Sialyltransferase of the 13762 Rat Mammary Ascites Tumor Cells1

[CANCER RESEARCH 44, 1148-1152, March 1984] Sialyltransferase of the 13762 Rat Mammary Ascites Tumor Cells1

ThÃ©rÃ¨sePrattand Anne P. Sherblom2

Department of Biochemistry, University of Maine, Orano, Maine 04469

ABSTRACT The MAT-B1 and MAT-C1 sublines of the 13762 rat mammary adenocarcinoma are a suitable system for studying sialic acid The MAT-B1 and MAT-C1 ascites sublines of the 13762 rat metabolism. The 2 cell lines, originally derived from the same mammary adenocarcinoma differ in morphology, agglutinability solid tumor, show marked differences in ability to be transplanted with concanavalin A, and xenotransplantability. Both cell lines into mice, agglutinability with concanavalin A, and total sialic acid contain a major mucin-type glycoprotein, but the MAT-C1 (xen- content (19). Greater than 70% of the protein-bound sialic acid otransplantable) subline contains a 3-fold-greater content of sialic in both cell lines is due to a high-molecular-weight mucin-type acid on the glycoprotein than does the MAT-B1 (nonxeno- glycoprotein, ASGP-1 (16). The 0-linked chains have a core transplantable) subline. structure Gal(01-Â»4)GlcNAc(01-Â»6)[Gal(|31-Â»3)]GalNAc3 where The present work indicates that whole cells of both lines both galactose residues may be substituted with sialic acids incorporate radioactivity from labeled CMP-sialic acid into a linked (Â«2â€”>3).4TheMAT-C1 subline contains much more of component which comigrates with the major glycoprotein by disialylated hexasaccharide than does the MAT-B1 subline,4 sodium dodecyl sulfate polyacrylamide gel electrophoresis, and whereas the MAT-B1 oligosaccharides are predominantly neutral that label incorporated by MAT-B1 cells is released by alkaline- but may contain sulfate as well as sialic acid (17). Thus, the borohydride treatment. Sialyltransferase can be purified from sublines do not appear to differ in the amount of ASGP-1 but, 250- to 400-fold by chromatography of a Triton X-100 extract of rather, in the relative proportion of sialylated O-linked oligosac microsomes on CDP-agarose. The purified fraction of both cell charides in ASGP-1. lines has a Km for CMP-sialic acid of 0.40 Â± 0.10 mw with This report describes studies of Sialyltransferase in MAT-B1 asialofetuin as the acceptor, and gives 35 to 40% of the activity and MAT-C1 cell lines. Preliminary reports of this work have with the acceptor asialotransferrin as with asialofetuin. When appeared (18, 20). assayed with a variety of acceptors, the MAT-C1 extract showed higher Sialyltransferase activity at a pH below 6.5 than did the MAT-B1 extract. Analysis of the products following incubation MATERIALS AND METHODS with lactose yields only 3'-sialyllactose for both cell lines. The All chemicals were reagent grade unless specified otherwise. [4-C14]- results indicate that the differences in MAT-B1 and MAT-C1 CMP-N-acetylneuraminic acid, with specific activity of 1.6 mCi/mmol, Sialyltransferase when assayed with glycoprotein acceptors are was obtained from New England Nuclear. Unlabeled CMP-NeuAc was not large enough to account for the differences in sialic acid prepared by the method of Kean and Roseman (7) and was additionally content of the two cell lines. purified by chromatography on Sephadex G-25 in ethanohwater (1:1). The glycoproteins fetuin and human transferrin were obtained from Sigma Chemical Co. and were desialylated by treatment with 0.05 M INTRODUCTION H2SOÂ«for 1 hr at 80Â°,neutralized, dialyzed against distilled water, and lyophilized. OSM was prepared by the method of Carlson ef al. (4) and Sialic acid, as a component of cell surface glycoproteins and was desialylated as described above. Galactosylated OSM was prepared glycolipids, is thought to play a role in cell-cell communication. by incubating asialo OSM with porcine submaxillary gland microsomes For example, the presence of sialic acid increases the lifetime of and UDP-galactose as described by Van den Eijnden ef al. (22). Carbo circulating glycoproteins and cells by preventing uptake by the hydrate analysis of the various acceptors (12) indicated that the standard blood clearance system (6). The role of sialic acid in other Sialyltransferase assay (0.1 mg acceptor) contained asialofetuin (15.5 nmol galactose), asialotransferrin (0.67 nmol galactose), asialo-OSM biological processes, including cellular adhesiveness, binding of (83.5 nmol GalNAc), or galactosylated OSM (17.2 nmol galactose and viruses to cells, activation of lymphocytes, and synaptic trans 77.6 nmol GalNAc). mission, has been reviewed (6, 13). Studies have indicated that Cells and Cell Fractions. MAT-B1 and MAT-C1 refer to ascites serum sialic acid levels are increased in cancer patients relative sublines of 13762 rat mammary adenocarcinoma which arose by pas to healthy individuals (21), and the sialic acid content of tumor sage of the MAT-B and MAT-C lines (Mason Research Laboratories, cells appears to correlate with metastatic ability (26). In mouse Worcester, MA) in the laboratory of K. L. Carraway. Tumor-bearing rats mammary tumor cells (TA3-Ha), a sialic acid-rich mucin, epigly- were kindly provided by K. L. Carraway, University of Miami School of canin, masks cell surface antigens, allowing the cells to grow in Medicine. Cells were maintained by weekly transfer and through frozen allogeneic hosts (5, 23). An understanding of the factors which stocks. The properties of these cell lines have been described previously regulate the content of cell surface sialic acid seems critical to (19). To ensure that no significant changes in the major glycoprotein had understanding viral infection, the immune response, tumorigen- 3The abbreviations used are: GalNAc, W-acetylgalactosamine; OSM, ovine esis, or metastasis at the molecular level. submaxillary mucin: Gal, galactose; GlcNAc, N-acetylglucosamme; SDS, sodium dodecyl sulfate; PBS, Dulbecco's phosphate-buffered saline containing 137 mw 1Support for this work was provided by a grant from Research Corp., a grant NaCI:2.6 rtiM KCI:8.0 rriM NaÃ®HPOÂ«7H2O:1.0rriM KHZPO., pH 7.4; NeuAc, N- from NIH (1 RO1 CA 33238-01), and the Maine Life Sciences and Agriculture acetylneuraminic acid. Experiment Station (Hatch Project 8404). 4 S. R. Hull, R. A. Laine, T. Kaizu, I. Rodriguez, and K. L. Carraway. Structures 2To whom requests for reprints should be addressed, at Department of Bio of the O-linked oligosaccharides of the major cell surface sialoglycoprotein of MAT- chemistry, Hitchner Hall, University of Maine, Orono, ME 04469. B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma, Received August 29, 1983; accepted December 1, 1983. submitted for publication.

