Sialyltransferase Assays Research Articles

A Rapid and Sensitive MicroPlate Assay (MPSA) Using an Alkyne-Modified CMP-Sialic Acid Donor to Evaluate Human Sialyltransferase Specificity.

Human sialyltransferases primarily utilize CMP-Sias, especially transferring Neu5Ac from CMP-Neu5Ac to various acceptors. Advances in chemical biology have led to the synthesis of novel CMP-Sia donors suitable for bioorthogonal reactions in cell-based assays. However, the compatibility of these donors with all human enzymes remains uncertain. We synthesized a non-natural CMP-Sia donor with an alkyne modification on the N-acyl group of Neu5Ac, which was effectively used by human ST6Gal I and ST3Gal I. A sensitive MicroPlate Sialyltransferase Assay (MPSA) was developed and expanded to a panel of 13 human STs acting on glycoproteins. All assayed enzymes tolerated CMP-SiaNAl, allowing for the determination of kinetic parameters and turnover numbers. This study enhances the biochemical characterization of human sialyltransferases and opens new avenues for developing sialyltransferase inhibitors.

Salmonid polysialyltransferases to generate a variety of sialic acid polymers

The human polysialyltransferases ST8Sia II and ST8Sia IV catalyze the transfer of several Neu5Ac residues onto glycoproteins forming homopolymers with essential roles during different physiological processes. In salmonids, heterogeneous set of sialic acids polymers have been described in ovary and on eggs cell surface and three genes st8sia4, st8sia2-r1 and st8sia2-r2 were identified that could be implicated in these heteropolymers. The three polysialyltransferases from the salmonid Coregonus maraena were cloned, recombinantly expressed in HEK293 cells and the ST8Sia IV was biochemically characterized. The MicroPlate Sialyltransferase Assay and the non-natural donor substrate CMP-SiaNAl were used to demonstrate enzyme activity and optimize polysialylation reactions. Polysialylation was also carried out with natural donor substrates CMP-Neu5Ac, CMP-Neu5Gc and CMP-Kdn in cell-free and cell-based assays and structural analyses of polysialylated products using the anti-polySia monoclonal antibody 735 and endoneuraminidase N and HPLC approaches. Our data highlighted distinct specificities of human and salmonid polysialyltransferases with notable differences in donor substrates use and the capacity of fish enzymes to generate heteropolymers. This study further suggested an evolution of the biological functions of polySia. C. maraena ST8Sia IV of particular interest to modify glycoproteins with a variety of polySia chains.

Engineering of CHO cells for the production of vertebrate recombinant sialyltransferases.

BackgroundSialyltransferases (SIATs) are a family of enzymes that transfer sialic acid (Sia) to glycan chains on glycoproteins, glycolipids, and oligosaccharides. They play key roles in determining cell–cell and cell-matrix interactions and are important in neuronal development, immune regulation, protein stability and clearance. Most fully characterized SIATs are of mammalian origin and these have been used for in vitro and in vivo modification of glycans. Additional versatility could be achieved by the use of animal SIATs from other species that live in much more variable environments. Our aim was to generate a panel of stable CHO cell lines expressing a range of vertebrate SIATs with different physicochemical and functional properties.MethodsThe soluble forms of various animal ST6Gal and ST3Gal enzymes were stably expressed from a Gateway-modified secretion vector in CHO cells. The secreted proteins were IMAC-purified from serum-free media. Functionality of the protein was initially assessed by lectin binding to the host CHO cells. Activity of purified proteins was determined by a number of approaches that included a phosphate-linked sialyltransferase assay, HILIC-HPLC identification of sialyllactose products and enzyme-linked lectin assay (ELLA).ResultsA range of sialyltransferase from mammals, birds and fish were stably expressed in CHO Flp-In cells. The stable cell lines expressing ST6Gal1 modify the glycans on the surface of the CHO cells as detected by fluorescently labelled lectin microscopy. The catalytic domains, as isolated by Ni Sepharose from culture media, have enzymatic activities comparable to commercial enzymes. Sialyllactoses were identified by HILIC-HPLC on incubation of the enzymes from lactose or whey permeate. The enzymes also increased SNA-I labelling of asialofetuin when incubated in a plate format.ConclusionStable cell lines are available that may provide options for the in vivo sialylation of glycoproteins. Proteins are active and should display a variety of biological and physicochemical properties based on the animal source of the enzyme.

