Purification and characterization of an O-acetylsialic acid-specific lectin from a marine crab Cancer antennarius

A sialic acid-binding lectin with high specificity for 9-O-acetyl- and 4-O-acetylsialic acids was purified from the hemolymph of the California coastal crab, Cancer antennarius, by affinity chromatography using bovine submaxillary mucin coupled to agarose. The binding specificity of the crab lectin distinguishes it from other known sialic acid-specific lectins from Limulus polyphemus and Limax flavus which show a broader range of specificity for sialic acids. The purified lectin is homogenous on sodium dodecyl sulfate-polyacrylamide electropherograms with a subunit molecular weight of about 36 kDa. The specificity of the lectin for O-acetylsialic acids appears to account for the fact that it agglutinates mouse, rat, rabbit, and horse erythrocytes, which contain O-acetylsialic acids on cell surface glycoconjugates, but not human monkey, sheep, goat, and chicken erythrocytes which contain only NeuAc or N-glycolylneuraminic acid (NeuGc). This conclusion was supported by the potent inhibition of hemagglutination by bovine and equine submaxillary mucins which contain 9(7,8)-O-acetyl- and 4-O-acetylsialic acids, respectively, and also by free 9-O-acetyl-N-acetylneuraminic acid (9-O-Ac-NeuAc) and 4-O-Ac-NeuAc relative to NeuAc and NeuGc. Further support for the role of O-Ac-sialic acids in hemagglutination of erythrocytes was obtained by enzymatic modification of human erythrocytes. Sialidase-treated erythrocytes were resialylated with purified sialyltransferases and various CMP-sialic acid donor substrates to contain NeuAc or NeuGc or 9-O-Ac-NeuAc in the Sia alpha 2,3Gal or Sia alpha 2,6Gal linkages. Cells resialylated to contain NeuAc or NeuGc were not agglutinated, but cells resialylated to contain 9-O-Ac-NeuAc were agglutinated with high titer, comparable to that of mice or horse erythrocytes.     

Prooxidant and antioxidant effects of trolox on ferric ion-induced oxidation of erythrocyte membrane lipids

Achatinin_H (ATN_H)is a lectin, isolated from the hemolymph ofAchatina fulica snail, which has been shown to have narrow specificity towards 9-O-acetyl sialic acid. Usually ATN_H does not agglutinate normal human erythrocytes, however, it is capable of agglutinating erythrocytes of patients suffering from acute lymphocytic and acute myelogenous leukemia. Determination of binding constants, numbers of binding sites and lectin overlay experiments using patients' erythrocytes ghost, have suggested that some alterations in erythrocyte cell surface sialoglycoproteins or more precisely appearance of some O-acetylated sialoglycoprotein as a result of pathological transformations has caused this change in the binding of ATN_H.Abbreviations ATN_H Achatinin_H - 9-OAc-NeuAc 9-O-acetyl N-acetyl neuraminic acid - BSM Bovine submaxillary mucin - TBS Tris-buffered saline - SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis - BSA Bovine serum albumin - HA Hemagglutination assay - ALL Acute lymphocytic leukemia - AML Acute myelogenuos leukemia - NP 40 Nonidet 40     

Immunological significance of metal induced conformational changes in the mitogenic achatininH binding to carbohydrate ligands

9-O-Acetyl neuraminic acid specific lectin (AchatininH) was isolated from the hemolymph of the land snail Achatina fulica by affinity chromatography on sheep submaxillary mucin (SSM) coupled cyanogen bromide activated Sepharose 4B. The molecular weight of the native protein was 2.42 kDa. UV-Vis absorption, fluorescence and circular dichroism spectroscopic studies on AchatininH revealed the importance of divalent metal ions (Ca2 +, Mg2+ and Mn2+) on lectin conformational change associated with activity of lectins. The binding of these cations changes lambdamax to shorter wavelength in the far UV region (blue shift) and longer wavelength in UV region (red shift), indicating substantial contribution of aromatic side chain in the far UV region on binding with metal ions. The results infer that divalent cations cause conformational changes in lectin which may be responsible for affinity with their carbohydrate moiety.     

Purification and characterization of a sialic acid specific lectin from the hemolymph of the freshwater crab Paratelphusa jacquemontii.

