Subsarcolemmal mitochondria isolated with the proteolytic enzyme nagarse exhibit greater protein specific activities and functional coupling

• Exposure of mitochondria to nagarse removes non-mitochondrial proteins
• Exposure of mitochondria to nagarse removes protein of damaged mitochondria
• Treating mitochondria with nagarse improves indices of functional integrity

Skeletal muscle mitochondria are arranged as a reticulum. Insight into the functional characteristics of such structure is achieved by viewing the network as consisting of “subsarcolemmal” (SS) and “intermyofibrillar” (IMF) regions. During the decades, most, but not all, published studies have reported higher (sometimes over 2-fold) enzyme and enzyme-pathway protein-specific activities in IMF compared to SS mitochondria. We tested the hypothesis that non-mitochondrial protein contamination might account for much of the apparently lower specific activities of isolated SS mitochondria. Mouse gastrocnemii (n=6) were suspended in isolation medium, minced, and homogenized according to procedures typically used to isolate SS mitochondria. However, the supernatant fraction, collected after the first slow-speed (800g) centrifugation, was divided equally: one sample was exposed to nagarse (MITO+), while the other was not (MITO−). Nagarse treatment reduced total protein yield by 25%, while it increased protein-specific respiration rates (nmol O2 min−1 mg−1), by 38% under “resting” (state 4) and by 84% under maximal (state 3) conditions. Nagarse therefore increased the respiratory control ratio (state 3/state 4) by 30%. In addition, the ADP/O ratio was increased by 9% and the activity of citrate synthase (U/mg) was 49% higher. Mass spectrometry analysis indicated that the MITO+ preparation contained less contamination from non-mitochondrial proteins. We conclude that nagarse treatment of SS mitochondria removes not only non-mitochondrial proteins but also the protein of damaged mitochondria, improves indices of functional integrity, and the resulting protein-specific activities.