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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-. </p></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK594057_"><span class="title" itemprop="name">Enzyme assay for mammalian sialidases</span></h1><div class="contrib half_rhythm"><span itemprop="author">Taeko Miyagi</span>, MD, PhD<div class="affiliation small">Miyagi
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Cancer Center,
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Res. Inst.<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="moc.ytfin@at-igayim" class="oemail">moc.ytfin@at-igayim</a></div></div><div class="small">Corresponding author.</div></div><div class="contrib half_rhythm"><span itemprop="author">Koji Yamamoto</span>, PhD<div class="affiliation small">Saitama Med. Univ.<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.dem-amatias@ok-amaY" class="oemail">pj.ca.dem-amatias@ok-amaY</a></div></div></div><div class="contrib half_rhythm"><span itemprop="author">Kazuhiro Shiozaki</span>, PhD<div class="affiliation small">Kagoshima Univ.<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-amihsogak.hsif@ikazoihS" class="oemail">pj.ca.u-amihsogak.hsif@ikazoihS</a></div></div></div><p class="small">Created: <span itemprop="datePublished">October 7, 2021</span>; Last Revision: <span itemprop="dateModified">March 22, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g36-assaysialidase.Introduction"><h2 id="_g36-assaysialidase_Introduction_">Introduction</h2><p>Sialic acids are acidic monosaccharides generally found in terminal positions on various glycoproteins, glycolipids, and oligosaccharides. Sialidases catalyze the removal of α-glycosidically linked sialic acid residues from these glycoconjugates as an initial step in degradation and are involved in various biological processes by influencing chemical and biological features of the molecules. These enzymes exist widely in vertebrates and microorganisms, but their enzymatic properties and functional significance vary greatly between these groups. Although the primary sequences are not particularly similar, alignment studies have shown that they contain several Asp boxes (-Ser-X-Asp-X-Gly-X-Thr-Trp-) and an Arg-Ile-Pro sequence conserved from microorganisms throughout evolution. Recent progress in mammalian sialidase research has accumulated evidence that these enzymes are of great importance for various cellular functions along with lysosomal catabolism, whereas microbial sialidases play roles limited to nutrition and infection.</p><p>Four types of mammalian sialidases have been identified and characterized to date (<a class="bk_pop" href="#g36-assaysialidase.REF.1">1</a>), designated as Neu1, Neu2, Neu3, and Neu4. They differ in enzymatic properties and subcellular sites and in their primary structures and chromosomal localization (<a class="figpopup" href="/books/NBK594057/table/g36-assaysialidase.T.properties_of_four/?report=objectonly" target="object" rid-figpopup="figg36assaysialidaseTpropertiesoffour" rid-ob="figobg36assaysialidaseTpropertiesoffour">Table 1</a>). Among human sialidase orthologs, the overall amino acid identity of NEU1 to the other forms is relatively low (19%–24%), while NEU2, NEU3, and NEU4 show 34%–40% homology to each other. Regarding comparative expression levels of human sialidases, NEU1 generally shows the highest expression, 10–20 times higher than those of NEU3 and NEU4, whereas NEU2 expression is extremely low, only one-fourth to ten-thousandth of the NEU1 value at the most in various tissues as assessed by quantitative real-time reverse transcription–polymerase chain reaction (RT–PCR) (<a class="bk_pop" href="#g36-assaysialidase.REF.2">2</a>). NEU1 mainly localized in lysosomes is a target gene for sialidosis (sialidase deficiency) and is associated with a protective protein (carboxypeptidase A) and β-galactosidase as a complex in lysosomes. The dissociation of the complex leads to sialidase inactivation (<a class="bk_pop" href="#g36-assaysialidase.REF.3">3</a>). The expression of NEU3 is markedly increased in various human cancers, leading to the enhancement of malignant properties, including suppression of apoptosis and invasion (<a class="bk_pop" href="#g36-assaysialidase.REF.4">4</a>). NEU4 is localized in two sites: one is the lysosomal lumen and the other is the mitochondria and microsomal membranes. Unlike other human sialidases, the enzyme possesses broad substrate specificity with sensitivity to mucin <i>in vitro</i>. Concerning subcellular localization, sialidases can functionally move to sites other than their major locations, such as the cell surface, depending on physiological conditions.