scholarly journals Development of a New Assay for Complex I of the Respiratory Chain

2000 ◽  
Vol 46 (3) ◽  
pp. 345-350 ◽  
Author(s):  
Hilary Brooks ◽  
Stephan Krähenbühl
Keyword(s):  
Skeletal Muscle ◽  
Complex I ◽  
Human Skeletal Muscle ◽  
Specific Activity ◽  
Mitochondrial Diseases ◽  
Spectrophotometric Analysis ◽  
Isolated Mitochondria ◽  
Tissue Homogenates ◽  
Multiple Samples ◽  
Complex I Activity

Abstract Background: Measurement of complex I activity has been hampered by the large amounts of tissue required and the resulting turbidity of the assay solution, which makes spectrophotometric analysis difficult. We have developed a new assay for measuring the activity of complex I in isolated mitochondria that is also applicable to skeletal muscle homogenate in patients with suspected mitochondrial diseases. Methods: The method was a radioenzymatic assay based on the preferential oxidation of the 4B hydrogen of NADH by complex I. We prepared tritiated isoforms of NADH for both the respective 4A-3H and 4B-3H positions. Enzyme in the form of purified mitochondria or homogenate was prepared from rat or human skeletal muscle and incubated with the respective radioisotopes. The product (3H2O) was collected after charcoal adsorption of unreacted NADH and taken as an indicator of NADH oxidation. Sensitivity to rotenone was used as a measure of complex I specific activity. Results: The assay was linear with time and protein for isolated mitochondria and tissue homogenates from rats and humans. The Vmax and Km values obtained for 4B-NADH with isolated rat skeletal muscle mitochondria were 35 μmol/L and 90 μmol · min−1 · mg protein−1, respectively. The assay was reproducible and useable for routine measurements in human skeletal muscle. The sensitivity was >10-fold higher than the sensitivities of spectrophotometric techniques. Conclusions: The results of our studies demonstrate the successful development of a new assay for complex I that is rapid, easy to perform, and that enables the processing of multiple samples at one time.

scholarly journals Maturation of Mitochondrial and other Isoenzymes of Creatine Kinase in Skeletal Muscle of Preterm Born Infants

1992 ◽  
Vol 29 (3) ◽  
pp. 302-306 ◽  
Author(s):  
J Smeitink ◽  
W Ruitenbeek ◽  
T v Lith ◽  
R Sengers ◽  
F Trijbels ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Creatine Kinase ◽  
Protein Content ◽  
Human Skeletal Muscle ◽  
Specific Activity ◽  
Prenatal Development ◽  
Quadriceps Muscle ◽  
Mitochondrial Creatine Kinase ◽  
Total Creatine ◽  
Preterm Born

We studied pre- and postnatal changes in total creatine kinase (CK) activity, mitochondrial creatine kinase (Mi-CK) activity and immunochemical reactivity with anti-Mi-CK antibodies in skeletal muscle specimens from 12 infants, 10 of them preterm born, after a pregnancy varying between 28 and 40 weeks. Our results demonstrate that Mi-CK is present in fetal human quadriceps muscle and that the specific activity of Mi-CK increases during prenatal development from week 28 to 40 by a factor about two. Generally, adult levels have not been reached at birth, indicating a further postnatal increase of the activity of the enzyme. The Mi-CK protein content also increases during prenatal development. These results suggest that in human skeletal muscle the expression and accumulation of Mi-CK starts at mid-gestation, later than is known to occur for cytosolic CK.

scholarly journals Lower oxygen consumption and Complex I activity in mitochondria isolated from skeletal muscle of fetal sheep with intrauterine growth restriction

2020 ◽  
Vol 319 (1) ◽  
pp. E67-E80
Author(s):  
Alexander L. Pendleton ◽  
Andrew T. Antolic ◽  
Amy C. Kelly ◽  
Melissa A. Davis ◽  
Leticia E. Camacho ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Oxygen Consumption ◽  
Intrauterine Growth Restriction ◽  
Complex I ◽  
Tca Cycle ◽  
Growth Restriction ◽  
Fetal Sheep ◽  
Intrauterine Growth ◽  
Tca Cycle Enzymes ◽  
Complex I Activity

