A rapid and highly sensitive method for measuring enzyme activities in single mycorrhizal tips using 4-methylumbelliferone-labelled fluorogenic substrates in a microplate system.
Identifieur interne : 003717 ( Main/Corpus ); précédent : 003716; suivant : 003718A rapid and highly sensitive method for measuring enzyme activities in single mycorrhizal tips using 4-methylumbelliferone-labelled fluorogenic substrates in a microplate system.
Auteurs : K. Pritsch ; S. Raidl ; E. Marksteiner ; H. Blaschke ; R. Agerer ; M. Schloter ; A. HartmannSource :
- Journal of microbiological methods [ 0167-7012 ] ; 2004.
English descriptors
- KwdEn :
- Chitinases (analysis), Fluorescent Dyes (MeSH), Hydrogen-Ion Concentration (MeSH), Hymecromone (analogs & derivatives), Mycology (methods), Mycology (statistics & numerical data), Mycorrhizae (enzymology), Phosphoric Monoester Hydrolases (analysis), Reproducibility of Results (MeSH), Sensitivity and Specificity (MeSH), Substrate Specificity (MeSH), beta-Glucosidase (analysis).
- MESH :
- chemical , analogs & derivatives : Hymecromone.
- chemical , analysis : Chitinases, Phosphoric Monoester Hydrolases, beta-Glucosidase.
- chemical : Fluorescent Dyes.
- enzymology : Mycorrhizae.
- methods : Mycology.
- statistics & numerical data : Mycology.
- Hydrogen-Ion Concentration, Reproducibility of Results, Sensitivity and Specificity, Substrate Specificity.
Abstract
A microplate fluorimetric assay was developed for measuring potential activities of extracellular enzymes of individual ectomycorrhizal (EM) roots using methylumbelliferone (MU)-labelled fluorescent substrate analogues and microsieves to minimise damage due to manipulation of excised mycorrhizal roots. Control experiments revealed that enzyme activities remained stable over the whole time of the experiment suggesting a strong affinity of the studied enzymes to the fungal cell walls. The same mycorrhizal tips thus could be used repeatedly for enzyme detection and subsequently analysed for the projection area by automated image analysis. The developed system was evaluated on four different EM species measuring pH optimum and substrate saturation of phosphatase, chitinase and beta-glucosidase. The four EM species studied were Lactarius subdulcis, Russula ochroleuca, Cortinarius obtusus and Xerocomus cf. chrysenteron. Depending upon the enzyme, each species exhibited different levels of enzymatic activities as well as enzyme kinetics and showed also differences in pH optima.
DOI: 10.1016/j.mimet.2004.04.001
PubMed: 15234521
Links to Exploration step
pubmed:15234521Le document en format XML
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Blaschke</name></author><author><name sortKey="Agerer, R" sort="Agerer, R" uniqKey="Agerer R" first="R" last="Agerer">R. Agerer</name></author><author><name sortKey="Schloter, M" sort="Schloter, M" uniqKey="Schloter M" first="M" last="Schloter">M. Schloter</name></author><author><name sortKey="Hartmann, A" sort="Hartmann, A" uniqKey="Hartmann A" first="A" last="Hartmann">A. Hartmann</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2004">2004</date><idno type="RBID">pubmed:15234521</idno><idno type="pmid">15234521</idno><idno type="doi">10.1016/j.mimet.2004.04.001</idno><idno type="wicri:Area/Main/Corpus">003717</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Corpus" wicri:corpus="PubMed">003717</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">A rapid and highly sensitive method for measuring enzyme activities in single mycorrhizal tips using 4-methylumbelliferone-labelled fluorogenic substrates in a microplate system.