</p><p>The optimum pH and good substrates for the activity assay are summarized in <a class="figpopup" href="/books/NBK594057/table/g36-assaysialidase.T.properties_of_four/?report=objectonly" target="object" rid-figpopup="figg36assaysialidaseTpropertiesoffour" rid-ob="figobg36assaysialidaseTpropertiesoffour">Table 1</a>. To evaluate the expression of the four isoforms, measurement of the mRNA level using RT–PCR gives helpful information about which form is responsible for the activity (<b>Note 1</b>). There has often been a tendency of the sialidase activity level being parallel with the mRNA level.</p></div><div id="g36-assaysialidase.Protocol"><h2 id="_g36-assaysialidase_Protocol_">Protocol</h2><p>For the activity assays, subcellular localization, substrate specificity, and the optimal pH of each sialidase must be considered. As the enzyme source from cells and tissues, membrane fractions are often used for Neu1, Neu3, Neu4, and cytosolic fractions for Neu2. Generally, 4MU-NeuAc is capable of being used as a substrate to determine Neu1, Neu2, and Neu4 activities, but gangliosides for Neu3 show ten-fold higher activity than the former. To determine released sialic acids, thiobarbituric acid (TBA) methods and high-performance liquid chromatography (HPLC) with fluorescence detection have often been applied for natural substrates, including glycoproteins, gangliosides, and oligosaccharides, as described below. Radioactive [3-<sup>3</sup>H] GD1a has also been used as a ganglioside substrate (<a class="bk_pop" href="#g36-assaysialidase.REF.5">5</a>) (<b>Note 2</b>).</p><p>Furthermore, to determine the desialylated products, an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody followed by treatment with a horseradish peroxidase-conjugated second antibody has also been employed, the amount of bound peroxidase then being measured in a colorimetric enzymatic assay (<a class="bk_pop" href="#g36-assaysialidase.REF.6">6</a>). In the case of GD1a as substrate, the cholera toxin B subunit conjugated to horseradish peroxidase can be used due to specific binding to GM1, the desialylated product of GD1a (<a class="bk_pop" href="#g36-assaysialidase.REF.7">7</a>). To determine the amount of galactose exposed by sialidase reactions, a galactose oxidase-coupling method has been used in ELISA assays, where galactose oxidase reacts with galactose to produce hydrogen peroxide resulting in a colorimetric or fluorometric product in the presence of horseradish peroxidase (commercially available, Molecular probes; Sigma-Aldrich).</p><div id="g36-assaysialidase.Materials"><h3>Materials</h3><p>1. Buffers: sodium acetate buffer (pH 4.5 or 5.5) and sodium phosphate buffer (pH 6.5).</p><p>2. Substrates: 4-Methylumbelliferyl N-acetylneuraminic acid (4MU-NeuAc, Nacalai Tesque, Inc., Kyoto, Japan), Gangliosides (mixed gangliosides from bovine brain) or GM3 (NeuAcα2-3Galβ1-4Glcβ1-1Cer) (Calbiochem, San Diego), Fetuin (Funakoshi, Tokyo), and 3’-Sialyllactose (Funakoshi)</p><p>3. Triton X-100 (Calbiochem)</p><p>4. Sodium cholate (Calbiochem)</p><p>5. Bovine serum albumin (Fraction V, Sigma-Aldrich, St. Louis, MO)</p><p>6. 4-methylumbelliferylone (4-MU)</p><p>7. Glycine-NaOH (pH 10.4)</p><p>8. Sodium metaperiodate</p><p>9. Sodium arsenite</p><p>10. TBA</p><p>11. Cyclohexanone</p><p>12. Sialic acid as the standard</p><p>13. Malononitrile</p><p>14. Sodium tetraborate</p><p>15. Methanol</p><p>16. Ammonium acetate buffer (pH 5.5) or 1,2-Diamino-4, 5-methylenedioxybenzene (DMB) kit (Takara Bio Inc., Otsu, Japan)</p><p>17. Dye-binding assay kit (Bio-Rad Laboratories, Hercules, CA)</p></div><div id="g36-assaysialidase.Instruments"><h3>Instruments</h3><p>1. Incubator (37°C)</p><p>2. Voltex mixer</p><p>3. Centrifuge</p><p>4. Spectrophotometer</p><p>5. Fluorescence detector</p><p>6. Reverse phase Luna 5u C18 column (Phenomenex, Torrance, CA)</p></div><div id="g36-assaysialidase.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Sialidase activity assays with ganglioside substrates</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Place the reaction mixture containing 5 μmol of sodium acetate buffer (pH 4.5), 10 nmol of gangliosides GM3, 50 μg of Triton X-100, 50 μg of bovine serum albumin, and enzyme (0.25mg of protein) in 50 μL in a test tube (<b>Note 3</b>).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Incubate for 0.5–2 h at 37°C.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Stop the incubation by freezing or immediately determine released sialic acid by TBA or HPLC method.</p></dd><dt>d.</dt><dd><p class="no_top_margin">One unit was defined as the amount of enzyme cleaving 1 nmol of sialic acid/h.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Protein concentrations were determined by dye-binding assay (Bio-Rad Laboratories).