Con A-Sepharose Affinity Chromatography Research Articles

The Degradation of Intramuscular Connective Tissue In Vitro with Purified Cathepsin L from Bovine Pancreas.

To investigate the possible degradation of the intramuscular connective tissue (IMCT) with cathepsin L, isolated IMCTs were incubated with purified cathepsin L in vitro. Here, we prepared purified cathepsin L from bovine pancreas by using DEAE Sephacel, Sephacryl S-100 HR, SP Sepharose FF, and con A-Sepharose affinity chromatography in sequence. An SDS-PAGE analysis of CNBr-digested peptides showed that the degradation of collagen in IMCT could take place on terminal non-helical peptides rather than the triple helix region. Decorin (DCN) was clearly degraded at a pH of 5.0. The TP and TO of intramuscular connective tissue decreased to 41.41 °C and 43.79 °C, respectively. In the cathepsin L treatment of pH 5.0, the decreases in the TP and TO of IMCT were more sensitive than they were at pH 5.5~6.5.

The phospholipid monolayer associated with perilipin-enriched lipid droplets is a highly organized rigid membrane structure

The significance of lipid droplets (LD) in lipid metabolism, cell signaling, and membrane trafficking is increasingly recognized, yet the role of the LD phospholipid monolayer in LD protein targeting and function remains unknown. To begin to address this issue, two populations of LD were isolated by ConA sepharose affinity chromatography: 1) functionally active LD enriched in perilipin, caveolin-1, and several lipolytic proteins, including ATGL and HSL; and 2) LD enriched in ADRP and TIP47 that contained little to no lipase activity. Coimmunoprecipitation experiments confirmed the close association of caveolin and perilipin and lack of interaction between caveolin and ADRP, in keeping with the separation observed with the ConA procedure. The phospholipid monolayer structure was evaluated to reveal that the perilipin-enriched LD exhibited increased rigidity (less fluidity), as shown by increased cholesterol/phospholipid, Sat/Unsat, and Sat/MUFA ratios. These results were confirmed by DPH-TMA, NBD-cholesterol, and NBD-sphingomyelin fluorescence polarization studies. By structure and organization, the perilipin-enriched LD most closely resembled the adipocyte PM. In contrast, the ADRP/TIP47-enriched LD contained a more fluid monolayer membrane, reflecting decreased polarizations and lipid order based on phospholipid fatty acid analysis. Taken together, results indicate that perilipin and associated lipolytic enzymes target areas in the phospholipid monolayer that are highly organized and rigid, similar in structure to localized areas of the PM where cholesterol and fatty acid uptake and efflux occur.

A lectin from the rhizomes of turmeric (Curcuma longa L.) and its antifungal, antibacterial, and α-glucosidase inhibitory activities

A Curcuma longa L. lectin was purified by aqueous extraction, 80% ammonium sulfate precipitation and ConA Sepharose affinity chromatography. Its specific activity was of 64,566 HU/mg protein for a yield of 41.2% total protein. The molecular weight is of 17.3 kDa. It has hemagglutinating activity against human blood group B, rabbit, mouse, rat, guinea pig, geese, and sheep erythrocytes. The optimum pH is between 6–7, and stable up to 40°C. Activity was stimulated by Ca2+ and Mn2+. The internal sequence indicated similarity with legume lectin family. Moreover, at concentration of 47 and 94 mg/0.3 cm2 disc showed antifungal activity against Exserohilum turicicum, Fusarium oxysporum, and Colectrotrichum cassiicola. The minimal inhibitory concentration were 0.002, 0.005, 0.011, 0.09, and 0.046 mg/mL Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Escheriehia coli, and Candida albicans, respectively. Additionally, it contains a high α-glucosidase inhibitory activity with an IC50 of 8 mg/mL.

Isolation and characterization of a dual function protein from Allium sativum bulbs which exhibits proteolytic and hemagglutinating activities