1148 CANCER RESEARCH VOL. 44

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1984 American Association for Cancer Research. fiaf Mammary Tumor Sialyltransferase occurred, ASGP-1 from each cell line was isolated, and the carbohydrate acceptor lactose (58 mg/ml) were prepared as described above, except composition was determined (16). No significant differences in the current that a 3-fold-greater volume of all reagents was used. Following a 20- analysis and the findings reported previously were detected. In particular, min incubation of 37Â°and heat inactivation, the samples were treated the MAT-C1 glycoprotein still contained significantly more sialic acid as described by Paulson et al. (10); i.e., the reaction was quenched by (1.49 mol per mol of GalNAc) than did the MAT-B1 line (0.69 mol per addition of 1 ml of cold 5 ITIMsodium phosphate, pH 6.8. The mixture mol of GalNAc). was applied to a small column of Dowex 1-X8 (phosphate) to remove Cells were washed and homogenized as described previously (16). unreacted CMP-sialic acid. The eluate was applied to a Dowex 1-X8 Immediately after homogenization, the suspension was brought to a 3 (acetate; 1 ml) column; washed with 2 ml of water; eluted with 2 ml 0.5 rriM concentration in Mg2+ by addition of 30 mw MgCI2 and 100 HIM NaCI. M pyridine acetate, pH 5.0; and lyophilized. The sample was taken up in The homogenate was centrifuged at 1,000 x g for 1 min, and the 25 n\ of distilled water containing 100 nmol Sialyllactose (mixed isomers) supernatant was centrifuged at 10,000 x g for 10 min. The supernatant and 25 nmol A/-acetylneuraminic acid, and the entire sample was injected. following the second centrifugation is referred to as "crude extract," and Separation of the oligosaccharides was performed on a Waters Model the pellet following 100,000 x g x 60 min centrifugation of crude extract 272 liquid Chromatograph with Model 480 variable-wave length detector. is referred to as "microsomes." Triton X-100 extracts of both fractions A column of Bondapak NH (Waters radial cartridge) was used for all were prepared by incubation with 1% Triton X-100 and 10 ITIMTris, pH separations with conditions as described by Bergh et al. (2). Separation 8.0, on ice for 30 min, and centrifugation at 100,000 x g for 60 min. of Sialyllactose isomers was performed isocratically with acetonitrile:15 Sialyltransferase Assay. The assay mixture contained the following rtiM potassium phosphate, pH 5.2 (72:28) with a flow rate of 2 ml/min. components in a total volume of 70 /il: (a) [14C]CMP-A/-acetylneuraminic The absorbance of the eluent was monitored at 195 nm. Beginning at acid (63 nmol; 220,000 dpm); (b) sodium cacodylate buffer (10 nmol); (c) 10 to 15 min postinjection, fractions were collected at 0.5-min intervals. bovine serum albumin (20 ^g); and (d) either a glycoprotein acceptor (0.1 The absorbance trace was aligned with the radioactivity profile by mg; see above) or distilled water. The pH of the sodium cacodylate injection of the standard [14C]NeuAc. buffer was adjusted with acetic acid at room temperature prior to the assay, and the standard assay was performed in the presence of RESULTS asialofetuin at a pH of 6.75. The assay mixture was incubated at 37Â°for 10 or 20 min, and the Endogenous Acceptor of Cellular Sialyltransferase. Incu reaction was quenched with 1% phosphotungstic acid and 0.5 M MCI. bation of MAT-B1 and MAT-C1 cells with [3H]CMP-sialic acid Samples were filtered and counted as described by Paulson ef al. (9) resulted in incorporation of 3H into material precipitable with 1% using a Beckman LS7500 scintillation counter. Under these conditions, at least 40% of the major glycoprotein ASGP-1 in both cell lines was phosphotungstic acid:0.5 M MCI. Preliminary experiments dem onstrated that the incorporation is linear for up to 20-min incu precipitatale. Duplicate values typically did not differ by more than 20%. Protein was estimated by the method of Lowry e/ al. (8). bation, and that the endogenous activity of MAT-B1 cells is Affinity Chromatography. CDP-agarose (6-carbon spacer; Sigma), greater than that of MAT-C1 cells (3.2 versus 