Hydrodynamic parameters and isolation of mitochondria, microperoxisomes and microsomes of rat heart

1. Analytical differential centrifugation of rat heart homogenates revealed a single population of mitochondria and microperoxisomes. Using cytochrome c oxidase, malate dehydrogenase and amine oxidase as mitochondrial marker enzymes, the s-value of mitochondria was estimated to s = 10326 +/- 406 S (average for the three marker enzymes). The s-value of microperoxisomes was found to be s = 1381 +/- 40 S using catalase as the marker enzyme. The s-value for the two organelles did not change significantly when the isoosmotic sucrose medium was substituted by an isoosmotic mannitol medium. 2. Analytical differential centrifugation revealed a polydispercity of the microsomal fraction using glucose-6-phosphatase and NADPH-cytochrome c reductase as the marker enzymes. The s-values were found to be sH1 = 1569 +/- 412 S (NADPH-cytochrome c reductase), sH2 = 1195 +/- 400 S (glucose-6-phosphatase) and sL = 153 +/- 28 S (NADPH-cytochrome c reductase and glucose-6-phosphatase). The recovery of marker enzymes in the isolated subcellular fractions was in the range of 84-94%. 3. When the mitochondrial and microperoxisomal fractions were subjected to isopycnic gradient centrifugation, using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.25 M sucrose medium, buoyant densities of 1.10 g/cm3 (main fraction of mitochondria) and 1.06 g/cm3 (main fraction of microperoxisomes) were obtained. The density gradient centrifugation separated microperoxisomes from contaminating lysosomes of high specific activity in acid phosphatase. A value 1.04 g/cm3 was found for the density of the microsomal fraction. 4. Based on the estimated s-values, an optimal procedure is described for the isolation of mitochondrial and microperoxisomal fractions from rat heart muscle.