A dual function protein was isolated from Allium sativum bulbs and was characterized. The protein had a molecular mass of 25–26 kDa under non-reducing conditions, whereas two polypeptide chains of 12.5 ± 0.5 kDa were observed under reducing conditions. E-64 and leupeptin inhibited the proteolytic activity of the protein, which exhibited characteristics similar to cysteine peptidase. The enzyme exhibited substrate specificity and hydrolyzed natural substrates such as α-casein ( K m: 23.0 μM), azocasein, haemoglobin and gelatin. It also showed a high affinity for synthetic peptides such as Cbz-Ala-Arg-Arg-OMe-β-Nam ( K m: 55.24 μM, k cat: 0.92 s −1). The cysteine peptidase activity showed a remarkable stability after incubation at moderate temperatures (40–50 °C) over a pH range of 5.5–6.5. The N-terminus of the protein displayed a 100% sequence similarity to the sequences of a mannose-binding lectin isolated from garlic bulbs. Moreover, the purified protein was retained in the chromatographic column when Con-A Sepharose affinity chromatography was performed and the protein was able to agglutinate trypsin-treated rabbit red cells. Therefore, our results indicate the presence of an additional cysteine peptidase activity on a lectin previously described.

Cloning and characterization of two alleles of the murine extracellular superoxide dismutase gene

We have recently documented the existence of a second allele of ecSOD in mice. Thus far, this allele was only found in the 129P3/J strain. It is characterized by two point mutations leading to amino acid changes as well as a 10 bp deletion from the 3′ UTR. We have also shown that the phenotype is profoundly affected by the genotype. In order to obtain a tool to investigate the differences in the properties as well as the posttranscriptional regulation of expression of the two alleles we now describe the creation and characterization of stably transfected CHO-K1 cell lines expressing either of these alleles. CHO-K1 cells were chosen because they do not express endogenous ecSOD and are easy to transfect. We demonstrate that the transfected cells secrete substantial amounts of glycosylated ecSOD, detected by Western blot analyses, ConA–Sepharose affinity chromatography and activity measurements.

Carp liver DNase—isolation, further characterization and interaction with endogenous actin

Deoxyribonuclease I (DNase I)-like enzyme from the liver of the carp ( Cyprinus carpio) was purified to homogeneity and further characterized. Ion exchange chromatography on DEAE-cellulose, molecular filtration on Sephacryl S-300 and Con A-Sepharose affinity chromatography were applied for enzyme isolation. Carp liver DNase, similarly to DNase I from bovine pancreas, was found to be an endonuclease that hydrolyses linear DNA from salmon sperm as well as circular DNA forms—plasmid and cosmid. The purified enzyme is a glycoprotein and shows microheterogeneity, as observed in DNase zymograms prepared after native and two-dimensional electrophoresis (2D-PAGE). The composition of sugar component of the enzyme was characterized. Special attention was focused on the ability of carp liver DNase to interact with carp liver actin. The carp liver enzyme was inhibited by endogenous actin. The estimated binding constant of carp liver DNase to carp liver actin was calculated to be 1.1×10 6 M −1.

Purification of heat shock protein 70-associated tumor peptides and their antitumor immunity to hepatoma in mice.

To purify the heat shock protein (HSP) 70-associated tumor peptides and to observe its non-MHC-I molecule restrictive antitumor effect. By ConA-sepharose affinity chromatography, ADP-agarose affinity chromatography, and DEAE anion exchange chromatography, we were able to purify HSP70-associated peptides from mouse hepatoma (HCaF) cells treated in heat shock at 42 degrees. Specific active immunization and adoptive cellular immunization assay were adopted to observe the immunoprotective effect elicited by HSP70-associated peptide complexes isolated from HcaF. The finally purified HSP-associated peptides had a very high purity and specificity found by SDS-PAGE and Western blot. Mice immunized with HSP70-associated peptide complexes purified from HCaF cells were protected from HCaF living cell challenge. This effect was dose dependent. Adoptive immunization of immune spleen cells of mice immunized with HSP70-associated peptide complexes could elicit immunity against HCaF challenge, and the tumor-free mice could resist repeated challenges. This effect could be continuously enhanced by repeated challenge with HCaF living cells. The tumor-free mice could tolerate the challenge for as high as 1 x 10(7) HCaF cells. The mice immunized once with spleen cells pulsed with HSP70-associated peptide complexes in vitro could also result in a certain adoptive immunity against HCaF. High purity and specificity of HSP70-associated peptides could be achieved from tumor cells by the low-pressure affinity chromatography method used in this study. HSP70-associated peptide complexes derived from the HCaF can elicit non-MHC-I molecule restrictive immunoprotective effect against HCaF. This effect can be transferred by adoptive immunization to mice and enhanced by repeated challenge with HCaF live cells.

Humoral immune response in aspergillosis: an immunodominant glycoprotein of 35 kDa from Aspergillus flavus.

