Journal: International Journal of Molecular Sciences

Article Title: Proline Oxidation Supports Mitochondrial ATP Production When Complex I Is Inhibited

doi: 10.3390/ijms23095111

Figure Lengend Snippet: Effect of proline concentration on Δ Ψ mt and NADH autofluorescence of isolated mouse liver mitochondria subjected to targeted inhibition of electron transfer (ET) pathways. No substrates were present prior to the addition of mitochondria. Glutamate (G), malate (M), and proline (Pro) were added at concentrations indicated. ( A – D ) Time course of safranine O signal indicative of Δ Ψ mt (arbitrary units, A.U.). SF6847 (SF, 1 µM). ( A ) No targeted inhibition of electron transfer pathways. ( B ) 1 µM rotenone (Rot) present in the buffer prior to addition of mitochondria; succinate (S, 5 mM). ( C ) As in B, but 1 µM atpenin A5 replaced Rot. ( D ) As in B, but myxothiazol (Myx) replaced Rot. ( E – H ) Concentrations of Pro: black (a): 0 mM, red (b): 0.25 mM, green (c): 0.5 mM, orange (d): 2 mM, blue (e): 5 mM. ( E ) NADH autofluorescence (arbitrary units A.U.) measured in the Hitachi F-7000 fluorescence spectrophotometer. ( F – H ) Oxygen consumption, NADH autofluorescence, and rhodamine 123 fluorescence indicative of Δ Ψ mt (arbitrary units A.U.), respectively, recorded simultaneously from the same liver mitochondria using the NextGen-O2k and aligned on the dashed grey lines.

Article Snippet: Fluorescence was recorded using a Hitachi F-7000 spectrofluorimeter (Hitachi High Technologies, Maidenhead, UK) at a 5-Hz acquisition rate, at 495 nm and 585 nm excitation and emission wavelengths, respectively, or the Oroboros O2k (Oroboros Instruments, Innsbruck, Austria) equipped with the O2k-Fluo LED2-Module, or the NextGen-O2k prototype equipped with the O2k-Fluo Smart Module, with optical sensors including an LED (465 nm; <505 nm short-pass excitation filter), a photodiode, and specific optical filters (>560 nm long-pass emission filter) [ ].

Techniques: Concentration Assay, Isolation, Inhibition, Fluorescence, Spectrophotometry

Journal: International Journal of Molecular Sciences

Article Title: Proline Oxidation Supports Mitochondrial ATP Production When Complex I Is Inhibited

doi: 10.3390/ijms23095111

Figure Lengend Snippet: Effect of proline concentration on Δ Ψ mt and NADH autofluorescence of isolated mouse liver mitochondria subjected to targeted inhibition of electron transfer (ET) pathways. No substrates were present prior to the addition of mitochondria. Glutamate (G), malate (M), and proline (Pro) were added at concentrations indicated. ( A – D ) Time course of safranine O signal indicative of Δ Ψ mt (arbitrary units, A.U.). SF6847 (SF, 1 µM). ( A ) No targeted inhibition of electron transfer pathways. ( B ) 1 µM rotenone (Rot) present in the buffer prior to addition of mitochondria; succinate (S, 5 mM). ( C ) As in B, but 1 µM atpenin A5 replaced Rot. ( D ) As in B, but myxothiazol (Myx) replaced Rot. ( E – H ) Concentrations of Pro: black (a): 0 mM, red (b): 0.25 mM, green (c): 0.5 mM, orange (d): 2 mM, blue (e): 5 mM. ( E ) NADH autofluorescence (arbitrary units A.U.) measured in the Hitachi F-7000 fluorescence spectrophotometer. ( F – H ) Oxygen consumption, NADH autofluorescence, and rhodamine 123 fluorescence indicative of Δ Ψ mt (arbitrary units A.U.), respectively, recorded simultaneously from the same liver mitochondria using the NextGen-O2k and aligned on the dashed grey lines.

