Direct Imaging of Dehydrogenase Activity within Living Cells Using Enzyme-Dependent Fluorescence Recovery after Photobleaching (ED-FRAP)
Reduced nicotine adenine dinucleotide (NADH) is a key metabolite involved in cellular energy conversion and many redox reactions. We describe the use of confocal microscopy in conjunction with enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH as a topological assay of NAD...
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| Veröffentlicht in: | Biophysical journal 2001-04, Vol.80 (4), p.2018-2028 |
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| creator | Combs, C.A. Balaban, R.S. |
| description | Reduced nicotine adenine dinucleotide (NADH) is a key metabolite involved in cellular energy conversion and many redox reactions. We describe the use of confocal microscopy in conjunction with enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH as a topological assay of NADH generation capacity within living cardiac myocytes. Quantitative validation of this approach was performed using a dehydrogenase system, in vitro. In intact cells the NADH ED-FRAP was sensitive to temperature (Q
10 of 2.5) and to dehydrogenase activation by dichloroacetate or cAMP (twofold increase for each). In addition, NADH ED-FRAP was correlated with flavin adenine dinucleotide (FAD
+) fluorescence. These data, coupled with the cellular patterns of NADH ED-FRAP changes with dehydrogenase stimulation, suggest that NADH ED-FRAP is localized to the mitochondria. These results suggest that ED-FRAP enables measurement of regional dynamics of mitochondrial NADH production in intact cells, thus providing information regarding region-specific intracellular redox reactions and energy metabolism. |
| doi_str_mv | 10.1016/S0006-3495(01)76172-3 |
| format | Article |
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10 of 2.5) and to dehydrogenase activation by dichloroacetate or cAMP (twofold increase for each). In addition, NADH ED-FRAP was correlated with flavin adenine dinucleotide (FAD
+) fluorescence. These data, coupled with the cellular patterns of NADH ED-FRAP changes with dehydrogenase stimulation, suggest that NADH ED-FRAP is localized to the mitochondria. These results suggest that ED-FRAP enables measurement of regional dynamics of mitochondrial NADH production in intact cells, thus providing information regarding region-specific intracellular redox reactions and energy metabolism.</description><identifier>ISSN: 0006-3495</identifier><identifier>EISSN: 1542-0086</identifier><identifier>DOI: 10.1016/S0006-3495(01)76172-3</identifier><identifier>PMID: 11259315</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Animals ; Cellular biology ; Cyclic AMP - metabolism ; Dichloroacetic Acid - metabolism ; Enzymes ; Flavin-Adenine Dinucleotide - metabolism ; Fluorescence ; Glutamate Dehydrogenase - metabolism ; Image Processing, Computer-Assisted ; Microscopy, Confocal ; Mitochondria - metabolism ; Models, Chemical ; Myocardium - cytology ; NAD - metabolism ; Oxidoreductases - chemistry ; Perfusion ; Rabbits ; Spectrometry, Fluorescence ; Temperature ; Time Factors</subject><ispartof>Biophysical journal, 2001-04, Vol.80 (4), p.2018-2028</ispartof><rights>2001 The Biophysical Society</rights><rights>Copyright Biophysical Society Apr 2001</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c490t-f3b8e80616c472519cf6122672f896a8319003eef55d243d2d4fc7458ac18f693</citedby><cites>FETCH-LOGICAL-c490t-f3b8e80616c472519cf6122672f896a8319003eef55d243d2d4fc7458ac18f693</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1301391/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://dx.doi.org/10.1016/S0006-3495(01)76172-3$$EHTML$$P50$$Gelsevier$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,3550,27924,27925,45995,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11259315$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Combs, C.A.</creatorcontrib><creatorcontrib>Balaban, R.S.</creatorcontrib><title>Direct Imaging of Dehydrogenase Activity within Living Cells Using Enzyme-Dependent Fluorescence Recovery after Photobleaching (ED-FRAP)</title><title>Biophysical journal</title><addtitle>Biophys J</addtitle><description>Reduced nicotine adenine dinucleotide (NADH) is a key metabolite involved in cellular energy conversion and many redox reactions. We describe the use of confocal microscopy in conjunction with enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH as a topological assay of NADH generation capacity within living cardiac myocytes. Quantitative validation of this approach was performed using a dehydrogenase system, in vitro. In intact cells the NADH ED-FRAP was sensitive to temperature (Q
10 of 2.5) and to dehydrogenase activation by dichloroacetate or cAMP (twofold increase for each). In addition, NADH ED-FRAP was correlated with flavin adenine dinucleotide (FAD
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These results suggest that ED-FRAP enables measurement of regional dynamics of mitochondrial NADH production in intact cells, thus providing information regarding region-specific intracellular redox reactions and energy metabolism.</description><subject>Animals</subject><subject>Cellular biology</subject><subject>Cyclic AMP - metabolism</subject><subject>Dichloroacetic Acid - metabolism</subject><subject>Enzymes</subject><subject>Flavin-Adenine Dinucleotide - metabolism</subject><subject>Fluorescence</subject><subject>Glutamate Dehydrogenase - metabolism</subject><subject>Image Processing, Computer-Assisted</subject><subject>Microscopy, Confocal</subject><subject>Mitochondria - metabolism</subject><subject>Models, Chemical</subject><subject>Myocardium - cytology</subject><subject>NAD - metabolism</subject><subject>Oxidoreductases - chemistry</subject><subject>Perfusion</subject><subject>Rabbits</subject><subject>Spectrometry, Fluorescence</subject><subject>Temperature</subject><subject>Time Factors</subject><issn>0006-3495</issn><issn>1542-0086</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>8G5</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNqFkd1u0zAUgCMEYmXwCCCLC7RdZPjEsZPcgKr-wKRKTINdW65z0nhK42InReEJeGycthqDG658LH_nz18UvQZ6BRTE-6-UUhGztOAXFC4zAVkSsyfRBHiaxJTm4mk0eUDOohfe31MKCafwPDqDEBQM-CT6NTcOdUeut2pj2g2xFZljPZTObrBVHslUd2ZvuoH8MF1tWrIKt8DNsGk8ufNjvGh_DluM57jDtsS2I8umtw69xlYjuUVt9-gGoqoOHbmpbWfXDSpdj7kXi3m8vJ3eXL6MnlWq8fjqdJ5Hd8vFt9nnePXl0_Vsuop1WtAurtg6x5wKEDrNEg6FrgQkiciSKi-EyhkUlDLEivMySVmZlGmls5TnSkNeiYKdRx-OdXf9eotlmLFzqpE7Z7bKDdIqI_9-aU0tN3YvgVFgBYQC704FnP3eo-_k1oRVm0a1aHsvM1HkWcbzAL79B7y3vWvDcjIBnkHBWRYgfoS0s947rB4mASpH0fIgWo4WJQV5EC1ZyHvzeI0_WSezAfh4BDB85t6gk16bUUh5EC5La_7T4jfYuLjz</recordid><startdate>20010401</startdate><enddate>20010401</enddate><creator>Combs, C.A.