A glycoprotein preparation containing 70% carbohydrates and 30% proteins was isolated from the mycelium of two strains of Aspergillus flavus and fractionated by ConA-Sepharose affinity chromatography. An immunodominant 35-kDa antigen was detected in a ConA-bound fraction (B fraction). It contained mannose and galactose in a 1.4:1.0 ratio. This antigen seems to be able to elicit an antibody response in patients with aspergillosis and in rabbits immunized with A. flavus whole cells. The carbohydrate units of the BF fraction appeared to be responsible for the antigenicity, since treatment with periodate removed most of the antibody binding capacity.

Invertase inhibitors from sweet potato (Ipomoea batatas): purification and biochemical characterization.

Two proteinaceous invertase inhibitors, designated ITI-L and ITI-R, were purified to electrophoretic homogeneity. ITI-L was purified from acetone powder of sweet potato leaves through sequential steps entailing buffer extraction, acid treatment, DEAE-Sephacel ion-exchange chromatography, and Sephacryl S-100 gel filtration. ITI-R was purified from sweet potato tuberous roots by sequentially applying buffer extraction, Con A-Sepharose affinity chromatography, DEAE-Sephacel ion-exchange chromatography, Sephacryl S-200, and Superose 12 gel filtration. The optimal pHs for interaction between ITI-L and ITI-R and acid invertase from sweet potato leaves were 5.5 and 5.0, respectively. The molecular masses of ITI-L and ITI-R were 10 and 22 kDa, respectively, as estimated by both gel filtration and SDS-PAGE. Both inhibitors were thermostable (90% of the activity remained after incubation at 100 degrees C for 20 min), and Western blotting showed them to be immunologically related.

Concanavalin A and the motile behaviour of the ciliate Tetrahymena

Concanavalin A (Con A) is a plant lectin that inhibits the persistent swimming behaviour of Tetrahymena cells by increasing ciliary reversals. Con A also increases ciliary reversals following chemosensory adaptation when it acts as a chemoattractant. The Con A effect is inhibited by mannose suggesting that Con A binds to glycoproteins in cellular membranes. In order to isolate these glycoproteins membrane proteins were fractionated by Con A-Sepharose affinity chromatography. A ciliary membrane protein denoted p66 was purified by two-step affinity chromatography from a whole cell Triton X-100 extract of Tetrahymena thermophila.

Three-step purification of gp96 from human liver tumor tissues suitable for isolation of gp96-bound peptides

Glycoprotein 96 (gp96), a member of heat shock protein (HSP) family, plays important roles in both innate immunity by itself and in T cell adaptive immunity in complex with antigenic-specific peptides. Using ammonium sulfate precipitation, ConA-Sepharose affinity chromatography and anion exchange chromatography, we have successfully purified gp96 from human liver tumor tissues. Subsequently, the gp96-associated peptides were successfully isolated from the gp96 preparation in quantities sufficient for micro-sequencing and identification of the peptides [Lancet 357 (2001) 528]. This three-step purification method might be used as a universal technique for the easy and reproducible isolation of antigenic heat shock protein gp96 from liver tissue or even other tissues of different sources. This will inevitably trigger the search for antigenic-specific peptides bound to gp96. Here, we report the method in detail.

Purification and properties of acetylcholinesterase from human brain.

Acetylcholinesterase from human caudate nucleus and partial thalamus was purified by using Con A-Sepharose, short-arm and long-arm ligand Sepharose affinity chromatographies. SDS-PAGE of the purified AChE under the reduced condition showed one main band, corresponding to a molecular weight of 66 kD. The purified AChE with a specific activity of 3384 U/mg protein represented 20% activity of the homogenate supernatant. Analysis of purified AChE by gradient slab PAGE and DISC-PAGE with activity staining revealed the existence of monomer, dimer, tetramer, hexamer and octomer of the enzyme. The isoelectric point of AChE ranged between pH 5.6 and 6.0. Con A-Sepharose affinity chromatography retained most of the applied AChE activity implying that the enzyme is a kind of glycoprotein. The isolated human brain AChE had no cross-immunoreactivity with 3F3 and weak cross-immunoreactivity with 2G8 and 1H11 anti-Torpedo AChE antibodies. Balb/c mice were immunized with human cerebellum AChE purified with Con A and short-arm ligand affinity chromatographies. The antiserum produced showed strong cross-immunoreactivity with Torpedo AChE but weak cross-immunoreactivity with human RBC membrane AChE. The purified human brain striatum AChE was reduced and alkylated, and then hydrolyzed by immobilized TPCK-treated trypsin. Trypsin peptides in the hydrolysate was separated by RP-HPLC. Several large peptide peaks and numbers of small peaks were observed. The large peaks showed obvious immunoreactivity with the mouse anti human cerebellum AChE antiserum.