A naturally occurring hemagglutinin was detected in the serum of the freshwater crab, Paratelphusa jacquemontii (Rathbun). Hemagglutination activity with different mammalian erythrocytes suggested a strong affinity of the serum agglutinin for horse and rabbit erythrocytes. The most potent inhibitor of hemagglutination proved to be bovine submaxillary mucin. The lectin was purified by affinity chromatography using bovine submaxillary mucin-coupled agarose. The molecular mass of the purified lectin was 34 kDa as determined by SDS/PAGE. The hemagglutination of purified lectin was inhibited by N-acetylneuraminic acid but not by N-glycolylneuraminic acid, even at a concentration of 100 mm. Bovine submaxillary mucin, which contains mainly 9-O-acetyl- and 8,9 di-O-acety-N-acetyl neuraminic acid was the most potent inhibitor of the lectin. Sialidase treatment and de-O-acetylation of bovine submaxillary mucin abolished its inhibitory capacity completely. Also, asialo-rabbit erythrocytes lost there binding specificity towards the lectin. The findings indicated an O-acetyl neuraminic acid specificity of the lectin.     

Infectious salmon anemia virus specifically binds to and hydrolyzes 4-O-acetylated sialic acids

Infectious salmon anemia virus (ISAV) is the causative agent of infections in farmed Atlantic salmon. ISAV presumably represents a new genus within the Orthomyxoviridae. ISAV has been shown earlier to exhibit a receptor-destroying activity, which was defined as an acetylesterase with unknown specificity. We have analyzed the substrate specificity of the ISAV esterase in detail. Purified ISAV hydrolyzed free 5-N-acetyl-4-O-acetyl neuraminic acid. In addition, the purified 9-O-acetylated sialic acid derivative was also hydrolyzed, but at lower rates. When we used a glycosidically bound substrate, ISAV was unable to hydrolyze 9-O-acetylated sialic acid, which represents the major substrate for the influenza C virus esterase. ISAV completely de-O-acetylated glycoprotein-bound 5-N-acetyl-4-O-acetyl neuraminic acid. Thus, the enzymatic activity of the hemagglutinin-esterase of ISAV is comparable to that of the sialate-4-O-esterases of murine coronaviruses and related group 2 coronaviruses. In addition, we found that ISAV specifically binds to glycoproteins containing 4-O-acetylated sialic acids. Both the ISAV esterase and recombinant rat coronavirus esterase specific for 4-O-acetylated sialic acids hydrolyzed ISAV receptors on horse and rabbit erythrocytes, indicating that this sialic acid represents a receptor determinant for ISAV.     

Purification and properties of a Ca2+-independent sialic acid-binding lectin from human placenta with preferential affinity to O-acetylsialic acids

A Ca2+-independent sialic acid-specific lectin from two developmental stages of human placenta was similarly purified to apparent homogeneity by DEAE-cellulose chromatography, affinity chromatography on bovine submaxillary mucin, and gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration disclosed a molecular mass of 53 kDa. The specificity of the lectin for O-acetylsialic acids was substantiated by the dependence of hemagglutination on the presence of acetylated sialic acids on the surface of mammalian erythrocytes of various sources, by hapten inhibition in hemagglutination assays with protease-treated rabbit erythrocytes and by hapten inhibition of binding of labeled N-acetylneuraminic acid-bovine serum albumin to the lectin in a solid-phase assay. Bovine and equine submaxillary mucins that contain 9(7,8)-O-acetyl and 4-O-acetylsialic acids were potent inhibitors in contrast to the non-acetylated sialic acids of ovine submaxillary mucin. Absence of inhibitory efficiency of other negatively charged substances like phosphorylated sugars, glucuronic acid, heparin, or oligodeoxynucleotides emphasized the importance of structural features instead of simple ionic interaction. In the presence of acetylation, the pattern of inhibition by gangliosides in the solid-phase assay indicated a preference to alpha-2,8- or alpha-2,6-linked sialic acids in comparison to alpha-2,3-linked moieties. Chemical modification of the lectin by group-specific reagents allowed to emphasize the role of primarily lysine residues, but also, although less pronounced, arginine, tryptophan, and carboxyl groups for ligand binding and/or maintenance of the active conformational state. Application of reagents, specific for histidine or tyrosine residues, failed to affect lectin activity.     

