CS activity

Mitochondrial content was analyzed by CS enzyme activity as previously described. ¹⁸Briefly, following muscle fiber collection for oxygen consumption experiments, the remainder of the gastrocnemius muscle was chopped into fine pieces and mixed. Approximately 40 mg of muscle was weighed and further homogenized in 33 mM phosphate buffer (pH 7.4) at a muscle to buffer ratio of 1:40 using a glass tissue grinder. Homogenate was incubated with 5,5′-dithio-bis(2-nitrobenzoic) acid (DTNB, 0.773 mM), acetyl CoA (0.116 mM), and oxaloacetate (0.441 mM) in 100 mM Tris buffer (pH 8.0). Activity of CS was monitored from the reduction of DTNB over time by measurement of absorbance at 412 nm.