1.0 pmol/min/mg which had been equilibrated with 10 HIM sodium cacodylate, pH 6.5:50 cell protein). The endogenous activity decreased 3- to 10-fold by mÂ«NaCI: 1% Triton X-100 (Buffer A), was used as the affinity absorbent. the addition of 1% Triton X-100. Crude extract or microsomal extract from approximately 1 to 2 x 109 Analysis of the endogenous acceptor by SDS:polyacrylamide ascites cells (25 to 40 ml; protein, 5 to 10 mg/ml) was mixed with 5 to gel electrophoresis (Fig. 1) indicated that the major acceptor in 10 ml of CDP-agarose, allowed to stand at 4Â°for 3 hr, and packed in a both MAT-B1 and MAT-C1 cells was a high-molecular-weight 0.7-cm column. Following elution of the sample, the column was washed with 10 bed-volumes of 10 HIM sodium cacodylate, pH 6.5:50 mw component with the same mobility as the major glycoprotein, ASGP-1. Further analysis of the MAT-B1 acceptor by chroma- NaCI:1% Triton X-100 (Buffer B) and 3 to 5 bed-volumes of 10 mw tography of the alkaline-borohydride-treated product (Chart 1) sodum cacodylate, pH 6.5:100 HIM NaCI:0.3 rriM CTP:1% Triton X-100 showed that radioactivity from [14C]CMP-sialic acid had been (Buffer C). Fractions of 1.5 ml were collected and assayed for Sialyltrans ferase activity and protein. incorporated into alkali-labile chains. Characterization of Endogenous Acceptor of Sialyltransferase. Activity of Various Fractions Following Cell Homogeniza Ascites cells were washed 3 times with PBS and 1 x 107 cells in a total tion. Homogenates and fractions from MAT-B1 and MAT-C1 volume of 0.25 ml were incubated in PBS with 0.4 ^mol each of MnCI2 cells were assayed for Sialyltransferase activity at pH 6.75 in the and MgCI2 with [3H]CMP-sialic acid (2.3 ^Ci; 1 nmol) at 37Â°for 10 or 20 presence of Triton X-100 with asialofetuin as the acceptor. min. Cold PBS (0.2 ml) was added, and the sample was immediately Typically, the incorporation with homogenates of both cell lines solubilized by addition of 0.4 ml of sample buffer (0.25 M Tris, pH 6.8:3% SDS:5% 0-mercaptoethanol:10% glycerol) and immersion in a was not stimulated by the addition of asialofetuin, whereas boiling water bath. Labeling of cells by periodate:[3H]borohydride, microsomes gave a specific activity of 12.5 (MAT-B1) or 11.9 (MAT-C1) pmol/min/mg with asialofetuin when corrected for SDS:polyacrylamide gel electrophoresis, and fluorography were per formed as described previously (19). endogenous activity. At least 50% of the activity remained in the In a separate experiment, MAT-B1 ascites cells were incubated with supernatant during the centrifugation used to pellet the micro [MC]CMP-sialic acid (0.3 nC\; 0.19 ^mol) for 20 min under the conditions somes. Thus, further studies were performed on microsomes as described above, and were solubilized with sample buffer. The sample well as on the crude extract which contained both microsomal was chromatographed on a Sephadex G-50-40 column (1.0 x 40 cm) in and cytosolic material. 0.5% deoxycholate:10 mw Tris, pH 8.0, to separate the product from Affinity Chromatography on CDP-Agarose. The pattern unreacted [14C]CMP-sialic acid. Void volume fractions were pooled, shown in Chart 2 was obtained when crude extract from MAI- dialyzed/concentrated against distilled water with a collodion bag appa ratus, treated for 16 hr at 45Â° with 0.05 M NaOH:1 M NaBHÂ«,and EI cells was chromatographed on CDP-agarose. Essentially 3 chromatographed on a Bio-Gel P-4 column (1 x 100 cm) in 0.1 M pyridine peaks of activity were obtained: (a) Peak I, eluting with 1 to 2 acetate, pH 6.0. For comparison, MAT-B1 ASGP-1 was purified from bed volumes of 50 rriM NaCI; (b) Peak II, eluting with 8 to 10 cells which had been labeled with [3H]glucosamine (16) and chromato bed-volumes of 50 mw NaCI; and (c) Peak III, eluting with 2 to 3 graphed on the same Bio-Gel P-4 column following alkaline-borohydride bed volumes of 0.1 M NaCI:0.3 ITIMCTP. These same 3 peaks treatment. of activity were observed for virtually all of the samples examined, Analysis of Sialyllactose Product. Assay mixtures containing the and the relative recoveries of activity in the 3 peaks were 15 to