Novel Zebrafish Mono-α2,8-sialyltransferase (ST8Sia VIII): An Evolutionary Perspective of α2,8-Sialylation.

The mammalian mono-α2,8-sialyltransferase ST8Sia VI has been shown to catalyze the transfer of a unique sialic acid residues onto core 1 O-glycans leading to the formation of di-sialylated O-glycosylproteins and to a lesser extent to diSia motifs onto glycolipids like GD1a. Previous studies also reported the identification of an orthologue of the ST8SIA6 gene in the zebrafish genome. Trying to get insights into the biosynthesis and function of the oligo-sialylated glycoproteins during zebrafish development, we cloned and studied this fish α2,8-sialyltransferase homologue. In situ hybridization experiments demonstrate that expression of this gene is always detectable during zebrafish development both in the central nervous system and in non-neuronal tissues. Intriguingly, using biochemical approaches and the newly developed in vitro MicroPlate Sialyltransferase Assay (MPSA), we found that the zebrafish recombinant enzyme does not synthetize diSia motifs on glycoproteins or glycolipids as the human homologue does. Using comparative genomics and molecular phylogeny approaches, we show in this work that the human ST8Sia VI orthologue has disappeared in the ray-finned fish and that the homologue described in fish correspond to a new subfamily of α2,8-sialyltransferase named ST8Sia VIII that was not maintained in Chondrichtyes and Sarcopterygii.

Remodeling of Marrow Hematopoietic Stem and Progenitor Cells by Non-self ST6Gal-1 Sialyltransferase

Glycans occupy the critical cell surface interface between hematopoietic cells and their marrow niches. Typically, glycosyltransferases reside within the intracellular secretory apparatus, and each cell autonomously generates its own cell surface glycans. In this study, we report an alternate pathway to generate cell surface glycans where remotely produced glycosyltransferases remodel surfaces of target cells and for which endogenous expression of the cognate enzymes is not required. Our data show that extracellular ST6Gal-1 sialyltransferase, originating mostly from the liver and released into circulation, targets marrow hematopoietic stem and progenitor cells (HSPCs) and mediates the formation of cell surface α2,6-linked sialic acids on HSPCs as assessed by binding to the specific lectins Sambucus nigra agglutinin and Polysporus squamosus lectin and confirmed by mass spectrometry. Marrow HSPCs, operationally defined as the Lin-c-Kit+ and Lin-Sca-1+c-Kit+ populations, express negligible endogenous ST6Gal-1. Animals with reduced circulatory ST6Gal-1 have marrow Lin-Sca-1+c-Kit+ cells with reduced S. nigra agglutinin reactivity. Bone marrow chimeras demonstrated that α2,6-sialylation of HSPCs is profoundly dependent on circulatory ST6Gal-1 status of the recipients and independent of the ability of HSPCs to express endogenous ST6Gal-1. Biologically, HSPC abundance in the marrow is inversely related to circulatory ST6Gal-1 status, and this relationship is recapitulated in the bone marrow chimeras. We propose that remotely produced, rather than the endogenously expressed, ST6Gal-1 is the principal modifier of HSPC glycans for α2,6-sialic acids. In so doing, liver-produced ST6Gal-1 may be a potent systemic regulator of hematopoiesis.

Rec. ST6Gal-I variants to control enzymatic activity in processes of in vitro glycoengineering

