Difference between revisions of "Kunz 1994 Anal Biochem"
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{{Publication | {{Publication | ||
|title=Kunz WS, Kuznetsov AV, Winkler K, Gellerich FN, Neuhof S, Neumann HW (1994) Measurement of fluorescence changes of NAD(P)H and fluorescent flavoproteins in saponin-skinned fibers. | |title=Kunz WS, Kuznetsov AV, Winkler K, Gellerich FN, Neuhof S, Neumann HW (1994) Measurement of fluorescence changes of NAD(P)H and fluorescent flavoproteins in saponin-skinned fibers. Anal Biochem 216:322-7. | ||
|info=[http://www.ncbi.nlm.nih.gov/pubmed/8179187 PMID: 8179187] | |info=[http://www.ncbi.nlm.nih.gov/pubmed/8179187 PMID: 8179187] | ||
|authors=Kunz WS, Kuznetsov AV, Winkler K, Gellerich FN, Neuhof S, Neumann HW | |authors=Kunz WS, Kuznetsov AV, Winkler K, Gellerich FN, Neuhof S, Neumann HW | ||
|year=1994 | |year=1994 | ||
|journal= | |journal=Anal Biochem | ||
|abstract=Saponin-skinned human muscle fibers from M. vastus lateralis were immobilized in a quartz capillary to detect the fluorescence changes of NAD(P)H and of fluorescent flavoproteins. To get sufficient intense fluorescence signals from a small amount of muscle tissue the NAD(P)H fluorescence was excited by means of an HeCd laser at 325 nm and the flavoprotein fluorescence by an argon-ion laser at 454 nm or by the second wavelength of a HeCd laser at 442 nm. Using this experimental setup the fluorescence spectra of NAD(P)H, of ฮฑ-lipoamide dehydrogenase and of electron-transfer flavoprotein were detected in saponin-skinned human muscle fibers. These fibers behaved identically to isolated mitochondria: (i) The addition of substrates caused an increase in reduction of mitochondrial NAD+, (ii) the addition of ADP caused its reoxidation, and (iii) the addition of respiratory chain inhibitors led to an almost complete reduction of NAD+. It was observed that the redox state of the NAD(P) system and of the ฮฑ-lipoamide dehydrogenase reached after addition of 1 mM ADP correlates with the rate of active state respiration with NAD-dependent substrates. Therefore, this fluorimetric method is suitable to compare the mitochondrial oxidation capacities of NAD-dependent substrates in less then 5 mg wet weight muscle tissue. Moreover, the maximal changes in fluorescence of NAD(P)H and flavoproteins correlate with the amount of mitochondrial marker enzymes per milligram muscle tissue. Using this method a myopathy caused by a diminished content of mitochondria per milligram muscle tissue was observed. | |abstract=Saponin-skinned human muscle fibers from M. vastus lateralis were immobilized in a quartz capillary to detect the fluorescence changes of NAD(P)H and of fluorescent flavoproteins. To get sufficient intense fluorescence signals from a small amount of muscle tissue the NAD(P)H fluorescence was excited by means of an HeCd laser at 325 nm and the flavoprotein fluorescence by an argon-ion laser at 454 nm or by the second wavelength of a HeCd laser at 442 nm. Using this experimental setup the fluorescence spectra of NAD(P)H, of ฮฑ-lipoamide dehydrogenase and of electron-transfer flavoprotein were detected in saponin-skinned human muscle fibers. These fibers behaved identically to isolated mitochondria: (i) The addition of substrates caused an increase in reduction of mitochondrial NAD+, (ii) the addition of ADP caused its reoxidation, and (iii) the addition of respiratory chain inhibitors led to an almost complete reduction of NAD+. It was observed that the redox state of the NAD(P) system and of the ฮฑ-lipoamide dehydrogenase reached after addition of 1 mM ADP correlates with the rate of active state respiration with NAD-dependent substrates. Therefore, this fluorimetric method is suitable to compare the mitochondrial oxidation capacities of NAD-dependent substrates in less then 5 mg wet weight muscle tissue. Moreover, the maximal changes in fluorescence of NAD(P)H and flavoproteins correlate with the amount of mitochondrial marker enzymes per milligram muscle tissue. Using this method a myopathy caused by a diminished content of mitochondria per milligram muscle tissue was observed. | ||
|mipnetlab= | |mipnetlab=DE Magdeburg Gellerich FN, AT Innsbruck Oroboros | ||
|discipline=Biomedicine | |discipline=Biomedicine | ||
|articletype=Protocol; Manual | |articletype=Protocol; Manual | ||
}} | }} | ||
{{Labeling | {{Labeling | ||
| | |area=Respiration | ||
|organism=Human | |organism=Human | ||
|tissues=Skeletal muscle | |tissues=Skeletal muscle | ||
|preparations=Permeabilized tissue | |preparations=Permeabilized tissue | ||
|couplingstates=OXPHOS | |couplingstates=OXPHOS | ||
|instruments=Oxygraph-2k | |||
|additional=Spectrophotometry; Spectrofluorimetry | |additional=Spectrophotometry; Spectrofluorimetry | ||
|discipline=Biomedicine | |discipline=Biomedicine | ||
|articletype=Protocol; Manual | |articletype=Protocol; Manual | ||
}} | }} |
Latest revision as of 10:43, 23 January 2019
Kunz WS, Kuznetsov AV, Winkler K, Gellerich FN, Neuhof S, Neumann HW (1994) Measurement of fluorescence changes of NAD(P)H and fluorescent flavoproteins in saponin-skinned fibers. Anal Biochem 216:322-7. |
Kunz WS, Kuznetsov AV, Winkler K, Gellerich FN, Neuhof S, Neumann HW (1994) Anal Biochem
Abstract: Saponin-skinned human muscle fibers from M. vastus lateralis were immobilized in a quartz capillary to detect the fluorescence changes of NAD(P)H and of fluorescent flavoproteins. To get sufficient intense fluorescence signals from a small amount of muscle tissue the NAD(P)H fluorescence was excited by means of an HeCd laser at 325 nm and the flavoprotein fluorescence by an argon-ion laser at 454 nm or by the second wavelength of a HeCd laser at 442 nm. Using this experimental setup the fluorescence spectra of NAD(P)H, of ฮฑ-lipoamide dehydrogenase and of electron-transfer flavoprotein were detected in saponin-skinned human muscle fibers. These fibers behaved identically to isolated mitochondria: (i) The addition of substrates caused an increase in reduction of mitochondrial NAD+, (ii) the addition of ADP caused its reoxidation, and (iii) the addition of respiratory chain inhibitors led to an almost complete reduction of NAD+. It was observed that the redox state of the NAD(P) system and of the ฮฑ-lipoamide dehydrogenase reached after addition of 1 mM ADP correlates with the rate of active state respiration with NAD-dependent substrates. Therefore, this fluorimetric method is suitable to compare the mitochondrial oxidation capacities of NAD-dependent substrates in less then 5 mg wet weight muscle tissue. Moreover, the maximal changes in fluorescence of NAD(P)H and flavoproteins correlate with the amount of mitochondrial marker enzymes per milligram muscle tissue. Using this method a myopathy caused by a diminished content of mitochondria per milligram muscle tissue was observed.
โข O2k-Network Lab: DE Magdeburg Gellerich FN, AT Innsbruck Oroboros
Labels: MiParea: Respiration
Organism: Human
Tissue;cell: Skeletal muscle
Preparation: Permeabilized tissue
Coupling state: OXPHOS
HRR: Oxygraph-2k
Spectrophotometry; Spectrofluorimetry