Isopycnic-zonal centrifugation of plasma membrane, sarcoplasmic reticular fragments, lysosomes, and cytoplasmic proteins from phasic skeletal muscle.
Homogenates of the posterior latissimus dorsi muscle, a phasic muscle, were fractionated by a one-step zonal centrifugation technique into four major organelle populations and cytoplasmic constituents. These were: (1) Plasma membrane fragments with a modal equilibrium density of 1.10 and containing 5'-nucleotidase, alkaline phosphodiesterase, p-nitrophenylphosphatase and acid phosphatase (beta-glycerophosphate was used as the substrate). (2) Sarcoplasmic reticular fragments which could be further subdivided into calcium transport vesicles, with a model equilibrium density of 1.16, that exhibited calcium uptake; K+-ATPase; leucyl-bet-naphthylamidase; acid phosphodiesterase; acid phosphatase (using cytidine monophosphate as the substrate); and sarcoplasmic reticular lysosomes, with a model equilibrium density of 1.18, possessing dipeptidyl-aminopeptidase II, cathepsin D, alpha-glucosidase, N-acetyl-beta-glucosaminidase, and NADH oxidase activity. (3) Mitochondria with a modal equilibrium density of 1.21. (4) Catalase-containing vesicles with a modal equilibrium density of 1.22; and cytoplasmic constituents (modal density of 1.25) with phosphorylase, pyruvate kinase, myosin-ATPase, aldolase, and protein and RNA content. The purity of these organelles was equal to or better than previous efforts, with a 30-fold purification achieved for 5'-nucleotidase and alkaline phosphodiesterase. These results lend support to the hypothesis that the sarcoplasmic reticulum of phasic muscle, in addition to its specialized role in excitation-contraction coupling, represents a multifunctional membrane system, and that, similar to the smooth endoplasmic reticulum of other cells, it includes some membrane-bound lysosomal enzymes and NADH oxidase.
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000648:ultrastructure*; D002501:Centrifugation, Zonal / Q000379:methods; D002645:Chickens; D003593:Cytoplasm / Q000201:enzymology; D004798:Enzymes / Q000032:analysis*; D008247:Lysosomes / Q000648:ultrastructure*; D008854:Microscopy, Electron; D009132:Muscles / Q000032:analysis / Q000648:ultrastructure*; D012519:Sarcoplasmic Reticulum / Q000648:ultrastructure*
D004798:Enzymes; D002118:Calcium