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30% (Peak I), 5 to 15% (Peak II), and 3 to 10% (Peak III). No (MAT-C1) pmol/min/mg protein, showing a 250- to 450-fold significant differences were detected between microsomes and purification from microsomes. The Km for CMP-sialic acid with crude extract or between fractions from MAT-B1 and MAT-C1 asialofetuin as the acceptor was in the range of 0.4 Â±0.1 mivi cells. Peak III had a specific activity of 3200 (MAT-B1) or 5400 for Peak III from both MAT-B1 and MAT-C1 cells. Peak III from both cell lines also showed activity toward the acceptor asialotransferrin, yielding 35 to 40% of the activity obtained with asialofetuin. Activity with Various Acceptors as a Function of pH. Crude extracts were tested with various acceptors to determine whether the MAT-B1 and MAT-C1 sialyltransferases differed in ASGP-I- â€¢ specificity. With asialofetuin as the acceptor (Chart 3A), the 240 K- extracts from both cell lines showed a broad plateau of activity 220 K- from pH 6.0 to 7.5. Asialofetuin, which contains 3 O-linked oligosaccharides [Gal(/i1â€”>3)GalNAc]and 3 triantennary W-linked oligosaccharides [each with nonreducing terminus [Gal(/31â€”Â» 4)GlcNAcâ€”),can serve as an acceptor for a variety of sialyltrans-