Characterization of Aspergillus umbrosus carbohydrate antigens by biotinylated lectins and IgG response to mannan/mannoprotein antigens in patients with farmer's lung

Twenty-one different biotinylated lectins were used to recognize the carbohydrates of Aspergillus umbrosus, one of the most common microbes that patients with farmer's lung in Finland are exposed to. The glycoprotein fraction of A. umbrosus was bound especially well by Concanavalin A and consisted mainly of carbohydrates mannose and glucose. The carbohydrate fraction of A. umbrosus antigens were isolated from the crude extract of A. umbrosus with Con A-Sepharose affinity chromatography. Serum IgG antibodies to A. umbrosus mannoprotein fraction were determined in 57 patients with farmer's lung, 10 asymptomatic exposed farmers and 10 healthy controls by enzyme linked immunosorbent assay. Candida albicans and Saccharomyces cerevisiae mannan were used as controls. Patients had high levels, whereas asymptomatic exposed farmers showed moderate, and health controls low levels, of IgG antibodies to the A. umbrosus mannan/mannoprotein fraction. There were no significant differences in the mean antibody levels between the patients and controls against C. albicans or S. cerevisiae mannose fraction although in all groups more antibodies were detected against C. albicans than S. cerevisiae.

The proportion of symmetric and asymmetric IgG antibody molecules synthesized by a cellular clone (Hybridoma) can be regulated by placental culture supernatants

The purpose of the present work was to establish whether the placenta is producing factors favoring an increased synthesis of asymmetric IgG antibodies which are known to assume a protective effect upon paternal antigens to which they largely are specific. In this way they can contribute to fetal survival in the maternal uterine environment. The hybridoma cell lines OKT8 (anti-CD8) and 112B4 (anti-DNP) were used in this respect since they synthesize both symmetric and asymmetric molecules of the IgG2a and IgG1 subclasses, respectively, murine isotypes in which anti-paternal antibodies have been detected. The cells were cultured in RPMI 1640 medium supplemented with 10% BCS and different amounts (5, 10, and 20%) of human placental supernatant. After incubation for 3 days at 37 °C in a humid chamber containing 5% CO 2 the cells were centrifuged and the antibodies were obtained from the culture medium by a purification procedure involving precipitation at 50% ammonium sulfate saturation followed by DEAE-cellulose chromatography. Symmetric and asymmetric antibodies were separated by Con A-Sepharose affinity chromatography, the latter lectin retaining selectively only asymmetric IgG molecules. Both OKT8 and 112B4 hybridomas presenting a stable background synthesis of 15–17% of asymmetric antibodies have shown an increased level reaching 27–28% of these molecules in the presence of 5–10% placental supernatant added to the RPMI 1640 culture medium. These results clearly show that placental factors can up-regulate efficiently the synthesis of asymmetric IgG molecules of different isotypes secreted by plasma cells.

Goat sperm membrane: lectin-binding sites of sperm surface and lectin affinity chromatography of the mature sperm membrane antigens

The cell surface glycoproteins of goat epididymal maturing spermatozoa have been investigated using lectins as surface probes that interact with specific sugars with high affinity. Concanavalin A (ConA) and wheat-germ agglutinin (WGA) showed high affinity for mature cauda epididymal sperm agglutination, whereas RCA 2, kidney beans lectin and peanut agglutinin caused much lower or little agglutination of the cells. The mature sperm exhibited markedly higher efficacy than the immature caput epididymal sperm for binding both ConA and WGA, as evidenced by sperm agglutination and the binding of the fluorescence isothiocyanate (FITC)-labelled lectins. FITC-ConA binds uniformly to the entire mature sperm surface whereas FITC-WGA binds to the acrosomal cap region of the head. The FITC-RCA 2 mainly labelled the posterior head of mature cauda sperm. However, no WGA-specific glycoprotein receptors could be detected in sperm plasma membrane (PM) by WGA-Sepharose affinity chromatography. The data implied that the epididymal sperm maturation is associated with a marked increase in the ConA/WGA receptors and that WGA receptors may be glycolipids rather that glycoproteins. Analysis of the ConA receptors of cauda sperm PM identified by ConA-Sepharose affinity chromatography and subsequent resolution in SDS-PAGE demonstrated the presence of five glycopolypeptides of different concentrations (98, 96, 43, 27 and 17 kDa) of goat sperm membrane. The immunoblot of these ConA-specific glycopeptides with anti-sperm membrane antiserum showed that 98- and 96-kDa receptors are immunoresponsive.

