Isolation of skeletal muscle mitochondria from hamsters using an lonic medium containing ethylenediarninetetraacetic acid and nagarse

Abstract

An improved procedure is reported for the isolation of skeletal muscle mitochondria from hamsters and compared with our previous method. This procedure utilizes 20 mg % Nagarse in an ionic medium containing 100 m m sucrose, 10 m m EDTA, 100 m m Tris-HCl, 46 m m KCl, and 0.5% bovine serum albumin (BSA), at pH 7.4 (medium-B). Oxidative phosphorylation was studied by measuring ADP O ratio and respiratory control ratio (RCR) using NAD +-linked pyruvate-malate (PM), as well as FAD-linked succinate (SUCC) as substrates. The mitochondria isolated in medium-B exhibited high RCR and high ADP phosphorylation capacity, and were superior to those prepared by our previous method. Electron micrographs of organelles isolated in medium-B revealed intact mitochondrial membrane and structural integrity, whereas those isolated with medium-A containing 50 mg % Nagarse depicted considerable damage including swelling, ruptured membrane, and loss of intramitochondrial matrix. Previously, we used a nonionic medium containing 210 m m mannitol, 70 m m sucrose, 0.1 m m EDTA, 10 m m Tris-HCl, 50 mg % Nagarse, and 0.5% BSA, at pH 7.4 (medium-A). Mitochondria isolated with medium-B yielded mean RCR values of 7.3 to 8.3 with PM, and values of 3.7 to 4.7 with SUCC as substrates, compared to 1.6 and 1.8 with PM, and 1.4 and 1.7 with SUCC for the organelles isolated using medium-A, respectively. Likewise, the ADP O ratios were 2.6 to 2.7 with PM, and 1.6 to 1.7 with SUCC for medium-B preparations, compared to 1.5 and 1.8 with PM and 1.0 and 1.2 with SUCC for medium-A preparations, respectively. The overall significance of the present method, as well as the relative merits of various components of medium-B, are discussed.