Carbohydrate Recognition Properties of Human Ficolins: GLYCAN ARRAY SCREENING REVEALS THE SIALIC ACID BINDING SPECIFICITY OF M-FICOLIN*

Ficolins are oligomeric innate immune recognition proteins consisting of a collagen-like region and a fibrinogen-like recognition domain that bind to pathogen- and apoptotic cell-associated molecular patterns. To investigate their carbohydrate binding specificities, serum-derived L-ficolin and recombinant H- and M-ficolins were fluorescently labeled, and their carbohydrate binding ability was analyzed by glycan array screening. L-ficolin preferentially recognized disulfated N-acetyllactosamine and tri- and tetrasaccharides containing terminal galactose or N-acetylglucosamine. Binding was sensitive to the position and orientation of the bond between N-acetyllactosamine and the adjacent carbohydrate. No significant binding of H-ficolin to any of the 377 glycans probed could be detected, providing further evidence for its poor lectin activity. M-ficolin bound preferentially to 9-O-acetylated 2-6-linked sialic acid derivatives and to various glycans containing sialic acid engaged in a 2-3 linkage. To further investigate the structural basis of sialic acid recognition by M-ficolin, point mutants were produced in which three residues of the fibrinogen domain were replaced by their counterparts in L-ficolin. Mutations G221F and A256V inhibited binding to the 9-O-acetylated sialic acid derivatives, whereas Y271F abolished interaction with all sialic acid-containing glycans. The crystal structure of the Y271F mutant fibrinogen domain was solved, showing that the mutation does not alter the structure of the ligand binding pocket. These analyses reveal novel ficolin ligands such as sulfated N-acetyllactosamine (L-ficolin) and gangliosides (M-ficolin) and provide precise insights into the sialic acid binding specificity of M-ficolin, emphasizing the essential role of Tyr²⁷¹ in this respect.     

Estimation of pyruvate decarboxylation in perfused rat skeletal muscle

A total of 46 E. coli strains showing mannose-resistant, P-blood-group independent hemagglutination of human erythrocytes were tested for binding to neuraminic acid. Nine of the strains completely lost their hemagglutination activity after the erythrocytes were treated with neuraminidase. To characterize the receptor structure, different neuraminic acid containing glycoproteins, their desialylated derivatives and neuraminyl oligosaccharides were tested for hemagglutination inhibition. These studies showed that the nine strains had binding specificity for alpha 2-3 linked neuraminic acid.     

Binding specificity of influenza C-virus to variablyO-acetylated glycoconjugates and its use for histochemical detection ofN-acetyl-9-O-acetylneuraminic acid in mammalian tissues

The specificity of influenza C-virus binding to sialoglycoconjugates was tested with various naturallyO-acetylated gangliosides or syntheticallyO-acetylated sialic acid thioketosides, which revealed binding to 9-O-acetylatedN-acetylneuraminic acid. Binding was also observed with a sample of Neu5,7Ac₂-GD3, however at a lower degree. Sialic acids with two or threeO-acetyl groups in the side chain of synthetic sialic acid derivatives are not recognized by the virus. In these experiments, bound viruses were detected with esterase substrates. Influenza C-virus was also used for the histological identification of mono-O-acetylated sialic acids in combination with an immunological visualization of the virus bound to thin-sections. The occurrence of these sialic acids was demonstrated in bovine submandibular gland, rat liver, human normal adult and fetal colon and diseased colon, as well as in human sweat gland. Submandibular gland and colon also contain significant amounts of glycoconjugates with two or three acetyl esters in the sialic acid side chain, demonstrating the value of the virus in discriminating between mono- and higherO-acetylation at the same site. The patterns of staining showed differences between healthy persons and patients with colon carcinoma, ulcerative colitis or Crohn's disease. Remarkably, some human colon samples did not showO-acetyl sialic acid-specific staining. The histochemical observations were controlled by chemical analysis of tissue sialic acids.Abbreviations BSA bovine serum albumin - BSM bovine submandibular gland mucin - HAU haemagglutination units - HPLC high-performance liquid chromatography - HPTLC high-performance thin-layer chromatography - Neu5Ac N-acetylneuraminic acid - Neu5,9Ac₂ N-acetyl-9-O-acetylneuraminic acid - Neu5,7,9Ac₃ N-acetyl-7,9-di-O-acetylneuraminic acid - Neu5,7,8,9Ac₄ N-acetyl-7,8,9-tri-O-acetylneuraminic acid - PBS phosphate-buffered saline - TLC thin-layer chromatography Dedicated to Prof. Dr Nathan Sharon on the occasion of his 70th birthday.     