Article Snippet: Fluorescence was recorded using a Hitachi F-7000 spectrofluorimeter (Hitachi High Technologies, Maidenhead, UK) at a 5-Hz acquisition rate, at 495 nm and 585 nm excitation and emission wavelengths, respectively, or the Oroboros O2k (Oroboros Instruments, Innsbruck, Austria) equipped with the O2k-Fluo LED2-Module, or the NextGen-O2k prototype equipped with the O2k-Fluo Smart Module, with optical sensors including an LED (465 nm; <505 nm short-pass excitation filter), a photodiode, and specific optical filters (>560 nm long-pass emission filter) [ ].

Techniques: Concentration Assay, Isolation, Inhibition, Fluorescence, Spectrophotometry

Journal: International Journal of Molecular Sciences

Article Title: Proline Oxidation Supports Mitochondrial ATP Production When Complex I Is Inhibited

doi: 10.3390/ijms23095111

Figure Lengend Snippet: Coenzyme Q (Q) redox state recorded simultaneously with oxygen consumption rate and rhodamine 123 fluorescence in isolated mouse liver mitochondria using the NextGen-O2k. ( A ) Oxygen consumption. ( B ) Q redox state. ( C ) Rhodamine 123 fluorescence indicative of Δ Ψ mt (arbitrary units A.U.). No substrates were present. Succinate (S, 5 mM) and proline (Pro) were added at the concentrations indicated. 0.25 µM SF ( D – F ) as in A, B, and C, but rotenone was present in the medium. ( G – I ) As in A, B, and C, but 0.1 µM myxothiazol was present in the medium.

Article Snippet: Fluorescence was recorded using a Hitachi F-7000 spectrofluorimeter (Hitachi High Technologies, Maidenhead, UK) at a 5-Hz acquisition rate, at 495 nm and 585 nm excitation and emission wavelengths, respectively, or the Oroboros O2k (Oroboros Instruments, Innsbruck, Austria) equipped with the O2k-Fluo LED2-Module, or the NextGen-O2k prototype equipped with the O2k-Fluo Smart Module, with optical sensors including an LED (465 nm; <505 nm short-pass excitation filter), a photodiode, and specific optical filters (>560 nm long-pass emission filter) [ ].

Techniques: Fluorescence, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Proline Oxidation Supports Mitochondrial ATP Production When Complex I Is Inhibited

doi: 10.3390/ijms23095111

Figure Lengend Snippet: The effects of THFA and S-5-oxo on ProDH activity ( A ), on proline—mediated respiration rates in isolated mouse liver and kidney mitochondria (* p <0.05 ANOVA on Ranks (Liver) or Bonferroni (Kidney; comparisons are to controls, i.e., no proline or no ProDH inhibitor added)); ( B – E ), and on oxygen consumption, Q redox state, and rhodamine 123 fluorescence, which were recorded simultaneously from liver mitochondria using the NextGen-O2k ( F – K ). (A) Scatter plot depicting ProDH catalytic activity (pkat/mg protein of isolated mitochondria) with 10 mM proline and the effects of THFA (10 mM) and S-5-oxo (5 mM). ( B ) Bar graph depicting LEAK (red) and OXPHOS (green) oxygen consumption rates in liver mitochondria respiring on 5 mM proline as a function of THFA concentration. ( C ) As in B but using S-5-oxo instead of THFA. ( D ) As in B but with kidney mitochondria. ( E ) As in C but with kidney mitochondria. Data are SEM of at least three independent experiments. ( F – K ) Proline (Pro, 5 mM), ( F – H ) THFA, ( I – K ) S-5-oxo. GMβOH always present: glutamate (G), malate (M), and βOH (10 mM). Black (a): GMβOH; red (b): plus proline (Pro, 5 mM); green (c): plus Pro and THFA (2 mM) or S-5-oxo (2 mM); orange (d): plus Pro and THFA (5 mM) or S-5-oxo (5 mM); blue (e): plus Pro and THFA (10 mM) or S-5-oxo (10 mM). For B–E, * p <0.05 ANOVA (Bonferroni or on ranks).

