Aconitase and ATP synthase are targets of malondialdehyde modification and undergo an age-related decrease in activity in mouse heart mitochondria

Abstract

The main purpose of this study was to identify mitochondrial proteins that exhibit post-translational oxidative modifications during the aging process and to determine the resulting functional alterations. Proteins forming adducts with malondialdehyde (MDA), a product of lipid peroxidation, were identified by immunodetection in mitochondria isolated from heart and hind leg skeletal muscle of 6-, 16-, and 24-month-old mice. Aconitase, very long chain acyl coenzyme A dehydrogenase, ATP synthase, and alpha-ketoglutarate dehydrogenase were detected as putative targets of oxidative modification by MDA. Aconitase and ATP synthase from heart exhibited significant decreases in activity with age. Very long chain acyl coenzyme A dehydrogenase and alpha-ketoglutarate dehydrogenase activities were unaffected during aging in both heart and skeletal muscle. This suggests that the presence of a post-translational oxidative modification in a protein does not a priori reflect an alteration in activity. The biological consequences of an age-related decrease in aconitase and ATP synthase activities may contribute to the decline in mitochondrial bioenergetics evident during aging.

Fig. 1

Immunodetection and identification of MDA-modified proteins in heart and skeletal muscle mitochondria from mice of different ages. (A) Heart mitochondrial proteins from 6-, 16-, and 24-month-old mice were separated by SDS–PAGE, transferred to PVDF, and probed with antibodies against MDA. Left panel, proteins stained with Coomassie blue. Right panel, blot probed with anti-MDA antibodies. (B) Identification of proteins putatively modified by MDA. NCBI refers to the accession number; MW, molecular weight.

Fig. 2

Quantitation of bands immunodetected with antibodies to MDA. Top panel shows representative blots for skeletal muscle (left) and heart (right) mitochondrial proteins. The bottom panel shows the integrated optical density of the MDA-modified bands for 6- (black), 16- (white), and 24- (gray)-month-old mice. Data represent averages ± standard deviations of four independent mitochondrial preparations, each consisting of tissue pooled from three mice.

Fig. 3

Aconitase and ATP synthase activities in heart and skeletal muscle mitochondria from mice of different ages. Maximal activities of aconitase (A) and ATP synthase (B) in 6-, 16-, and 24-month-old mice were determined by at least triplicate assays of four independent preparations of heart and skeletal muscle mitochondria, as described in Materials and methods. Statistically significant alterations are indicated by an asterisk, **p < 0.001, *p < 0.05 for old vs. young, based on a t test: two-sample, assuming equal variance.

Fig. 4

α-Ketoglutarate dehydrogenase and very long chain acyl coenzyme A dehydrogenase activities in heart and skeletal muscle mitochondria from mice of different ages. Maximal activities for α-ketoglutarate dehydrogenase (A) and very long chain acyl coenzyme A dehydrogenase (B) in 6-, 16-, and 24-month-old mice were determined by at least triplicate assays of four independent preparations of heart and skeletal muscle mitochondria, as described in Materials and methods.