Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

Podsumowanie

This protocol describes a comparative assay, using mitochondrial complex activities CI+CIII and CII+CIII in the presence or absence of Na⁺, to study the existence of partially segmented functional CoQ pools.

Streszczenie

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision can be easily assessed by a comparative assay using NADH or succinate as electron donors in frozen-thawed mitochondria, in which cytochrome c (cyt c) reduction is measured. The assay relies on the effect of Na⁺ on the IMM, decreasing its fluidity. Here, we present a protocol to measure NADH-cyt c oxidoreductase activity and succinate-cyt c oxidoreductase activities in the presence of NaCl or KCl. The reactions, which rely on the mixture of reagents in a cuvette in a stepwise manner, are measured spectrophotometrically during 4 min in the presence of Na⁺ or K⁺. The same mixture is performed in parallel in the presence of the specific enzyme inhibitors in order to subtract the unspecific change in absorbance. NADH-cyt c oxidoreductase activity does not decrease in the presence of any of these cations. However, succinate-cyt c oxidoreductase activity decreases in the presence of NaCl. This simple experiment highlights: 1) the effect of Na⁺ in decreasing IMM fluidity and CoQ transfer; 2) that supercomplex I+III₂ protects ubiquinone (CoQ) transfer from being affected by lowering IMM fluidity; 3) that CoQ transfer between CI and CIII is functionally different from CoQ transfer between CII and CIII. These facts support the existence of functionally differentiated CoQ pools in the IMM and show that they can be regulated by the changing Na⁺ environment of mitochondria.

Wprowadzenie

Mitochondrial oxidative phosphorylation system (OXPHOS) is the main pathway driving adenosine triphosphate (ATP) synthesis, reactive oxygen species (ROS) production, and consumption of reducing equivalents, such as nicotinamide adenine dinucleotide (NADH) or succinate, by mitochondria. OXPHOS system is composed of five protein complexes: Complex I (CI) oxidizes NADH and reduces CoQ into ubiquinol (CoQH₂). Complex II (CII) oxidizes succinate into fumarate and reduces CoQ into CoQH₂. Complex III (CIII) oxidizes CoQH₂ back into CoQ, reducing cytochrome c (cyt c). Finally, complex IV (CIV) oxidizes cyt c and reduces oxygen to water. This o....

Protokół

All animal experiments were performed following the Guide for the Care and Use of Laboratory Animals and were approved by the institutional ethics committee of the Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Spain, in accordance with the European Union Directive of 22 September 2010 (2010/63/UE) and with the Spanish Royal Decree of 1 February 2013 (53/2013). All efforts were made to minimize the number of animals used and their suffering.

NOTE: This comparative assay to study the segmentation of mitochondrial CoQ pools is described as follows:

1. Protein quantification

Wyniki

Typical results from this protocol are represented below (Figure 3). As reduced cyt c absorbance locates at 550 nm, all uninhibited subsamples must show an increase in the absorbance at 550 nm. Inhibited subsamples ideally show a flat-line or slightly increasing slope (Figure 3). Slopes from inhibited subsamples are to be subtracted from uninhibited subsamples.

Samples A and B, both corrected by their correspondent inhibition and whic.......

Dyskusje

Though this protocol represents a very straightforward procedure to identify the existence of the partially segmented CoQ pools, there are a few critical steps to take into account. Substrates (i.e., NADH or succinate) are preferably added last since autooxidation of these compounds may occur. Cuvette's flipping must be careful in order to avoid the formation of bubbles which may interfere with the reading.

In addition, the present technique presents a few limitations which are worth menti.......

Materiały

Name	Company	Catalog Number	Comments
Antimycin A	Sigma-Aldrich	A8674
Bovine Serum Albumin (BSA)	Sigma-Aldrich	10775835001
Bradford protein assay	Bio-Rad	5000001
Cytochrome c from equine heart	Sigma-Aldrich	C7752
K2HPO4	Sigma-Aldrich	P3786
KCl	Sigma-Aldrich	P3911
Malonic acid	Sigma-Aldrich	M1296
MgCl2	Sigma-Aldrich	M8266
NaCl	Sigma-Aldrich	S9888
NADH	Roche	10107735001
Potassium cyanide	Sigma-Aldrich	207810
Rotenone	Sigma-Aldrich	R8875
Spectra Manager software	JASCO	version 2
Spectrophotometer	UV/VISJASCO
Succinate	Sigma-Aldrich	398055