</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Determination of released sialic acids (TBA method) (<a class="bk_pop" href="#g36-assaysialidase.REF.8">8</a>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Terminate the reaction (50 μL) by adding 25 μL of metaperiodate solution.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Stand for 20 min at room temperature.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Add 250 μL of arsenite solution, and vortex the tube until the yellow–brown color disappears.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Add 750 μL of TBA solution and vortex and heat in a boiling water bath for15 min.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Place in cold water for 5 min.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Add 1 mL of cyclohexanone and vortex and centrifuge at 3,000 rpm for 10 min.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Measure the absorbance of upper phase at 549 nm.</p></dd><dt>h.</dt><dd><p class="no_top_margin">If the absorbance ratio A<sub>549</sub>/A<sub>532</sub> for the sample is lower than the standard, calculate the value according to the formula: (0.9 × A<sub>549</sub> - 0.3 × A<sub>532</sub>), to compensate for the interference (<b>Note 4</b>).</p></dd></dl></dd><dt>3.</dt><dd><p class="no_top_margin">HPLC analysis with fluorometric detection (<a class="bk_pop" href="#g36-assaysialidase.REF.9">9</a>) (<b>Note 5</b>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Add 50 μL of malononitrile (0.8%) to the reaction mixture (25 μL).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Add 355 μL of tetraborate (0.15 M, pH 9.5).</p></dd><dt>c.</dt><dd><p class="no_top_margin">Warm for 20 min at 80°C.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Apply 10–20 μL to a reversed-phase column (C18) and separate with methanol and ammonium acetate buffer (15:85 v/v, pH 5.5).</p></dd><dt>e.</dt><dd><p class="no_top_margin">Measure fluorescence at 434 nm for emission and 357 nm for excitation.</p></dd></dl></dd><dt>4.</dt><dd><p class="no_top_margin">Sialidase activity assays with the 4MU-NeuAc substrate</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Place the reaction mixture containing 10 μmol of sodium acetate buffer (pH 4.5 or pH 5.5), 20 nmol of 4MU-NeuAc, 0.1 mg of bovine serum albumin, and enzyme in 0.1 mL.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Incubate for 20–60 min at 37°C.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Terminate the reaction by adding 1.25 mL of 0.25M glycine-NaOH (pH 10.4).</p></dd><dt>d.</dt><dd><p class="no_top_margin">Fluorometrically measure the 4-methylumbelliferylone(4-MU) released (emission 448 nm, excitation 365 nm).</p></dd></dl></dd></dl></div><div id="g36-assaysialidase.Notes"><h3>Notes</h3><p><b>1.</b> Quantification of the sialidase transcripts.</p><p>Quantitative analysis can be performed by real-time RT–PCR using a LightCycler rapid thermal cycler system (Roche, Basel) as described previously (<a class="bk_pop" href="#g36-assaysialidase.REF.10">10</a>). Total RNA is prepared using an RNeasy mini kit (Qiagen, Venlo, Netherland) and reverse transcribed with Super Script II (Invitrogen, Waltham, MA), and RT–PCR is performed using a QuantiTect SYBR Green PCR kit (Qiagen). To normalize for sample variation, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAP-DH), β-actin, or porphobilinogen deaminase can be determined as an internal control. For comparison of mRNA levels among the sialidases, a standard curve for each cDNA should be generated by serial dilution of the pBluescript vector containing the gene encoding the entire open reading frame. The primers for the four human and mouse sialidases have been listed elsewhere (<a class="bk_pop" href="#g36-assaysialidase.REF.10">10</a>,<a class="bk_pop" href="#g36-assaysialidase.REF.11">11</a>). Amplification of the cDNAs involves a 15-min denaturation step followed by 40–45 cycles at 94°C for 15 s, 60°C for 30 s, and 72°C for 30 s. Fluorescence from SYBR Green bound to the PCR product is detected, and the specificity of the reactions is confirmed by melting curve analysis and subsequently by agarose gel electrophoresis.</p><p><b>2.</b> Preparation of [3-3H(Sph18)]GD1a</p><p>Radioactive GD1a containing erythro-C18-sphingosine, isotopically tritium-labeled at position 3, and [3-3H(Sph18)]GD1a was prepared by the dichloro-dicyano-benzoquinone/sodium boro[3H] hydride method followed by reversed-phase HPLC purification (homogeneity >99% and specific radioactivity of 1.2 Ci/mmol) (<a class="bk_pop" href="#g36-assaysialidase.REF.12">12</a>).</p><p><b>3.