Fetal sheep with placental insufficiency-induced intrauterine growth restriction (IUGR) have lower hindlimb oxygen consumption rates (OCRs), indicating depressed mitochondrial oxidative phosphorylation capacity in their skeletal muscle. We hypothesized that OCRs are lower in skeletal muscle mitochondria from IUGR fetuses, due to reduced electron transport chain (ETC) activity and lower abundances of tricarboxylic acid (TCA) cycle enzymes. IUGR sheep fetuses ( n = 12) were created with mid-gestation maternal hyperthermia and compared with control fetuses ( n = 12). At 132 ± 1 days of gestation, biceps femoris muscles were collected, and the mitochondria were isolated. Mitochondria from IUGR muscle have 47% lower State 3 (Complex I-dependent) OCRs than controls, whereas State 4 (proton leak) OCRs were not different between groups. Furthermore, Complex I, but not Complex II or IV, enzymatic activity was lower in IUGR fetuses compared with controls. Proteomic analysis ( n = 6/group) identified 160 differentially expressed proteins between groups, with 107 upregulated and 53 downregulated mitochondria proteins in IUGR fetuses compared with controls. Although no differences were identified in ETC subunit protein abundances, abundances of key TCA cycle enzymes [isocitrate dehydrogenase (NAD+) 3 noncatalytic subunit β (IDH3B), succinate-CoA ligase ADP-forming subunit-β (SUCLA2), and oxoglutarate dehydrogenase (OGDH)] were lower in IUGR mitochondria. IUGR mitochondria had a greater abundance of a hypoxia-inducible protein, NADH dehydrogenase 1α subcomplex 4-like 2, which is known to incorporate into Complex I and lower Complex I-mediated NADH oxidation. Our findings show that mitochondria from IUGR skeletal muscle adapt to hypoxemia and hypoglycemia by lowering Complex I activity and TCA cycle enzyme concentrations, which together, act to lower OCR and NADH production/oxidation in IUGR skeletal muscle.

scholarly journals Nitric oxide increases cyclic GMP levels, AMP-activated protein kinase (AMPK)α1-specific activity and glucose transport in human skeletal muscle

Diabetologia ◽  
2010 ◽  
Vol 53 (6) ◽  
pp. 1142-1150 ◽  
Author(s):  
A. S. Deshmukh ◽  
Y. C. Long ◽  
T. de Castro Barbosa ◽  
H. K. R. Karlsson ◽  
S. Glund ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Skeletal Muscle ◽  
Protein Kinase ◽  
Glucose Transport ◽  
Cyclic Gmp ◽  
Human Skeletal Muscle ◽  
Specific Activity ◽  
Amp Activated Protein Kinase

scholarly journals Differential responses of targeted lung redox enzymes to rat exposure to 60 or 85% oxygen

2011 ◽  
Vol 111 (1) ◽  
pp. 95-107 ◽  
Author(s):  
Zhuohui Gan ◽  
David L. Roerig ◽  
Anne V. Clough ◽  
Said H. Audi
Keyword(s):  
Lung Tissue ◽  
Complex I ◽  
Complex Iii ◽  
Oxygen Species ◽  
Mitochondrial Complex ◽  
Complex Iv ◽  
Arterial Inflow ◽  
Tissue Homogenates ◽  
Species Production ◽  
Complex I Activity

Rat exposure to 60% O2 (hyper-60) or 85% O2 (hyper-85) for 7 days confers susceptibility or tolerance, respectively, of the otherwise lethal effects of exposure to 100% O2. The objective of this study was to determine whether activities of the antioxidant cytosolic enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) and mitochondrial complex III are differentially altered in hyper-60 and hyper-85 lungs. Duroquinone (DQ), an NQO1 substrate, or its hydroquinone (DQH2), a complex III substrate, was infused into the arterial inflow of isolated, perfused lungs, and the venous efflux rates of DQH2 and DQ were measured. Based on inhibitor effects and kinetic modeling, capacities of NQO1-mediated DQ reduction ( Vmax1) and complex III-mediated DQH2 oxidation ( Vmax2) increased by ∼140 and ∼180% in hyper-85 lungs, respectively, compared with rates in lungs of rats exposed to room air (normoxic). In hyper-60 lungs, Vmax1 increased by ∼80%, with no effect on Vmax2. Additional studies revealed that mitochondrial complex I activity in hyper-60 and hyper-85 lung tissue homogenates was ∼50% lower than in normoxic lung homogenates, whereas mitochondrial complex IV activity was ∼90% higher in only hyper-85 lung tissue homogenates. Thus NQO1 activity increased in both hyper-60 and hyper-85 lungs, whereas complex III activity increased in hyper-85 lungs only. This increase, along with the increase in complex IV activity, may counter the effects the depression in complex I activity might have on tissue mitochondrial function and/or reactive oxygen species production and may be important to the tolerance of 100% O2 observed in hyper-85 rats.