</title><author><name sortKey="Pritsch, K" sort="Pritsch, K" uniqKey="Pritsch K" first="K" last="Pritsch">K. Pritsch</name><affiliation><nlm:affiliation>Chair of Soil Ecology, Technische Universität Munich (TUM), D-85758 Oberschleissheim, Germany. pritsch@gsf.de</nlm:affiliation></affiliation></author><author><name sortKey="Raidl, S" sort="Raidl, S" uniqKey="Raidl S" first="S" last="Raidl">S. Raidl</name></author><author><name sortKey="Marksteiner, E" sort="Marksteiner, E" uniqKey="Marksteiner E" first="E" last="Marksteiner">E. Marksteiner</name></author><author><name sortKey="Blaschke, H" sort="Blaschke, H" uniqKey="Blaschke H" first="H" last="Blaschke">H. Blaschke</name></author><author><name sortKey="Agerer, R" sort="Agerer, R" uniqKey="Agerer R" first="R" last="Agerer">R. Agerer</name></author><author><name sortKey="Schloter, M" sort="Schloter, M" uniqKey="Schloter M" first="M" last="Schloter">M. Schloter</name></author><author><name sortKey="Hartmann, A" sort="Hartmann, A" uniqKey="Hartmann A" first="A" last="Hartmann">A. Hartmann</name></author></analytic><series><title level="j">Journal of microbiological methods</title><idno type="ISSN">0167-7012</idno><imprint><date when="2004" type="published">2004</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Chitinases (analysis)</term><term>Fluorescent Dyes (MeSH)</term><term>Hydrogen-Ion Concentration (MeSH)</term><term>Hymecromone (analogs & derivatives)</term><term>Mycology (methods)</term><term>Mycology (statistics & numerical data)</term><term>Mycorrhizae (enzymology)</term><term>Phosphoric Monoester Hydrolases (analysis)</term><term>Reproducibility of Results (MeSH)</term><term>Sensitivity and Specificity (MeSH)</term><term>Substrate Specificity (MeSH)</term><term>beta-Glucosidase (analysis)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analogs & derivatives" xml:lang="en"><term>Hymecromone</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analysis" xml:lang="en"><term>Chitinases</term><term>Phosphoric Monoester Hydrolases</term><term>beta-Glucosidase</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>Fluorescent Dyes</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Mycorrhizae</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Mycology</term></keywords><keywords scheme="MESH" qualifier="statistics & numerical data" xml:lang="en"><term>Mycology</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Hydrogen-Ion Concentration</term><term>Reproducibility of Results</term><term>Sensitivity and Specificity</term><term>Substrate Specificity</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">A microplate fluorimetric assay was developed for measuring potential activities of extracellular enzymes of individual ectomycorrhizal (EM) roots using methylumbelliferone (MU)-labelled fluorescent substrate analogues and microsieves to minimise damage due to manipulation of excised mycorrhizal roots. Control experiments revealed that enzyme activities remained stable over the whole time of the experiment suggesting a strong affinity of the studied enzymes to the fungal cell walls. The same mycorrhizal tips thus could be used repeatedly for enzyme detection and subsequently analysed for the projection area by automated image analysis. The developed system was evaluated on four different EM species measuring pH optimum and substrate saturation of phosphatase, chitinase and beta-glucosidase. The four EM species studied were Lactarius subdulcis, Russula ochroleuca, Cortinarius obtusus and Xerocomus cf. chrysenteron. Depending upon the enzyme, each species exhibited different levels of enzymatic activities as well as enzyme kinetics and showed also differences in pH optima.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">15234521</PMID><DateCompleted><Year>2004</Year><Month>09</Month><Day>17</Day></DateCompleted><DateRevised><Year>2016</Year><Month>11</Month><Day>24</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0167-7012</ISSN><JournalIssue CitedMedium="Print"><Volume>58</Volume><Issue>2</Issue><PubDate><Year>2004</Year><Month>Aug</Month></PubDate></JournalIssue><Title>Journal of microbiological methods</Title><ISOAbbreviation>J Microbiol Methods</ISOAbbreviation></Journal><ArticleTitle>A rapid and highly sensitive method for measuring enzyme activities in single mycorrhizal tips using 4-methylumbelliferone-labelled fluorogenic substrates in a microplate system.