Subfractionation of Rabbit Skeletal-Muscle Microsomal Fraction

In rabbit skeletal muscle a well-defined and highly organized system of internal membranes exists. These were first described by Retzius (1881) and Veratti (1902), subsequently overlooked and later rediscovered by Porter & Palade (1957) and Andersson-Cedergren (1959) and others. The skeletal-muscle cell is composed of three main structural components. These are, first, the myofibrils or contractile elements, the chief components of which are the thick and the thin filaments. The second group of structural components embraces the mitochondria, nuclei and ribosomes. Lysosomes have been reported in fractions prepared by the fractionation of skeletal muscle; however, their origin is obscure (Stagni & De Bernard, 1968). The internal membrane systems and the external membrane constitute the third group of structural components. The cell membrane or sarcolemma and the intracellular membrane systems together control the initiation of contraction and the subsequent relaxation of the muscle cell. Homogenization of skeletal muscle results in the formation of small rounded-off vesicles by an active pinching-off process of the various membrane systems (Palade & Siekevitz, 1956). These vesicles are to be found in the microsomal fraction (de Duve, 1964; Reid, 1967). The microsomal fraction would be expected to contain, in addition to the vesicles derived from the sarcoplasmic reticulum and from the transverse tubule system, elements of the outer cell membrane or sarcolemma and possibly vesicles arising by fragmentation of the mitochondria during the homogenization. Many enzymic activities have been demonstrated cytochemically to be associated with the internal membrane systems. Among these are ATPase* (EC 3.6.1.4) (Zebe, 1965) and acetylcholinesterase (EC 3.1.1.7) (Karnofsky, 1964). The ATPase activities associated with the internal membrane systems have been extensively studied since the discovery of the relaxing factor (Marsh, 1952). For a review of the various muscle ATPases see Duggan (1971). The ATP-dependent Ca2+-transporting system, which is responsible for the lowering of the sarcoplasmic Ca2+ concentration and thereby resulting in relaxation of the muscle, is present in the sarcoplasmic reticulum. Another microsomal ATPase activity has been found that is active in the absence of Ca2+ (Weber et al., 1966; Duggan, 1968b). This is the Ca2+-independent or basal ATPase. White skeletal muscle of the rabbit was homogenized in ice-cold 1 .OM-sucrose, forming a 25 % (w/v) homogenate. After a preliminary centrifugation for lOmin at 4°C at 4OOOg in an MSE High Speed 18 centrifuge (rotor no. 69179) the sediment was discarded. The supernatant, after filtration through several layers of cheesecloth, was diluted with half its volume of ice-cold distilled deionized water. This diluted supernatant was then centrifuged for 30min at 4°C at 15000g in the same centrifuge and rotor as used above. Again the sediment was discarded and the supernatant filtered through several layers of cheesecloth. This filtered supernatant served as the starting material for the preparation of concentrated microsomal suspensions. The preparation of concentrated microsomal suspensions was achieved by using the BXIV zonal rotor and a large sample volume (450ml). The microsomal material was sedimented from this sample into a lOOml layer of 1.0M-sucrose by centrifugation for 1 h at 4°C at 30000rev./min in an MSE Super Speed 50 centrifuge. After centrifugation the microsomal material was collected in four 25ml fractions. Analysis for the various enzymes and protein were carried out as described previously (Headon & Duggan, 1970). Fraction 2 was found to contain much of the total ATPase and Ca2+-uptake activities.