N-glycolylneuraminic acid specific lectin from Pila globosa snail

A N-glycolylneuraminic acid-specific lectin (PAL) has been purified from an albumin gland extract of the apple snail, Pila globosa. Purification is conducted on a bovine submaxillary mucin-Sepharose 4B affinity matrix followed by gel filtration on a Sepharose 6B column. The lectin agglutinates rabbit erythrocytes. The hemagglutination activity is dependent on Ca2+ concentration in a significant manner but with a remarkable behaviour. The lectin is a trimeric glycoprotein of native Mr 440 kDa with 25% carbohydrate and is composed of three nonidentical subunits of molecular weights 190, 145, and 105 kDa. It has a pI of 7.0. The lectin exhibits a unique and strict specificity toward N-glycolylneuraminic acid and this phenomenon discriminates it from other known sialic acid binding lectins. The uniqueness indicates the absolute need for a glycolyl substitution on the amino residue and of a glyceryl side chain on the exocyclic part and an axial -COOH group in neuraminic acid. The presence of an acetyl substitution on the exocyclic part impedes lectin-ligand interaction.     

Diversity of sialic acids revealed using gas chromatography/mass spectrometry of heptafluorobutyrate derivatives

The fine structural motifs of sialic acids, a frequent terminal monosaccharide of glycans, seem to contain essential biological properties. To identify such subtle structural differences, a reliable method was developed for the qualitative and quantitative identification of sialic acids present in different tissues and fluids. This method involved, after liberation of sialic acids by mild acid hydrolysis, their methyl esterification using diazomethane in the presence of methanol and the formation of volatile derivatives using heptafluorobutyric anhydride. The derivatives were analyzed by gas chromatography coupled to mass spectrometry in the electron impact mode. This technique allowed the separation and identification of a large variety of sialic acids, including different O-acylated forms of N-acetyl and N-glycolyl neuraminic acids and of 3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn). This method allowed also identifying 8-O-methylated and 8-O-sulfated derivatives, de-N-acetylated neuraminic acid, and 1,7-sialic acid lactones. Compounds present in very complex mixtures could be identified through their fragmentation patterns. Because of the stability of the heptafluorobutyrate derivatives, this method presents important improvements compared to the previous techniques, because it can be frequently applied on very small amounts of crude samples. This methodology will support progress in the field of the biology of sialic acids.     

Elderberry bark lectin-gold techniques for the detection of Neu5Ac (α2,6) Gal/GalNAc sequences: applications and limitations

Summary The lectin from the elderberry (Sambucus nigra L.) bark, shown to recognize the sequence neuraminic acid (2,6) galactose/N-acetylgalactosamine, was applied for detecting binding sites in Lowicryl K4M sections by light and electron microscopy. The lectin was used either directly complexed to colloidal gold or in a two-step cytochemical affinity technique. The lectin-gold complex proved to be superior and thus was extensively tested on rat liver, kidney and hepatoma cells as well as on sheep and bovine submandibular glands. Controls to establish specificity of lectin-gold binding included sugar and glycoprotein inhibition tests and enzymic removal of sialic acid. In agreement with biochemical data demonstrating the potentiating effect of sialic acid on the binding of the lectin to oligosaccharides, enzymic removal of sialic acid from liver sections resulted in abolition of lectin staining. However, in the submandibular glands, neuraminidase pretreatment of the sections had no effect on the subsequent lectin-gold binding. In rat kidney some structures became negative while others retained the lectin-gold staining due to binding to penultimate.N-acetylgalactosamine exposed after sialic acid removal. In line with this, spot blot analysis demonstrated that the lectin-gold complex reacted with both fetuin and asialofetuin. Taken together, these results suggest that, for cytochemical staining, the sialic acid and the galactose/N-acetylgalactosamine lectin combining subsites ofSambucus nigra L. lectin are equally reactive with cellular glycoconjugates and that neuraminidase predigestion of tissue sections is of utmost importance to ensure specificity of staining for the sequence neuraminic acid (2,6) galactose/N-acetylgalactosamine.     

Specificity of the binding site of the sialic acid-binding lectin from ovine placenta, deduced from interactions with synthetic analogues

The specificity of the sialic acid-binding lectin from ovine placenta was examined in detail by haemagglutination inhibition assays applying a panel of 32 synthetic sialic acid analogues. The carboxylic acid group is a prerequisite for the interaction with the lectin, the -anomer of the methyl glycoside is only a little more effective as an inhibitor than the -anomer and the most potent inhibitor was 9-deoxy-10-carboxylic acid Neu5Ac, followed by 4-oxo-Neu5Ac. In contrast to the majority of known sialic acid-binding lectins, the N-acetyl group of Neu5Ac is not indispensable for binding, neither is the hydroxyl group at C-9 since substitutions at this carbon atom are well tolerated. Furthermore, all sulfur-containing substituents at C-9 enhanced the affinity of the lectin. This is the first sialic acid-binding lectin found to strongly bind thio derivatives.     