</creator><creator>Balaban, R.S.</creator><general>Elsevier Inc</general><general>Biophysical Society</general><scope>6I.</scope><scope>AAFTH</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7QO</scope><scope>7QP</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AF</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8G5</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M2O</scope><scope>M2P</scope><scope>M7P</scope><scope>MBDVC</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>Q9U</scope><scope>S0X</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20010401</creationdate><title>Direct Imaging of Dehydrogenase Activity within Living Cells Using Enzyme-Dependent Fluorescence Recovery after Photobleaching (ED-FRAP)</title><author>Combs, C.A. ; Balaban, R.S.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c490t-f3b8e80616c472519cf6122672f896a8319003eef55d243d2d4fc7458ac18f693</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Animals</topic><topic>Cellular biology</topic><topic>Cyclic AMP - metabolism</topic><topic>Dichloroacetic Acid - metabolism</topic><topic>Enzymes</topic><topic>Flavin-Adenine Dinucleotide - metabolism</topic><topic>Fluorescence</topic><topic>Glutamate Dehydrogenase - metabolism</topic><topic>Image Processing, Computer-Assisted</topic><topic>Microscopy, Confocal</topic><topic>Mitochondria - metabolism</topic><topic>Models, Chemical</topic><topic>Myocardium - cytology</topic><topic>NAD - metabolism</topic><topic>Oxidoreductases - chemistry</topic><topic>Perfusion</topic><topic>Rabbits</topic><topic>Spectrometry, Fluorescence</topic><topic>Temperature</topic><topic>Time Factors</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Combs, C.A.</creatorcontrib><creatorcontrib>Balaban, R.S.</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Biotechnology Research Abstracts</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Agricultural Science Collection</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>STEM Database</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Research Library (Alumni Edition)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>Advanced Technologies & Aerospace Collection</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>Research Library Prep</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Agricultural Science Database</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Research Library</collection><collection>Science Database</collection><collection>Biological Science Database</collection><collection>Research Library (Corporate)</collection><collection>Advanced Technologies & Aerospace Database</collection><collection>ProQuest Advanced Technologies & Aerospace Collection</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>ProQuest Central Basic</collection><collection>SIRS Editorial</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Biophysical journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Combs, C.A.</au><au>Balaban, R.S.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Direct Imaging of Dehydrogenase Activity within Living Cells Using Enzyme-Dependent Fluorescence Recovery after Photobleaching (ED-FRAP)</atitle><jtitle>Biophysical journal</jtitle><addtitle>Biophys J</addtitle><date>2001-04-01</date><risdate>2001</risdate><volume>80</volume><issue>4</issue><spage>2018</spage><epage>2028</epage><pages>2018-2028</pages><issn>0006-3495</issn><eissn>1542-0086</eissn><abstract>Reduced nicotine adenine dinucleotide (NADH) is a key metabolite involved in cellular energy conversion and many redox reactions. We describe the use of confocal microscopy in conjunction with enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH as a topological assay of NADH generation capacity within living cardiac myocytes. Quantitative validation of this approach was performed using a dehydrogenase system, in vitro. In intact cells the NADH ED-FRAP was sensitive to temperature (Q
10 of 2.5) and to dehydrogenase activation by dichloroacetate or cAMP (twofold increase for each). In addition, NADH ED-FRAP was correlated with flavin adenine dinucleotide (FAD
+) fluorescence. These data, coupled with the cellular patterns of NADH ED-FRAP changes with dehydrogenase stimulation, suggest that NADH ED-FRAP is localized to the mitochondria. These results suggest that ED-FRAP enables measurement of regional dynamics of mitochondrial NADH production in intact cells, thus providing information regarding region-specific intracellular redox reactions and energy metabolism.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>11259315</pmid><doi>10.1016/S0006-3495(01)76172-3</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Animals Cellular biology Cyclic AMP - metabolism Dichloroacetic Acid - metabolism Enzymes Flavin-Adenine Dinucleotide - metabolism Fluorescence Glutamate Dehydrogenase - metabolism Image Processing, Computer-Assisted Microscopy, Confocal Mitochondria - metabolism Models, Chemical Myocardium - cytology NAD - metabolism Oxidoreductases - chemistry Perfusion Rabbits Spectrometry, Fluorescence Temperature Time Factors |
| title | Direct Imaging of Dehydrogenase Activity within Living Cells Using Enzyme-Dependent Fluorescence Recovery after Photobleaching (ED-FRAP) |
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