Background Glycosylation is an important posttranslational modification of proteins influencing protein folding, stability and regulation of the biological activity. The sialyl mojety (sialic acid, 5-N-acetylneuramic acid) is usually exposed at the terminal position of N-glycosylation and therefore, a major contributor to biological recognition and ligand function, e.g. IgG featuring terminal sialic acids were shown to induce less inflammatory response and increased serum half-life. The biosynthesis of sialyl conjugates is controlled by a set of sugar-active enzymes including sialyltransferases which are classified as ST3, ST6 and ST8 based on the hydroxyl position of the glycosyl acceptor the Neu5Ac is transferred to [1]. The ST6 family consists of 2 subfamilies, ST6Gal and ST6GalNAc. ST6Gal catalyzes the transfer of Neu5Ac residues to the hydroxyl group in C6 of a terminal galactose residue of type 2 disaccharide (Galb1-4GlcNAc). To our knowledge, the access to recombinant ST6GalI for therapeutic applications is still limited due to low expression and/or poor activity in various hosts (Pichia pastoris, Spodoptera frugiperda and E. coli). The present study describes the high-yield expression of two variants of human beta-galactoside alpha-2,6 sialyltransferase 1 (ST6Gal-I, EC 2.4.99.1; data base entry P15907) by transient gene expression in HEK293 cells with yields >100 mg/L featuring distinct mono(G2 +1SA) as well as bi(G2+2SA) sialylation activity. Materials and methods Two N-terminally truncated fragments of human ST6Gal-I (delta89, residues 89-406, and delta108, residues 109-406) were designed for transient gene expression (TGE): Instead of the natural leader sequence and N-terminal residues, both ST6Gal-I coding regions harbor the Erythropoietin (EPO) signal sequence in order to ensure correct processing of the polypeptides by the secretion machinery. Following cloning into pM1MT, expression of the ST6Gal-I coding sequences is under control of a hCMV promoter followed by an intron A. Sialyltransferase assays: 1. Asialofetuin was used as acceptor and CMP-9F-NANA as donor substrate. Enzymatic activity was determined by measuring the transfer of 9F-NANA to asialofetuin. 2. Recombinant humanized IgG1 and IgG4 monoclonal antibodies (mabs), characterized as G2+0SA, as well as desialylated EPO were used as targets in sialylation experiments (30 μg enzyme/300 μg target protein). Both enzyme variants of ST6Gal-I (delta89 and delta108) were used under identical reaction conditions and the sialylation status was analyzed by mass spectrometry.

Synthesis of CMP-9″-modified-sialic acids as donor substrate analogues for mammalian and bacterial sialyltransferases

Cytidine-5′-monophospho-sialic acid (CMP-Neu5Ac) derivatives bearing a phenyl group in which the tether length between the phenyl group and the 9-position of Neu5Ac varied were synthesized and evaluated as substrates for sialyltransferases. In the synthesis of the compounds, a coupling reaction between methyl 5-acetamido-4,7,8-tri-O-acetyl-9-azido-3,5,9-trideoxy-β-d-glycero-d-galacto-2-nonulopyranosonate and 2-cyanoethyl 2′,3′-O,N4, triacetylcytidine-5′-yl N,N-diisopropylphosphoramidite was carried out and the phosphite derivative thus obtained was oxidized and then deprotected to yield CMP-9″-azido-Neu5Ac. Modification of the 9-amino group prepared by reduction of the azido groups was performed by the use of several phenyl-substituted alkylcarboxylic acid derivatives. Using these CMP-9″-modified-Neu5Ac analogues bearing the phenyl-substituted alkyl-amide group, sialyltransferase assays were performed with both rat liver α-(2→6)-sialyltransferase and Photobacterium α-(2→6)-sialyltransferase. These 9-modified analogues could be transferred to disaccharide acceptors, and a practical enzymatic synthesis using CMP-9″-modified-Neu5Ac yielded sialoside analogues and sialylglycoproteins in good yield. These experiments demonstrate that the Photobacterium sialyltransferase can be used in the synthesis of sialoside analogues having a large substituent at the 9-position of Neu5Ac.

A rapid, semi-automated method for detection of Galbeta1-4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) activity using the lectin Sambucus nigra agglutinin.