Purification and molecular characterization of a sialic acid specific lectin from the phytopathogenic fungus Macrophomina phaseolina

A lectin was isolated and purified from the culture filtrate of the plant pathogenic fungus Macrophomina phaseolina by a combination of ammonium sulfate precipitation, affinity chromatography on fetuin-Sepharose 4B and ion-exchange chromatography on DEAE-A 50. The lectin designated MPL was homogeneous by PAGE and HPLC and a monomeric protein with a molecular weight of approximately 34 kDa as demonstrated by SDS-PAGE. It is a glycoprotein and agglutinated human erythrocytes regardless of the human blood type. Neuraminidase treatment of erythrocytes reduced the agglutination activity of the lectin. It is thermally stable and exhibits maximum activity between pH 6 and 7.2. Its carbohydrate binding specificity was investigated both by hapten inhibition of hemagglutination and by enzyme-conjugated lectin inhibition assay. Although, M. phaseolina lectin bound sialic acid, it exhibited binding affinity towards neuraminyl oligosaccharides of N-linked glycoproteins, alpha-Neu5Ac-(2-->3)-beta-Gal-(1-->4)-GlcNAc being maximum.     

Rapid analysis of O-acetylated neuraminic acids by matrix assisted laser desorption/ionization time-of-flight mass spectrometry

N-Acetylneuraminic acid (a sialic acid) occurs mainly as a terminal substituent of oligosaccharides of glycoconjugates. Derivatives of neuraminic acid occur widely, substituted in the amino and hydroxy side chains, as well in the C-9 carbon skeleton. These derivatives are responsible for specific functions of sialic acids during cell-cell, cell-substrate, or cell-virus interactions. The study of O-acetylated neuraminic acids is difficult, because only small amounts are extractable from natural sources and they are generally unstable to acids and bases. We report a new method for the rapid analysis of O-acetylated neuraminic acids, using a combination of reversed phase HPLC and MALDI-TOF mass spectrometry. A mixture of neuraminic acids from bovine submaxillary gland mucins was analysed, as well as neuraminic acids variously substituted in the amino and hydroxy side chains with acetyl and glycolyl groups, respectively. © 1998 Rapid Science Ltd     

The Murine Coronavirus Hemagglutinin-esterase Receptor-binding Site: A Major Shift in Ligand Specificity through Modest Changes in Architecture

The hemagglutinin-esterases (HEs), envelope glycoproteins of corona-, toro- and orthomyxoviruses, mediate reversible virion attachment to O-acetylated sialic acids (O-Ac-Sias). They do so through concerted action of distinct receptor-binding (“lectin”) and receptor-destroying sialate O-acetylesterase (”esterase”) domains. Most HEs target 9-O-acetylated Sias. In one lineage of murine coronaviruses, however, HE esterase substrate and lectin ligand specificity changed dramatically as these viruses evolved to use 4-O-acetylated Sias instead. Here we present the crystal structure of the lectin domain of mouse hepatitis virus (MHV) strain S HE, resolved both in its native state and in complex with a receptor analogue. The data show that the shift from 9-O- to 4-O-Ac-Sia receptor usage primarily entailed a change in ligand binding topology and, surprisingly, only modest changes in receptor-binding site architecture. Our findings illustrate the ease with which viruses can change receptor-binding specificity with potential consequences for host-, organ and/or cell tropism, and for pathogenesis.     

Identification of lectin isoforms in juvenile freshwater prawns Macrobrachium rosenbergii (DeMan, 1879)

From the serum of juvenile freshwater prawns, we isolated by affinity chromatography on glutaraldehyde-fixed rat erythrocytes stroma, immobilized in Sephadex G-25, a sialic acid specific lectin of 9.6[emsp4 ]kDa per subunit. Comparative analysis against adult organisms purified lectin, by chromatofocusing, showed that the lectin from juvenile specimens is composed by four main isoforms with a pl of 4.2, 4.6, 5.1, and 5.6, whereas the lectin from adults is eluted at pH 4.2. The amino acid composition of the lectin obtained from adult and juvenile stages suggest identity, but the compositions are not identical since a higher content of carbohydrates was found in the lectin from younger organisms. The freshwater prawn lectin showed specificity toward N-acetylated amino sugar residues such as GlcNAc, GalNAc, Neu5Ac and Neu5,9Ac; but in juvenile organisms the lectin showed three times less hemagglutinating activity than the lectin from adults. Both lectins agglutinated rat, rabbit and chicken erythrocytes, indicating that Neu5,9Ac in specific O-glycosydically linked glycans seems to be relevant for the interaction of M. rosenbergii lectins with their specific cellular receptor. Our results suggest that the physicochemical characteristics of the lectin from the freshwater prawn are regulated through maturation.     