Calcium content and respiratory control index of skeletal muscle mitochondria during exercise and recovery

1996 ◽  
Vol 271 (6) ◽  
pp. E1044-E1050 ◽  
Author(s):  
K. Madsen ◽  
P. Ertbjerg ◽  
M. S. Djurhuus ◽  
P. K. Pedersen
Keyword(s):  
Skeletal Muscle ◽  
Human Skeletal Muscle ◽  
Respiratory Control ◽  
Calcium Content ◽  
Exhaustive Exercise ◽  
Isolated Mitochondria ◽  
Skeletal Muscle Mitochondria ◽  
Before And After ◽  
The Relationship ◽  
Muscle Biopsies

The purpose of this study was to evaluate the relationship between mitochondrial Ca2+ concentration and the respiratory control index (RCI; state III/state IV) in isolated mitochondria before and after exhaustive exercise at 75% of maximal O2 consumption. Muscle biopsies of 100-150 mg from 12 moderately trained men were sampled at rest, immediately after exercise, and 30 or 60 min after exercise. The mitochondrial Ca2+ content after exhaustive exercise was significantly higher than the preexercise level [15.1 (range 39.4) vs. 11.6 (range 6.5) nmol/mg protein, respectively; P < 0.05], and RCI increased from 11.6 (range 14.4) at rest to 13.7 (range 15.0) at exhaustion (P < 0.05). After 60 min of recovery, the mitochondrial Ca2+ content was still high [18.8 (range 29.9) nmol/mg protein], but the RCI value was significantly depressed because of the increased state IV value and, in fact, was lower than the preexercise value [8.6 (range 5.1); P < 0.05]. Our results show that the mitochondrial Ca2+ content is increased in human skeletal muscle after prolonged exhaustive exercise and that this is followed by an elevated RCI value, with slightly increased state III and decreased state IV respiration. The restoration of the elevated mitochondrial Ca2+ level is slow and could be related to an increased state IV respiration, which together indicate uncoupled Ca2+ respiration during recovery.

scholarly journals Reoxygenation-dependent decrease in mitochondrial NADH:CoQ reductase (Complex I) activity in the hypoxic/reoxygenated rat heart

1991 ◽  
Vol 274 (1) ◽  
pp. 133-137 ◽  
Author(s):  
L Hardy ◽  
J B Clark ◽  
V M Darley-Usmar ◽  
D R Smith ◽  
D Stone
Keyword(s):  
Respiratory Function ◽  
Complex I ◽  
Ruthenium Red ◽  
Isolated Cardiomyocytes ◽  
Isolated Mitochondria ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Complex I Activity ◽  
Perfused Hearts ◽  
Total Tissue

Reoxygenation of the hypoxic myocardium results in a number of processes, including an O2-dependent increase in total tissue Ca2+ and cell lysis in which mitochondrial electron transport plays a key role. In the present study we have isolated mitochondria from perfused rat hearts subjected to hypoxia and found no change in their respiratory function relative to controls. In contrast, mitochondria isolated immediately after reoxygenation of hypoxic-perfused hearts exhibited a specific and significant decrease in NADH:CoQ reductase (Complex I; EC 1.6.5.3) activity, as measured both polarographically and spectrophotometrically. Isolated cardiomyocytes subjected to a similar protocol of hypoxia/reoxygenation also exhibited a specific decrease in Complex I activity. Myocardial perfusion with media containing Ruthenium Red protected against the reoxygenation-dependent loss of Complex I activity. These observations taken together suggest that mitochondrial Ca2+ uptake on reoxygenation is implicated in the mechanism of the specific loss of Complex I activity.

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