Biochemical and morphological characterization of subcellular fractions isolated from rabbit colon muscle.
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, mitochondrial 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 was 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 found to be 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 X 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. From these results we concluded that 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.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002784:Cholesterol / Q000378:metabolism; D003106:Colon / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure*; D003576:Electron Transport Complex IV / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism; D009130:Muscle, Smooth / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010042:Ouabain / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D011817:Rabbits; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure
D009124:Muscle Proteins; D010743:Phospholipids; D010042:Ouabain; D012313:RNA; D002784:Cholesterol; D009247:NADH, NADPH Oxidoreductases; D003576:Electron Transport Complex IV; D010727:Phosphoric Diester Hydrolases; D000251:Adenosine Triphosphatases; D000262:Adenylyl Cyclases; D007930:Leucine