90 K- I 1500-

1000- 45 K-

40 60 80 100 ELUTION VOLUME (ml) I7 K- Chart 2. Chromatography of MAT-B1 crude extract on CDP-agarose. The eluting buffer was Buffer A, was changed to Buffer B (left arrow) and to Buffer C (right arrow). Fractions were assayed for protein (A) and sialyltransferase activity (O). Radioactivity values were not corrected for endogenous activity. AB CDEFGH

Fig. 1. SDS:polyacrylamide gel electrophoresis of MAT-B1 and MAT-C1 cells. MAT-B1 (A. C, E, F) or MAT-C1 (B, D, G, H) cells were treated as described in text. A. B, Coomassie blue; C. D, fluorogram of cells labeled with periodate:(3H]- BH4; E, G, fluorogram of cells incubated 10 min with [3H)CMP-sialic acid; F, H, fluorogram of cells incubated 20 min with [3H]CMP-sialic acid.

8.0

40 SO Chart3. Sialyltransferase activity as a function of pH. The crude extract of ELUTION WEIGHT MAT-B1 (O; 0.13 mg protein) or MAT-C1 (A; 0.14 mg protein) cells was assayed Chart 1. Gel filtration on Bio-Gel P-4 following alkaline-borohydride treatment. for sialyltransferase activity as described in the text. Values, which represent cpm Details are given in the text. A, purified ASGP-1 from [3H]glucosamine-labeled cells; of MC incorporated from [14C]CMP-sialic acid, are the average of duplicates and B, endogenous acceptor of MAT-B1 sialyltransferase monitored by incorporation are corrected for endogenous activity. Each assay contained 0.1 mg of acceptors of "C from ("C]CMP-sialic acid. asialofetuin (A), asialotransferrin (B), asialo OSM (C). or galactosylated OSM (D).