Sialyltransferase activity has traditionally been studied by determining the rate at which the enzyme transfers a labeled donor sugar to an acceptor substrate. These types of assays can be difficult to quantitate, and the separation of untransfered donor sugar from the sialylated acceptor is time-consuming. The biosensor-based method described here is both rapid and semi-automated. The NeuAc-alpha2-6Gal-R-specific lectin Sambucus nigra agglutinin (SNA) immobilized to the carboxymethyl dextran surface of a BIAcore sensor chip was used to detect and measure the formation of the NeuAc-alpha2-6Gal-R moieties. The sialyltransferase assays were carried out using modified protocols based on the method described in Rearick, J.I., Sadler, J.E., Paulson, J.C., and Hill, R.L. (1979) Enzymatic characterization of betaD-galactoside alpha2-3 sialyltransferase from porcine submaxillary gland. J. Biol. Chem., 254, 4444-4451. The complete assay mixture was simply diluted before injection into the instrument. All injections were performed automatically using the robotics of the BIAcore instrument. Using this technique it is possible to detect product from 0.4 microU of commercial Galbeta1-4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) (ST6Gal I). One unit of sialyltransferase is defined as the quantity that will transfer 1 micromol of N-acetylneuraminic acid from cytidine monophosphate (CMP)-N-acetylneuraminic acid to asialofetuin per min at pH 6.5 and 37 degrees C. The method described here requires as little as 10 microl total assay volume, thus reducing the consumption of reagents. In addition, the sample is completely recoverable from the sensor chip surface, which allows for downstream analysis of the reaction product if desired. This method eliminates the need for labeled donor and acceptor molecules and does not require the separation of the substrates from the product before analysis. Although some kinetic properties of the enzyme can be estimated using this method, further development and validation is required. The method is most useful in determining qualitative estimates of ST6Gal I activity in tissue extracts and in characterizing the production of enzymes in cultured cell systems. The use of a microtiter plate assay format enables the rapid screening of multiple fractions for sialyltransferase activity.

Neoglycolipids derived from phosphatidylethanolamine serve as probes in cell culture studies on glycolipid metabolism.

The neoglycolipid (NeoGL) N-acetyl-1-deoxy-1-phosphatidylethanolamino lacto-N-tetraositol [Lc4Ose-PtdEtn(NAc)] and the radioactivly labeled analog [Lc4Ose-PtdEtn(N[14C]Ac)] were synthesized by coupling the corresponding oligosaccharide to phosphatidylethanolamine (dihexadecyl) via reductive amination and subsequent N-acetylation with unlabeled and [14C]acetic acid anhydride, respectively. Lc4Ose-PtdEtn(N[14C]Ac) was then incubated with homogenates of rat small intestine epithelial cells (IEC-6) at pH 4. The reaction products were shown to be the degradation products formed by glycosidases by fast atom bombardment mass spectrometry (FAB MS). On the other hand, incubation of Lc4Ose-PtdEtn(NAc) with IEC-6 cell homogenates in sialyltransferase assays yielded the corresponding sialylated product. When Lc4Ose-PtdEtn(N[14C]Ac) was fed to IEC-6 cells as BSA complex, up to 5% of the NeoGL administered were taken up by the cells. After extraction of the NeoGL and separation by thin layer chromatography (TLC) the catabolic products Lc3Ose-PtdEtn(N[14C]Ac), Lac-PtdEtn(N[14C]Ac), and Glc-PtdEtn(N[14C]Ac), as well as the main anabolic product NeuGc-Lc4Ose-PtdEtn(N[14C]Ac) could be identified by FAB MS. These results demonstrate that PtdEtn-derived NeoGL can be used as probes for studies on the metabolism of specific oligosaccharide structures in cell culture.

New sialyltransferase inhibitors based on CMP-quinic acid: development of a new sialyltransferase assay.

Quinic acid (4) was transformed into phosphitamides 6, 14, and 15, which could be readily linked to 5'-O-unprotected cytidine derivative 7; ensuing oxidation of the obtained phosphite triesters with tert-butylhydroperoxide furnished the corresponding phosphate triesters 8, 16, and 17, respectively. Hydrogenolytic debenzylation of the phosphate moiety, base catalysed removal of acetyl protective groups, and basic hydrolysis of the methylester of the quinic acid moiety furnished CMP-Neu5Ac analogues 1-3. In order to measure their inhibition of sialyltransferases, a nonradioactive sialyltransferase assay [employed for alpha(2-6)-sialyltransferase from rat liver (EC 2.4.99.1)] based on reversed-phase HPLC separation of UV-labelled acceptor 20 (p-nitrophenyl glycoside of N-acetyllactosamine) from the UV-labelled product 21 (p-nitrophenyl glycoside of sialyl alpha(2-6')-N-acetyllactosamine) and p-nitrophenylalanine as internal standard was developed. The assay reproduced the reported K(M) values for CMP-Neu5Ac and N-acetyllactosamine and the Ki values for CDP. 1 and 2 turned out to be potent sialyltransferase inhibitors.