</b> NEU2 activity assays with ganglioside as substrate</p><p>For NEU2 activity assays with ganglioside as substrate, pH 5.5–6.0 buffer and sodium cholate are used instead of Triton X-100. For the determination of released sialic acid by HPLC method, 25 μL of the reaction mixture can be used.</p><p><b>4.</b> Some interference in TBA methods</p><p>TBA methods have been generally used, but the color reaction is affected by other sugar compounds and is much less sensitive than HPLC methods with fluorogenic reagents. Chromophores generated from sialic acids can be quantitated with spectrophotometric or HPLC detection. With crude homogenates as the enzyme source, the TBA method may be performed after passage through an AG1X-2 mini-column to reduce interference by other sugar compounds, wherever necessary. To directly detect chromophores, another analytical method using HPLC (elution buffer, 2:3:5 v/v/v mixture of water/methanol/0.2% v/v orthophosphoric acid) connecting to ultraviolet detector can be applied.</p><p><b>5.</b> HPLC methods using DMB</p><p>DMB can also be used as a fluorogenic reagent for sialic acid determination (<a class="bk_pop" href="#g36-assaysialidase.REF.13">13</a>), but the reaction needs a longer time (2.5 h at 50°C in the dark). However, N-acetylneuraminic acid and N-glycolylneuraminic acid can be separately quantitated by the DMB method.</p></div></div><div id="g36-assaysialidase.References"><h2 id="_g36-assaysialidase_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g36-assaysialidase.REF.1">Miyagi T, Yamaguchi K. Mammalian sialidases physiological and pathological roles in cellular functions. <span><span class="ref-journal">Glycobiology. </span>2012 Jul;<span class="ref-vol">22</span>(7):880–96.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/22377912" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22377912</span></a>] [<a href="http://dx.crossref.org/10.1093/glycob/cws057" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g36-assaysialidase.REF.2">Koseki K, Wada T, Hosono M, Hata K, Yamaguchi K, Nitta K, Miyagi T. Human cytosolic sialidase NEU2— low general tissue expression but involvement in PC-3 prostate cancer cell survival. <span><span class="ref-journal">Biochem Biophys Res Commun. </span>2012;<span class="ref-vol">428</span>:142–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/23068092" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23068092</span></a>] [<a href="http://dx.crossref.org/10.1016/j.bbrc.2012.10.028" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g36-assaysialidase.REF.3">D’Azzo A, Hoogeveen A, Reuser A J, Robinson D, Galjaard H. Molecular defect in combined beta-galactosidase and neuraminidase deficiency in man. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>1982 Aug;<span class="ref-vol">79</span>(15):4535–9.</span> [<a href="/pmc/articles/PMC346709/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC346709</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/6812049" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 6812049</span></a>] [<a href="http://dx.crossref.org/10.1073/pnas.79.15.4535" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g36-assaysialidase.REF.4">Kakugawa Y, Wada T, Yamaguchi K, Yamanami H, Ouchi K, Sato I, Miyagi T. Up-regulation of plasma membrane-associated ganglioside sialidase (Neu3) in human colon cancer and its involvement in apoptosis suppression. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2002 Aug 6;<span class="ref-vol">99</span>(16):10718–23.</span> [<a href="/pmc/articles/PMC125023/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC125023</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/12149448" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12149448</span></a>] [<a href="http://dx.crossref.org/10.1073/pnas.152597199" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="g36-assaysialidase.REF.5">Papini N, Anastasia L, Tringali C, Croci G, Bresciani R, Yamaguchi K, Miyagi T, Preti A, Prinetti A, Prioni S, Sonnino S, Tettamanti G, Venerando B, Monti E. The plasma membrane-associated sialidase MmNEU3 modifies the ganglioside pattern of adjacent cells supporting its involvement in cell-to-cell interactions. <span><span class="ref-journal">J Biol Chem. </span>2004;<span class="ref-vol">279</span>:16989–95.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14970224" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14970224</span></a>] [<a href="http://dx.crossref.org/10.1074/jbM400881200" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="g36-assaysialidase.REF.6">Taki T, Nishiwaki S, Ishii K, Handa S. A simple and specific assay of glycosyltransferase and glycosidase activities by an enzyme-linked immunosorbent assay method, and its application to assay of galactosyltransferase activity in sera from patients with cancer. <span><span class="ref-journal">J Biochem. </span>1990 Mar;<span class="ref-vol">107</span>(3):493–8.