1150 CANCER RESEARCH VOL. 44

ferases. The product formed by incubation (pH 6.75) of MAT-B1 (10). The results confirmed the finding that both cell lines incor crude extract with [14C]CMP-sialic acid and asialofetuin was porated approximately twice as much radioactivity at pH 7.4 alkaline-borohydride-treated and chromatographed on Bio-Gel than at pH 6.55, although no differences in the specific activity P-4. Most of the radioactivity appeared in the void volume, of MAT-B1 and MAT-C1 microsomes toward lactose were de indicating that incorporation was predominantly onto A/-linked tected (data not shown). chains (data not shown). With the mucin acceptors, OSM (Chart 3C) and galactosylated OSM (Chart 3D), the MAT-B1 extract DISCUSSION showed optimal activity around pH 7.5, whereas the MAT-C1 The results demonstrate that, with asialofetuin as the accep extract gave moderate activity from pH 6.0 to 7.5. OSM contains tor, no significant differences in MAT-B1 and MAT-C1 sialyltrans predominantly the side chain GalNAc, whereas galactosylated OSM contains GalNAc (78%) and Gal(/31-Â»3)GalNAc (22%). ferase could be detected by: (a) specific activity of microsomes; (b) CDP-agarose chromatography; or (c) activity and Kmfor CMP- Analysis of the products with OSM as the acceptor was per formed by high-performance liquid chromatography following sialic acid of a partially purified sialyltransferase. Although MAT-C1 sialyltransferase appeared more active than alkaline borohydride treatment (2, 15). None of the product the MAT-B1 enzyme at a pH of 6.5 to 6.7, it is unclear whether NeuAc(a2â€”Â»6)GalNAcwas detected, although the formation of Gal(/31â€”>3)[NeuAc(a2â€”>6)]GalNAc could not be ruled out by this difference could result in increased sialylation under physio the analysis. Thus, it is likely that, with OSM, the Gal(/31-> logical conditions. With respect to specificity of the tumor cell sialyltransferases, both W-linked (asialotransferrin) and O-linked 3)GalNAc chains preferentially serve as the acceptor, even (OSM and galactosylated OSM) glycoproteins serve as accep though these chains comprise only about 5% of the total. With asialotransferrin, which contains /V-linked chains terminating in tors. Neither cell line catalyzes the transfer of sialic acid to GalNAc side chains of OSM, and the sole product with lactose Gal(01â€”Â»4)GlcNAc,bothcell lines showed moderate activity, with as the acceptor is 3'-sialyllactose. the MAT-C1 extract more active at low pH than the MAT-B1 Previous work has shown that rat mammary gland contains a extract (Chart 38). sialyltransferase which forms 3'-sialyllactose (3). However, this Lactose as the Acceptor. Since the sialyltransferase from both cell lines showed activity with both A/-linked and O-linked enzyme was less active with orosomucoid than with lactose and was not active with sialidase-treated submaxillary mucins (3). It chains, it was possible that enzymes capable of forming 2 types of linkages to galactose [(Â«2â€”>3)and(Â«2â€”Â»6)]werepresent in is unlikely that an enzyme with the same properties as normal the cell extracts. To test for these 2 classes of enzyme, micro- rat mammary gland sialyltransferase participates in the synthesis somes were incubated with lactose and [14C]CMP-sialic acid, of such large amounts of mucin as are found in the MAT-B1 and MAT-C1 cells. and the products were analyzed by high-performance liquid It is also unclear as to whether one enzyme catalyzes the chromatography. The results (Chart 4) demonstrate that, for both cell lines at pH 6.55 and pH 7.40,3'-sialyllactose is the only transfer of sialic acid to both the Gal(01-Â»3)GalNAc and Gal(/31-Â» 4)GlcNAc termini of ASGP-1. The Gal-R(Â«2^3) sialyltransferase isomer formed. Furthermore, both cell lines incorporated more active on the former acceptor was purified from porcine submax radioactivity (1.5 to 2 times as much) at pH 7.4 than at pH 6.55. Incorporation of radioactivity from [14C]CMP-sialic acid into illary glands (14) and was shown to be minimally active with the Gal(/31->4)GlcNAc termini of W-linked glycoproteins (11). Simi sialyllactose was determined as a function of pH for MAT-B1 larly, the Gal-R(Â«2â€”>3)enzymeactive on the latter acceptor as and MAT-C1 microsomes, using the method of Paulson et al. part of an W-linked oligosaccharide has been purified from rat liver (24) and shown to be inactive on antifreeze glycoprotein which contains Gal(,01â€”>3)GalNAcchains (25). Furthermore, a â€¢A 500 sialyltransferase in embryonic chicken brain catalyzes the trans 0.20 Â«JO fer of sialic acid to both glycolipid termini Gal(01â€”Â»4)GlcNAcand Gal(01â€”Â»3)GalNAc,but is not active on the O-linked Gal(01-> MO 0.10 3)GalNAc found in mucin (1). Thus, it appears that either the 200 MAT-B1 and MAT-C1 sialyltransferase is of unusually broad toc- specificity, accepting lactose and O-linked termini Gal(ÃŸ1â€”Â» ; o 3)GalNAc and Gal(/31^4)GlcNAc, or that 2 different enzymes 500- B are required in the synthesis of ASGP-1. The present work suggests that, with the acceptors used, no Â«00- 0-20 dramatic differences in sialyltransferase activity were detected 300' between the MAT-B1 and MAT-C1 cell lines. Other aspects of 0.10 sialic acid metabolism, including CMP-sialic acid synthetase and 200 neuraminidase, are currently under investigation. 100

20 25 20 25 30 REFERENCES TIME (min) 1. Basu, M., Basu, S., Stoffyn, A., and Stoffyn, P. Biosynthesis in vitro of Chart 4. Analysis of sialyllactose products by high-pressure liquid chromatog sialyl(a2â€”Â»3)neolactotetraosylceramide by a sialyltransferase from embryonic raphy. MAT-B1 (A, B) or MAT-C1 (C, 0) vesicles were incubated with ["CJCMP- chicken brain. J. Biol. Chem. 257:12765-12769, 1982. sialic acid and lactose at a pH of 6.55 (A, C) or 7.40 (B, D) as described in 'Materials 2. Bergh, M. L. E., Koppen, P., and Van den Eijnden, D. H. High-pressure liquid and Methods." The absorbance trace ( ) is from a mixture of N-acetylneuraminic chromatography of sialic acid-containing oligosaccharides. Carbohydr. Res., acid (7), 3'-sialyllactose (2), and 6'-sialyllactose (3) which was added to the sample, 94:225-229,1981. and the radioactive trace ( ) has been corrected for a heat-inactivated control. 3. Carlson, D. M., Jourdian, G. W., and Roseman, S. Synthesis of sialyl-lactose

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Susan P. Banks-Schlegel and Curtis C. Harris

Cancer Res 1984;44:1153-1157.

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