Characterization of two glycolipid: alpha 2-3sialyltransferases, SAT-3 (CMP-NeuAc:nLcOse4Cer alpha 2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer alpha 2-3sialyltransferase), from human colon carcinoma (Colo 205) cell line.

Sialyltransferase activities, SAT-3 (CMP-NeuAc:nLcOse4Cer alpha 2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer alpha 2-3sialyltransferase), in Colo 205 cells catalyze the transfer of sialic acid to the terminal galactose of GlcNc-- and GalNAc-containing glycolipid substrates, respectively. Competition kinetic studies with nLcOse4Cer and GM1 as substrates in a sialyltransferase assay show that these two activities are catalyzed by two different catalytic entities. The two enzymes were co-solubilized with taurochlorate and resolved by DEAE--Cibacron Blue--Sepharose column chromatography into two elution peaks. The column eluent with SAT-3 activity failed to transfer sialic acid to asialo alpha(1)-acid glycoprotein, indicating that this enzyme is different from the sialyltransferase (ST3N) that synthesizes NeuAc alpha 2-3Gal linkage in asparagine-linked oligosaccharides of glycoprotein. However, SAT-3 activity can be immunoprecipitated with a polyclonal antibody produced against a protein expressed in Escherichia coli as GST-fusion protein from an ECB cDNA homolog of an alpha 2-3 sialyltransferase SAT-3 or STZ) the has been cloned from human melanoma cell and human placenta. Thus a concentration-dependent decrease in the residual SAT-3 activity relative to SAT-4 activity was observed in the supernatant after precipitation of the immune complex. Expression of SAT-3 (STZ) cDNA was also detected in Colo 205 cell by RT-PCR, followed by sequence analysis of the RT-PCR product. Characterization of the catalytic reaction products of SAT-3 and SAT-4 with thin-layer chromatography, sialidase treatment, and binding to specific antibodies indicates that both SAT-3 and SAT-4 catalyze the formation of alpha 2-3 linkage between sialic acid and terminal galactose of glycolipid substrates.

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-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-9-fluoresceinyl-NeuAc was a suitable donor substrate for each enzyme, affording low K m values and V max values comparable in magnitude (15–100%) to that obtained with the parent CMP-NeuAc. Sensitivity was enhanced 200- to 1000-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 α2,6-sialyltransferase 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.

Both GA2, GM2, and GD2 synthases and GM1b, GD1a, and GT1b synthases are single enzymes in Golgi vesicles from rat liver.

Competition experiments using lactosylceramide, ganglioside GM3 and ganglioside GD3 as substrates, as well as mutual inhibitors for ganglioside N-acetylgalactosaminyltransferase, in Golgi vesicles derived from rat liver suggested that N-acetylgalactosamine transfer to these three respective compounds, leading to gangliosides GA2, GM2, and GD2, respectively, is catalyzed by one enzyme. Analogous studies with gangliosides GA1, GM1, and GD1b as glycolipid acceptors in sialyltransferase assays indicated GM1b, GD1a, and GT1b synthases to be identical. These results are incorporated into a model for ganglioside biosynthesis and its regulation.

Extended polysialic acid chains (n greater than 55) in glycoproteins from human neuroblastoma cells.