A sialic acid-binding lectin from ovine placenta: purification,specificity and interaction with actin

A sialic-acid-specific lectin from ovine placental cotyledons was purified by affinity chromatography on bovine submaxillary mucin-agarose followed by gel filtration, and it showed a molecular weight of 65 000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis. This lectin has the capacity to interact with actin, since in binds to actin-F in a cosedimentation assay and it acts as a mediator in the binding of action to the affinity column. The lectin agglutinated rabbit and rat erythrocytes, but not human A, B or O erythrocytes. Haemagglutination inhibition assays of different saccharides, glycoproteins and glycolipids indicate that this lectin has affinity for sialic acid, which is enhanced by its O-acetylation. The N-terminal sequence of the protein shows 92% identity with rabbit and porcine uterine calreticulin.     

A single step purification of a sialic acid binding lectin (AchatininH) from Achatina fulica snail

A sialic acid binding lectin, Achatinin_H, was purified in single step from the hemolymph of the land snail, Achatina fulica, by the affinity chromatography on sheep submaxillary mucin coupled to Sepharose 4B. The yield of the lectin was found to be 3 mg from 100 ml of hemolymph. The homogeneity of the lectin was established by alkaline gel electrophoresis, immunodiffusion, immunoelectrophoresis and analytical isoelectrophoresis. The molecular weight of the native protein was 242000, having identical subunits of Mr 15000. The lectin agglutinated rabbit erythrocytes in the presence of Ca^2–. The inhibition study clearly suggests that the binding site of the lectin recognizes sialic acid as the immunodominant sugar. This was further confirmed by the observation that there was a marked decrease of agglutinating activity of the lectin with neuraminidase treated rabbit erythrocytes and asialofetuin was unable to inhibit the activity of Achatinin_H. Among the inhibitors used the glycoconjugate containing 2-6 linkages of N-acetylneuraminic acid with subterminal galactopyranose or 2-acetamido-2-deoxy-galactopyranose residue was found to be better inhibitor than that containing 23 linkages of N-acetyl neuraminic acid. Besides that sialoglycoprotein containing both N and O type of glycosidic linkages plays an important role in binding with the lectin. Fetuin was found to be the best inhibitor.     

Analysis of sialidase and N-acetylneuraminate pyruvate-lyase substrate specificity by high-performance liquid chromatography

A rapid and sensitive assay by high-performance liquid chromatography for determination of the activity and substrate specificity of sialidase (EC 3.2.1.18) and N-acetylneuraminate lyase (EC 4.1.3.3) is described. Sialic acids were separated on a strong anion-exchange resin using 0.75 mM sodium sulfate as elution medium. This method allows the determination of a minimum amount of 200 pg (0.6 pmol) of sialic acid. Usually the enzyme mixtures were directly applied to the column without prior purification of substrates and products. The action of sialidase was studied either by the decrease of sialyllactose concentration or by the amount of sialic acid liberated. The relative hydrolysis rates of N-acetylneuraminyl-alpha(2-3)-lactose, N-glycolylneuraminyl-alpha(2-3)-lactose, N-acetylneuraminyl-alpha(2-6)-lactose, N-acetyl-9-O-acetylneuraminyl-alpha(2-3)-lactose, and N-acetyl-4-O-acetylneuraminyl-alpha(2-3)-lactose by Vibrio cholerae sialidase were 100, 88, 25, 12, and 0, respectively. The activity of N-acetylneuraminate lyase from Clostridium perfringens was determined by measuring the rate of disappearance of sialic acids or the formation of acylmannosamines, which is possible in the same chromatogram. Relative cleavage rates of N-acetylneuraminic acid, N-glycolylneuraminic acid, N-acetyl-9-O-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, and N-acetyl-4-O-acetylneuraminic acid were found to be 100, 67, 24, 3, and 0, respectively. Comparison of the substrate specificities shows that substituents on the neuraminic acid molecule influence the reactions of both enzymes in a similar way.