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/1692829" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 1692829</span></a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="g36-assaysialidase.REF.7">Ogura K, Ogura M, Anderson R L, Sweeley C.C. Peroxidase-amplified assay of sialifdase activity toward gangliosides. <span><span class="ref-journal">Anal Biochem. </span>1992 Jan;<span class="ref-vol">200</span>(1):52–7.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/1595901" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 1595901</span></a>] [<a href="http://dx.crossref.org/10.1016/0003-2697(92)90276-d" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="g36-assaysialidase.REF.8">Warren L. The thiobarbituric acid assay of sialic acids. <span><span class="ref-journal">J Biol Chem. </span>1959 Aug;<span class="ref-vol">234</span>(8):1971–5.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/13672998" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 13672998</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="g36-assaysialidase.REF.9">Li K. Determination of sialic acids in human serum by reversed-phase liquid chromatography with fluorimetric detection. <span><span class="ref-journal">J Chromatogr. </span>1992 Sep 2;<span class="ref-vol">579</span>(2):209–13.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/1429968" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 1429968</span></a>] [<a href="http://dx.crossref.org/10.1016/0378-4347(92)80384-3" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="g36-assaysialidase.REF.10">Miyagi T, Wada T, Yamaguchi K. Human sialidase as a cancer marker. <span><span class="ref-journal">Proteomics. </span>2008 Aug;<span class="ref-vol">8</span>(16):3303–11.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18651674" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18651674</span></a>] [<a href="http://dx.crossref.org/10.1002/pmic.200800248" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="g36-assaysialidase.REF.11">Shiozaki K, Koseki K, Yamaguchi K, Shiozaki M, Narimatsu H, Miyagi T. Developmental change of sialidase Neu4 expression in murine brain and its involvement in the regulation of neuronal cell differentiation. <span><span class="ref-journal">J Biol Chem. </span>2009 Aug 7;<span class="ref-vol">284</span>(32):21157–64.</span> [<a href="/pmc/articles/PMC2755838/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2755838</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19506080" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19506080</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.M109.012708" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>12.</dt><dd><div class="bk_ref" id="g36-assaysialidase.REF.12">Sonnino S, Ghidoni R, Gazzotti G, Kirschner G, Galli G, Tettamanti G. High performance liquid chromatography preparation of the molecular species of GM1 and GD1a gangliosides with homogeneous long chain base composition. <span><span class="ref-journal">J Lipid Res. </span>1984 Jun;<span class="ref-vol">25</span>(6):620–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/6747465" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 6747465</span></a>]</div></dd><dt>13.</dt><dd><div class="bk_ref" id="g36-assaysialidase.REF.13">Hara S, Yamaguchi M, Takemori Y, Nakamura M, Ohkura Y. Highly sensitive determination of N-acetyl- and N-glycolylneuraminic acids in human serum and urine and rat serum by reversed-phase liquid chromatography with fluorescence detection. <span><span class="ref-journal">J Chromatogr. </span>1986;<span class="ref-vol">377</span>:111–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/3711202" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 3711202</span></a>] [<a href="http://dx.crossref.org/10.1016/s0378-4347(00)80766-5" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK594057_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g36-assaysialidase.FN1"><p class="no_top_margin">The authors declare no competing or financial interests.</p></div></dd></dl><div class="bk_prnt_sctn"><h2>Tables</h2><div class="whole_rhythm bk_prnt_obj bk_first_prnt_obj"><div id="g36-assaysialidase.T.properties_of_four" class="table"><h3><span class="label">Table 1: </span></h3><div class="caption"><p>Properties of four mammalian sialidases.