</ArticleTitle><Pagination><MedlinePgn>233-41</MedlinePgn></Pagination><Abstract><AbstractText>A microplate fluorimetric assay was developed for measuring potential activities of extracellular enzymes of individual ectomycorrhizal (EM) roots using methylumbelliferone (MU)-labelled fluorescent substrate analogues and microsieves to minimise damage due to manipulation of excised mycorrhizal roots. Control experiments revealed that enzyme activities remained stable over the whole time of the experiment suggesting a strong affinity of the studied enzymes to the fungal cell walls. The same mycorrhizal tips thus could be used repeatedly for enzyme detection and subsequently analysed for the projection area by automated image analysis. The developed system was evaluated on four different EM species measuring pH optimum and substrate saturation of phosphatase, chitinase and beta-glucosidase. The four EM species studied were Lactarius subdulcis, Russula ochroleuca, Cortinarius obtusus and Xerocomus cf. chrysenteron. Depending upon the enzyme, each species exhibited different levels of enzymatic activities as well as enzyme kinetics and showed also differences in pH optima.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Pritsch</LastName><ForeName>K</ForeName><Initials>K</Initials><AffiliationInfo><Affiliation>Chair of Soil Ecology, Technische Universität Munich (TUM), D-85758 Oberschleissheim, Germany. pritsch@gsf.de</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Raidl</LastName><ForeName>S</ForeName><Initials>S</Initials></Author><Author ValidYN="Y"><LastName>Marksteiner</LastName><ForeName>E</ForeName><Initials>E</Initials></Author><Author ValidYN="Y"><LastName>Blaschke</LastName><ForeName>H</ForeName><Initials>H</Initials></Author><Author ValidYN="Y"><LastName>Agerer</LastName><ForeName>R</ForeName><Initials>R</Initials></Author><Author ValidYN="Y"><LastName>Schloter</LastName><ForeName>M</ForeName><Initials>M</Initials></Author><Author ValidYN="Y"><LastName>Hartmann</LastName><ForeName>A</ForeName><Initials>A</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>Netherlands</Country><MedlineTA>J Microbiol Methods</MedlineTA><NlmUniqueID>8306883</NlmUniqueID><ISSNLinking>0167-7012</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D005456">Fluorescent Dyes</NameOfSubstance></Chemical><Chemical><RegistryNumber>3T5NG4Q468</RegistryNumber><NameOfSubstance UI="D006923">Hymecromone</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.1.3.2</RegistryNumber><NameOfSubstance UI="D010744">Phosphoric Monoester Hydrolases</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.2.1.14</RegistryNumber><NameOfSubstance 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derivatives</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D009172" MajorTopicYN="N">Mycology</DescriptorName><QualifierName UI="Q000379" MajorTopicYN="Y">methods</QualifierName><QualifierName UI="Q000706" MajorTopicYN="N">statistics & numerical data</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D038821" MajorTopicYN="N">Mycorrhizae</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="Y">enzymology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D010744" MajorTopicYN="N">Phosphoric Monoester Hydrolases</DescriptorName><QualifierName UI="Q000032" MajorTopicYN="N">analysis</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D015203" MajorTopicYN="N">Reproducibility of Results</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D012680" MajorTopicYN="N">Sensitivity and Specificity</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D013379" MajorTopicYN="N">Substrate Specificity</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D001617" MajorTopicYN="N">beta-Glucosidase</DescriptorName><QualifierName UI="Q000032" MajorTopicYN="N">analysis</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2004</Year><Month>03</Month><Day>11</Day></PubMedPubDate><PubMedPubDate PubStatus="revised"><Year>2004</Year><Month>04</Month><Day>02</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2004</Year><Month>04</Month><Day>02</Day></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2004</Year><Month>7</Month><Day>6</Day><Hour>5</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2004</Year><Month>9</Month><Day>21</Day><Hour>5</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate 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