Purification and Characterization of Macromolecular Phagocytosis Activators Released From Platelets

Substantial amounts of macromolecular activators of phagocytosis from platelets (MAPPs) were released in response to exposure of platelets to the specific agonists thrombin and collagen and to calcium ionophore A23187. Centrifugation of the platelets in culture medium also resulted in a release of MAPPs, but not when the platelets were frozen and thawed prior to centrifugation. In an experiment using outdated platelet concentrates, larger and smaller MAPPs (1-MAPP and s-MAPP, respectively) were purified from the thrombin stimulated supernatant (PRPr-plasma) by sequential steps of ammonium sulfate precipitation, delipidation with ethylacetate, ConA-Sepharose affinity chromatography, MONO Q anion exchange chromatography, and Superose 12 gel filtration. This procedure yielded 59,500-fold and 63,600-fold purified 1-MAPP (0.95 mg) and s-MAPP (0.41 mg), respectively, from 1,320 ml PRPr-plasma containing 84,500 mg protein. By gradient polyacrylamide gel electrophoresis the respective molecular weights (MW) of 1-MAPP and s-MAPP were 290-320 kd and 140-160 kd; isoelectric points were 5.6 and 4.6. Both MAPPs activated neutrophil phagocytosis of IgG-SRBC at concentrations in the range of 1 pM-100 nM. Indirect enzyme-linked immunosorbent assay (ELISA) and comparisons of the concentrations required for phagocytosis activation suggested that the MAPPs were not derived from fibronectin.

Natural killer cell cytolytic granule-associated enzymes. I. Purification, characterization, and analysis of function of an enzyme with sulfatase activity.

An enzyme with sulfatase activity has been isolated from the granules of a rat NK leukemia cell line, CRNK-16. The enzyme has been purified from crude preparation, with a specific activity of 52 nmol/min/mg of protein, by DEAE ion exchange and Con A-Sepharose affinity chromatography, resulting in a specific activity of 230 nmol/min/mg of protein. The molecular mass of the purified enzyme was estimated to be 40 kDa by gel filtration chromatography at pH 7.4, but the enzyme had the ability to complex to molecular masses of greater than 300 kDa at low pH when crude granule extract was used as the starting sample, suggesting that it associates with other granule components. The enzyme was determined to be an arylsulfatase by its ability to (a) hydrolyze p-nitrophenyl sulfate (Km = 26.0 mM) and p-nitrocatechol sulfate (pNC sulfate) (Km = 1.1 mM) and (b) be inhibited by sulfite (Ki = 6.0 x 10(-7) M), sulfate (Ki = 1 x 10(-3) M), and phosphate (Ki = 4 x 10(-5) M) in a competitive manner. The pH optimum for enzymatic activity was determined to be 5.6. The role of this enzyme in cytolytic function was investigated by examining the effect of its substrates and inhibitors on granule- and cell-mediated lysis. pNC sulfate was shown to cause a dose-dependent inhibition of target cell lysis by isolated cytolytic granules (complete inhibition at 12.5 mM). Sulfite induced an incomplete inhibition (50% at 1 mM), whereas phosphate was essentially without inhibitory effect. Sulfate, on the other hand, altered lytic activity in a biphasic manner, inasmuch as it induced an inhibition of lysis at high concentrations and an increase of lysis at low concentrations. Cell-mediated lysis was inhibited by pNC sulfate in a dose-dependent fashion at concentrations greater than 2.5 mM, with nearly complete inhibition at 50 mM. Sulfate also altered the lytic activity by intact cells in a biphasic manner, although the effect was much less pronounced. Sulfite and phosphate caused only a 30% inhibition of lytic activity. These results suggest that the sulfatase enzyme is involved in NK cytolytic function, presumably at the lethal hit stage.