Polysialic acid-containing glycoproteins consisting of extended chains of at least 55 sialyl residues (DP55, where DP represents degree of polymerization) are expressed on human neuroblastoma cells, CHP-134. The strategy used for detecting these unique carbohydrate structures was based on the use of two highly specific prokaryotic-derived enzyme systems and an anti-polysialosyl antibody (H.46). These probes were developed for the detection of polysialic acid on neural cell adhesion molecules (Troy, F. A., Hallenbeck, P. C., McCoy, R. D., and Vimr, E. R. (1987) Methods Enzymol. 138, 169-185). Proof for the presence of long chain multimers of sialic acid was based on two types of experiments which utilized: 1) a glycopeptide fraction of CHP-134 cells, labeled metabolically with D-[3H]GlcN and 2) a membrane fraction from CHP-134 cells which served as an exogenous acceptor of [14C] NeuNAc residues in an Escherichia coli K1 sialyltransferase assay. In vitro, this enzyme CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase catalyzes the transfer of [14C]NeuNAc from CMP-[14C]NeuNAc to exogenous acceptors containing at least 3 sialyl residues. In the first series of experiments, endo-N-acetylneuraminidase (Endo-N), a bacteriophage-derived enzyme specific for hydrolyzing poly-alpha-2,8-sialosyl chains containing a minimum of 5 sialyl residues was used. Limit Endo-N digestion of the 3H-glycopeptides from the [3H] GlcN-labeled cells released short [3H]sialyl oligomers [( 3H]DP1-6) which were degraded to [3H]NeuNAc by exosialidase. Partial Endo-N digestion released a series of [3H]sialyl oligomers extending up to DP55. The longer (DP20-55) and intermediate sized (DP10-20) oligomers were isolated and converted to short oligomers ((3H]DP1-6) by retreating with Endo-N, thus confirming their identity as homo-oligomers of alpha-2,8-linked [3H]NeuNAc residues. In the second series of experiments, a membrane fraction of CHP-134 cells was radiolabeled in vitro with [14C]NeuNAc by E. coli K1 sialyltransferase. The membrane fraction had a major portion of radioactivity that was high Mr and polydisperse (Mr 100,000-250,000) as demonstrated in sodium dodecyl sulfate-polyacrylamide gels. Using Western blotting, pre-existing material of similar size was shown to react with antibody H.46.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative rates of transfer of N-acetylneuraminic acid to acceptors bearing one or more Gal(beta 1-4)GlcNAc terminus by the Gal(beta 1-4)GlcNAc(NeuAc-Gal) (alpha 2-6)-sialyltransferase from embryonic chicken liver. Utilization of oligosaccharides as acceptors in sialyltransferase assays.

Using a number of branched and unbranched oligosaccharides, glycoproteins and artificial glycoproteins bearing Gal(beta 1-4)GlcNAc-R termini as acceptors (where R represents H, oligosaccharide, oligosaccharide-protein or fatty acid-protein), the comparative rates of transfer of NeuAc by the Gal(beta 1-4)GlcNAc(NeuAc-Gal) (alpha 2-6)-sialyltransferase of embryonic chicken liver were determined. Acceptor substrates were utilized at levels approximating physiological, near the Km value of the best acceptor, desialylated alpha 1 acid glycoprotein. The sialyltransferase has a marked preference for multi-branched acceptors. From the specificity data, it is concluded that the enzyme binds at least two Gal(beta 1-4)GlcNAc termini of an acceptor molecule, and that the relative orientation of the branches is an important factor determining the rate of catalysis by the enzyme. The use of oligosaccharides as acceptors to study sialyltransferase catalyses is emphasized. Results are discussed in the context of the mode of assembly of sialoside termini of known glycoprotein structures in vivo.

Sialyltransferase activities of aging diploid fibroblasts

Sialyltransferase activity and cell-cell adhesion rates of aging WI-38 cells were studied to determine the possible basis for a previously described decrease in membrane bound sialic acid and loss of proliferation of senescent cells. Ectosialyltransferase was demonstrated on the surface of both young and old WI-38 cells. The sialyltransferase assays consist of an enzyme source which is either the surface of intact cells (ectoenzyme) or a Triton X-100 cell homogenate, the nucleotide sialic acid donor (cytidine monophosphate-N- acetylneuraminic acid), and an asialo-acceptor which may be endogenous to the enzyme preparation or may be added exogenously. When sialyltransferase activity is measured in the absence of exogenous acceptors, there is a greater amount of sialic acid transferred by old cells. However, when exogenous acceptors are provided, the amount of transfer is stimulated to a greater extent in young cells equalizing the amount of sialic acid incorporated into young and old cells. This suggests that there are fewer asialoglycoproteins and that acceptor concentration is a limiting factor in assays of young cell sialyltransferase. The end result of this may be the previously described decreased amount of membrane-bound sialic acid of old cells. A change in the adhesiveness of old cells described which may be related to the altered cell surface.