending on the glycoprotein or glycolipid employed. Despite low incorporation rates (lo-40 pU/ml), kinetic data could be determined for the donor and the acceptor substrates. In order to establish the optimum assay conditions, the influence of several additives on plasma sialyltransferase activity was determined: divalent cations, detergent, anticoagulants, sodium chloride and DTE. No significant influence of on the plasma sialyltransferase Mg2+, Mn2+ or sodium EDTA up to 5 mmol/l activity was obtained in accordance with most previous reports [ll], and with the properties of purified human and rat liver Gal/31,4GlcNAc cu2,6_sialyltransferase [21,22]. Employing heparin instead of sodium EDTA to prevent coagulation, a strong inhibition of plasma sialyltransferase was measured. An identical influence of heparin was found on activity of both a2,6_sialyltransferases purified from human and rat liver (unpubl. results, [21]). The results obtained in this study clearly indicate the existence of at least two sialyltransferases differing in acceptor specificity: one acting on terminal galactose of complex type glycan structures (activity towards asialo-a,-acid glycoprotein), and the other acting on subterminal Nacetylgalactosamine of O-linked glycans (activity towards agalactoantifreeze glycoprotein). Considering the kinetic data, at saturating donor concentration both plasma enzymes surprisingly come up to almost identical sialyltransfer rates. The kinetic constants determined of the complex type specific plasma enzyme for CMP-9-fluoresceinyl-NeuAc and for asialo-cu,-acid glycoprotein (Table III) were in good accordance to the values obtained with the corresponding purified human and rat liver sialyltransferases (Table III). The donor K, values determined with the 0-glycan specific plasma enzyme and a purified porcine submaxillary GalNAc cY2,6_sialyltransferase were identical (Table III). There is some correlation between the human plasma sialyltransferase acting on complex type glycans and a purified human liver Gal/31,4GlcNAc a2,6sialyltransferase; i.e. donor and acceptor Km-values, influence of heparin and divalent cations. The origin of the 0-glycan specific enzyme remains speculative. The level of enzyme activity measured in this study with asialofetuin (35-55 pU/ml) was higher than the value described previously [ll] using CMP-( 3H)NeuAc as substrate; the activity obtained with the fluorogenic lactose derivative [33] was identical (38-50 pU/ml). It should, however, be noted that, in general, assay conditions for determination of human plasma sialyltransferase in previous reports are completely at variance, and thus the results are difficult to compare. Additionally, it is important to consider that plasma sialyltransferase activity depends on the anticoagulation method used, and only EDTA-plasma retains the full activity.

246

The results described in this paper represent an approach to define activity level, specificity and kinetic data of human plasma sialyltransferases by an easy, sensitive fluorometric assay. The new procedure should be suitable for a comparison, on the basis of kinetic properties and specificity, of normal and pathological plasma sialyltransferases, and thus may be useful in answering some of the many open questions regarding the clinical importance of sialyltransferases. AcknowIedgements

The excellent technical assistance of Jean Michel Krause is gratefully acknowledged. We are indebted to Dr. Karl Schmid (Boston), Dr. Robert E. Feeney (Davis) and Dr. J.C. Paulson (Los Angeles) for generously supplying glycoproteins and sialyltransferases. We also thank Petra Krapp for secretarial help. References 1 Jeanloz RW, Codington JF. The biological role of sialic acid at the surface of the cell. In: Rosenberg A, Schengrund, C-L, eds. Biological roles of sialic acid. New York: Plenum Press, 1976;201-238. 2 Corfield AP, Schauer R. Metabolism of sialic acids. In: Schauer R, ed. Sialic acids: chemistry, metabolism and function. Cell biology monographs, Vol. 10. Vienna: Springer Verlag, 1982;195-261. 3 Yogeeswaran G, Salk P. Metastatic potential is positively correlated with cell surface sialylation of cultured murine tumor cell lines, Science 1981;212:1514-1516. 4 Hakomori S. Tumor-associated carbohydrate antigens. AMU Rev Immunol 1984;2:103-126. 5 Fukuda M. Cell surface glycoconjugates as onto-differentiation markers in hematopoietic cells. B&him Biophys Acta 1985;780:119-150. 6 Grimes WJ. Sialic acid transferases and sialic acid levels in normal and transformed cells. Biochemistry 1970,8:5083-5091. 7 Alhadeff JA, Holzinger RT. Sialyltransferase, sialic acid and sialoglycoconjugates in metastatic tumor and human liver tissue. Int J Biochem 1982;14:119-126. 8 Berge P-G, Wilhelm A, Schriewer H. Sialyltransferase activity in tumor tissues. Khn Wochenschr 1984;62:331-336. 9 Baker MA, et al. Increased activity of a specific sialyltransferase in chronic myelogenous leukemia. Blood 1985;66:1068-1071. 10 Cohen AM, et al. Sialyltransferase activity and sialic acid levels in multiple myeloma and monoclonal gammopathy. Eur J Haematol 1989;42:289-292. 11 Weiser MM, Klohs WD, Podolsky DK, Wilson JR. Glycosyltransferases in cancer. In: Horowitz MI, ed. The glycoconjugates, Vol IV B. 1982;301-333. 12 Ronquist G, Nou E. Serum sialyltransferase and fucosyltransferase activities in patients with bronchial carcinoma. Cancer 1983;52:1679-1683. 13 Frithz G, Ronquist G, Ericsson P. Serum sialyltransferases and fucosyltransferase activities in patients with multiple myeloma. Eur J Cancer Clin Oncol 1985;21:913-917. 14 Gross HJ, Sticher U, Brossmer R. A highly sensitive fluorometric assay for sialyltransferase activity using CMP-9-fluoresceinyl-NeuAc as donor. Anal Biochem 1990;186:127-134. 15 Ckoss HJ, Btinsch A, Paulson JC, Brossmer R. Activation and transfer of novel synthetic 9-substituted sialic acids. Eur J Biochem 1987;168:595-602. 16 Gross HJ, Brossmer R. Enzymatic ~tr~u~tion of a fluorescent sialic acid into oligosaccharide chains of glycoproteins. Em J B&hem 1988;177:583-589. I7 S&mid K. a,-Acid glycoprotein. In: Putnam FW, ed. The plasma proteins, Vol. I, 2nd ed. New York: Academic Press, 1975;183-228. 18 DeVries AL, Komatsu SK, Feeney RE. Chemical and physical properties of freezing point-depressive glycoproteins fron antarctic fishes. J Biol Chem 1970;245:2901-2908.

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of

a2,6_sialyltransferase

from

rat

liver

by dye

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