Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels

Abstract

Blue native-polyacrylamide gel electrophoresis is a powerful technique that enables the separation of intact multi-subunit complexes. However, positive identification of particular enzymes generally requires further separation in a second dimension on a denaturing polyacrylamide gel. Histochemical staining is widely used to demonstrate enzyme activities in tissues, including oxidative phosphorylation enzymes. In this report, we demonstrate that the two techniques can be combined to quantify in situ mitochondrial enzymes, separated on nondenaturing polyacrylamide gels. The method gives quantitative results with human skeletal muscle as well as heart that contains higher mitochondrial numbers. Comparison of muscle from patients with oxidative phosphorylation enzyme deficiencies, such as those of two riboflavin-responsive patients, before and after vitamin treatment, gives results in agreement with those obtained by analyzing the activity of the mitochondrial enzymes in muscle homogenates.

Supramolecular organizations in the aerobic respiratory chain of Escherichia coli

“…The activities of NADH:NBT and succinate:NBT oxidoreductase were detected in gel [36] as well as b-type hemes [37]. Solubilized membranes (12.5 mg) were also applied on top of continuous sucrose gradients (0.3e1.5 M and 1e1.5 M) in a buffer containing 15 mM Tris/HCl pH 7, 20 mM KCl and 0.2% digitonin, resolved by ultra-centrifugation at 4 C (20 h, 150000 Â g) [38] and 1 mL fractions were collected, frozen in liquid nitrogen and stored at À80 C.…”

Biochemical and electron microscopic characterization of the F1F0 ATP Synthase from the hyperthermophilic eubacterium Aquifex aeolicus

“…This gel was sliced into individual lanes for histochemical staining. The enzyme activities were measured as described by Zarbetto [27] and Dabbeni-Sala et al [28] with minor modifications. The gel slices were incubated in 35 mM Tris-HCI (pH 7.8), 270 mM glycine, 1.4 mM MgSO 4 , 0.8 mM ATP, and 0.1 % Pb(NO 3 ) 2 at 32°C overnight.…”

Evaluation by blue native polyacrylamide electrophoresis colorimetric staining of the effects of physical exercise on the activities of mitochondrial complexes in rat muscle

“…The method of sample preparation for BN-PAGE was that originally described by Zerbetto et al (27), with the following modifications: muscle samples (30 mg) were minced and homogenized in 1 ml of a cooled solution containing 20 mM MOPS, 440 mM sucrose, 1 mM EDTA and 5 mM PMSF, pH 7.2, in a Kinematica Polytron Homogenizer (Westbury, NY, USA) with a generator diameter of 7 mm at the highest speed for 10 s. After centrifugation at 20,000 g for 20 min the supernatants were submitted to the enzyme assay (citrate synthase), as described by Srere (29). The pellet was re-suspended in 80 µl of 1 M 6-aminocaproic acid and 50 mM Bis-Tris, pH 7.0, containing 5 mM PMSF.…”

“…Complex I activity was analyzed by incubating the gel slices in a solution containing 2 mM Tris-HCl, pH 7.4, 0.1 mg/ ml NADH and 2.5 mg/ml NBT, diluted 1:10 (w:v) at room temperature (27). The gel was incubated in 50% methanol and 10% acetic acid for 15 min to fix the color of complex I reacting bands and then preserved in 10% acetic acid.…”

“…For this purpose we used the quantification of skeletal muscle OXPHOS enzymes by colorimetric enzymatic staining of BN-PAGE. This is a method recently developed for analyzing membrane complexes (26)(27)(28), which has the important advantage of using small quantities of muscle for analysis and which does not require the isolation of mitochondria. Moreover, it specifically measures the OXPHOS enzymes whereas most spectrophotometric assays also detect other cellular activities (27,28).…”