Article Snippet: Fluorescence was recorded using a Hitachi F-7000 spectrofluorimeter (Hitachi High Technologies, Maidenhead, UK) at a 5-Hz acquisition rate, at 495 nm and 585 nm excitation and emission wavelengths, respectively, or the Oroboros O2k (Oroboros Instruments, Innsbruck, Austria) equipped with the O2k-Fluo LED2-Module, or the NextGen-O2k prototype equipped with the O2k-Fluo Smart Module, with optical sensors including an LED (465 nm; <505 nm short-pass excitation filter), a photodiode, and specific optical filters (>560 nm long-pass emission filter) [ ].

Techniques: Activity Assay, Isolation, Fluorescence, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Proline Oxidation Supports Mitochondrial ATP Production When Complex I Is Inhibited

doi: 10.3390/ijms23095111

Figure Lengend Snippet: The effect of proline on CAT-induced changes in respiration ( A , D ), Q redox state ( B , E ), and rhodamine 123 fluorescence ( C , F ), which are indicative of Δ Ψ mt (arbitrary units A.U.) and were measured simultaneously in the same mouse liver mitochondria using the NextGen-O2k—aligned on the dashed grey lines. GM was always contained in the medium; 0.25 µM SF. ( A – C ) GM and increasing concentrations of Pro; black (a): 0 mM; red (b): 0.25 mM; green (c): 0.5 mM; orange (d): 2 mM; blue (e): 5 mM; magenta (f): 10 mM. ( D – F ) 5 mM βOH is additionally present in traces b–f and increasing concentrations of Pro; black (a): 0 mM; red (b): 0 mM; green (c): 0.25 mM; orange (d): 0.5 mM; blue (e): 2 mM; magenta (f): 5 mM.

Article Snippet: Fluorescence was recorded using a Hitachi F-7000 spectrofluorimeter (Hitachi High Technologies, Maidenhead, UK) at a 5-Hz acquisition rate, at 495 nm and 585 nm excitation and emission wavelengths, respectively, or the Oroboros O2k (Oroboros Instruments, Innsbruck, Austria) equipped with the O2k-Fluo LED2-Module, or the NextGen-O2k prototype equipped with the O2k-Fluo Smart Module, with optical sensors including an LED (465 nm; <505 nm short-pass excitation filter), a photodiode, and specific optical filters (>560 nm long-pass emission filter) [ ].

Techniques: Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Proline Oxidation Supports Mitochondrial ATP Production When Complex I Is Inhibited

doi: 10.3390/ijms23095111

Figure Lengend Snippet: No effect of proline on Q redox state of mitochondria inhibited at CIII (myxothiazol, 0.1 µM) or under anoxia. Oxygen consumption ( A ) or concentration ( D ), Q redox state ( B , E ), and rhodamine 123 fluorescence ( C , F ) are indicative of Δ Ψ mt (arbitrary units A.U.) and were measured simultaneously in the same mouse liver mitochondria using the NextGen-O2k—aligned on the dashed grey lines. Black traces: GM (and 10 mM βOH in D – F ); red trace: GM plus 5 mM proline where indicated (& 10 mM βOH in D – F ). 0.25 µM SF.

Article Snippet: Fluorescence was recorded using a Hitachi F-7000 spectrofluorimeter (Hitachi High Technologies, Maidenhead, UK) at a 5-Hz acquisition rate, at 495 nm and 585 nm excitation and emission wavelengths, respectively, or the Oroboros O2k (Oroboros Instruments, Innsbruck, Austria) equipped with the O2k-Fluo LED2-Module, or the NextGen-O2k prototype equipped with the O2k-Fluo Smart Module, with optical sensors including an LED (465 nm; <505 nm short-pass excitation filter), a photodiode, and specific optical filters (>560 nm long-pass emission filter) [ ].

Techniques: Concentration Assay, Fluorescence