</p></div><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK594057/table/g36-assaysialidase.T.properties_of_four/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__g36-assaysialidase.T.properties_of_four_lrgtbl__"><table class="no_bottom_margin"><thead><tr><th id="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_1" scope="col" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></th><th id="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_2" scope="col" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Neu1</th><th id="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_3" scope="col" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Neu2</th><th id="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_4" scope="col" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Neu3</th><th id="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_5" scope="col" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Neu4</th></tr></thead><tbody><tr><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_1" scope="row" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
||
<b>Major subcellular</b>
|
||
<br />
|
||
<b>localization</b>
|
||
</td><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Lysosome</td><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cytosol</td><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Plasma membrane<br />Endosome</td><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Lysosome<sup>1)</sup><br />Mitochondria and ER <sup>1)</sup></td></tr><tr><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_1" scope="row" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
||
<b>Good substrates</b>
|
||
<br />
|
||
<b>in activity assays</b>
|
||
<br />
|
||
<b>
|
||
<i>in vitro</i>
|
||
</b>
|
||
</td><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Oligosaccharides<br />Glycopeptides<br />4MU-NeuAc</td><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Oligosaccharides<br />Glycoproteins<br />Gangliosides<br />4MU-NeuAc</td><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Gangliosides</td><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Oligosaccharides<br />Glycoproteins<br />Gangliosides<br />4MU-NeuAc</td></tr><tr><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_1" scope="row" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
||
<b>Optimum PH</b>
|
||
</td><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4.4-4.6</td><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">6.0-6.5</td><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4.6-4.8<br />6.0-6.5<sup>2)</sup></td><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4.4-4.5</td></tr><tr><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_1" scope="row" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
||
<b>Endogenous substrates</b>
|
||
<br />
|
||
<b>identified in cellular level</b>
|
||
</td><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">LAMP-1<br />IR, IGFR,<br />PDGFR, Integrinβ4</td><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Sialyl-Le<sup>x</sup><br />GM3</td><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">GM3<br />GD3, GD1a<br />EGFR</td><td headers="hd_h_g36-assaysialidase.T.properties_of_four_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Sialyl-Le<sup>x</sup><br />Sialyl-Le<sup>a</sup><br />PolySia-NCAM<br />GD1a</td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt><sup>1)</sup>
|
||
</dt><dd><div id="g36-assaysialidase.TF.1.1"><p class="no_margin">For the subcellular localization of human NEU4, two different results have been described.</p></div></dd><dt><sup>2)</sup>
|
||
</dt><dd><div id="g36-assaysialidase.TF.1.2"><p class="no_margin">Human NEU3 has two peaks in pH curve. The second peak shows approx. 70% of the first peak.</p></div></dd></dl></div></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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<div class="post-content"><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a><p class="small">Licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 Unported license. To view a copy of this license, visit <a href="http://creativecommons.org/licenses/by-nc-nd/4.0/" ref="pagearea=meta&targetsite=external&targetcat=link&targettype=uri">http://creativecommons.org/licenses/by-nc-nd/4.0/</a>.</p></div><div class="small"><span class="label">Bookshelf ID: NBK594057</span><span class="label">PMID: <a href="https://pubmed.ncbi.nlm.nih.gov/37590779" title="PubMed record of this page" ref="pagearea=meta&targetsite=entrez&targetcat=link&targettype=pubmed">37590779</a></span></div><div style="margin-top:2em" class="bk_noprnt"><a class="bk_cntns" href="/books/n/glycopodv2/">Contents</a><div class="pagination bk_noprnt"><a class="active page_link prev" href="/books/n/glycopodv2/g35-enzymeassayacetyl/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/glycopodv2/g37-enzymeassayendo/" title="Next page in this title">Next ></a></div></div></div></div>
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