Inflammatory response induced with an isolated syngeneic activation antigen shared by normal lymphoblasts and YAC lymphoma cells

An immunogenic protein with an identical M r (64 kDa) was isolated from syngeneic concanavalin A-induced lymphoblasts (syn-Con A-blasts) and YAC lymphoma cells, both derived from A mice. The 64-kDa protein was purified by a sequence of biochemical steps: Sephadex G-100 gel filtration, ion-exchange chromatography in a fast protein luquid chromatography system, Con A-Sepharose affinity chromatography, and preparative gel electrophoresis. The immunogenic fraction isolated in each step was moved to the next one, and so on. The immunogenicity of the separated fractions was measured by a lymph node proliferation (LNP) assay, which is indicative of a delayed-type hypersensitivity response. For instance, the final 64-kDa isolated protein of the syn-Con A-blasts induced an efficient LNP response in A mice which was detected after challenge with the final 64-kDa isolated protein of YAC cells. In addition to their identical molecular weight, both proteins were eluted at the same ionic strength and both expressed affinity to Con A-Sepharose beads, suggesting that they were glycosilated. Similar 64-kDa proteins were isolated by a different purification procedure, which was performed in the presence of protease inhibitors, excluding the possibility that the final antigen was an autodigested product. As the 64-kDa protein is immunogenic in the syngeneic host, it may be employed as a immunotherapeutic reagent against the original tumor and perhaps against other tumors expressing the same antigen.

Immunoblotting analysis of concanavalin A ‐isolated allergens of Candida albicans

The carbohydrate-containing fraction of Candida albicans was isolated from the crude extract with ConA Sepharose affinity chromatography and studied by IgE-immunoblotting with individual and pooled sera from C. albicans-allergic subjects. In the ConA-bound fraction there was a diffuse IgE binding in the high molecular weight area which also gave a carbohydrate stain (PAS). A distinct band corresponding to a molecular weight of 70 kD bound specific IgE antibodies. This glycoprotein, presumably a mannoprotein, gave a weak carbohydrate staining and a strong protein staining. Further biochemical studies are needed to reveal the exact nature of the epitopes in the ConA-bound mannose-containing fraction of C. albicans.

O-acetylation and de-O-acetylation of sialic acids: Purification, characterization, and properties of a glycosylated rat liver esterase specific for 9-O-acetylated sialic acids

We have previously described the preparation and use of 9-O-[acetyl-3H]acetyl-N-acetylneuraminic acid to identify sialic acid O-acetylesterases in tissues and cells (Higa, H. H., Diaz, S., and Varki, A. (1987) Biochem. Biophys. Res. Commun. 144, 1099-1108). All tissues of the adult rat showed these activities, with the exception of plasma. Rat liver contained two major sialic acid esterases: a cytosolic nonglycosylated enzyme and a membrane-associated glycosylated enzyme. The two enzymes were found in similar proportions and specific activities in a buffer extract of rat liver acetone powder. By using the latter as a source, the two enzymes were separated, and the glycosylated enzyme was purified to apparent homogeneity by multiple steps, including ConA-Sepharose affinity chromatography and Procion Red-agarose chromatography (yield, 13%; fold purification, approximately 3000). The homogeneous enzyme is a 61.5-kDa disulfide-linked heterodimeric protein, whose serine active site can be labeled with [3H]diisopropyl fluorophosphate. Upon reduction, two subunits of 36 kDa and 30 kDa are generated, and the 30-kDa subunit carries the [3H]diisopropyl fluorophosphate label. The protein has N-linked oligosaccharides that are cleaved by Peptide N-glycosidase F. These chains are cleaved to a much lesser extent by endo-beta-N-acetylglycosaminidase H, indicating that they are mainly complex-type glycans. The enzyme activity has a broad pH optimum range between 6 and 7.5, has no divalent cation requirements, is unaffected by reduction, and is inhibited by the serine active site inhibitors, diisopropyl fluorophosphate (DFP) and diethyl-p-nitrophenyl phosphate (Paraoxon). Kinetic studies with various substrates show that the enzyme is specific for sialic acids and selectively cleaves acetyl groups in the 9-position. It shows little activity against a variety of other natural compounds bearing O-acetyl esters. It appears to deacetylate di-O-acetyl- and tri-O-acetyl-N-acetylneuraminic acids by first cleaving the O-acetyl ester at the 9-position. The 7- and 8-O-acetyl esters then undergo spontaneous migration to the 9-position, where they can be cleaved, resulting in the production of N-acetylneuraminic acid. In view of its interesting substrate specificity, complex N-linked glycan structure, and neutral pH optimum, it is suggested that this enzyme is involved in the regulation of O-acetylation in membrane-bound sialic acids.