Biochemical and Morphological Characterization of Subcellular Fractions Isolated from Rabbit Colon Muscle

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Abstract
From a homogenate of rabbit colon muscle subcellular fractions were isolated by differential centrifugation. The crude microsomal fraction could be separated into subfractions, a fraction of vesicular microsomes at 35% sucrose, a fraction containing sarcolemma, mitochondria fragments and microsomal vesicles at 35-45% sucrose and a small protein fraction at 45-55% sucrose. Their biochemical properties and their morphological characterization were investigated. The cholesterol and the phospholipid content were equally distributed between the microsomal fractions 35% and 35-45% while the RNA was localized to the mitochondria and the microsomal fraction 35%. The enzyme cytochrome-c oxidase was concentrated in the mitochondria while a high contamination was found in the microsomal fractions 35-45%. The NADH-oxidase activity was highest in the 35% fraction and the 5''-nucleotidase activity in the 40,000 .times. g supernatant. The microsomal subfractions contained the enzymes ATPase, adenylate cyclase and phosphodiesterase. In the 35% fraction Ca stimulated the hydrolysis of ATP. The binding of [3H]-ouabain and the incorporation of [3H]-leucine was most pronounced in the 35% fraction. In a K+-free Krebs Ringer medium the binding of the glucoside was stimulated in all the fractions. The fraction 35% sucrose may be mainly derived from the endoplasmic reticulum and the plasma membrane while the 35-45% originates from the plasma membrane, mitochondria and to a lesser extent the endoplasmic reticulum.