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Analytical subcellular fractionation of normal human skeletal muscle by sucrose density gradient centrifugation
Author(s) -
MARTIN F. C.,
LEVI A. J.,
SLAVIN G.,
PETERS T. J.
Publication year - 1983
Publication title -
european journal of clinical investigation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.164
H-Index - 107
eISSN - 1365-2362
pISSN - 0014-2972
DOI - 10.1111/j.1365-2362.1983.tb00064.x
Subject(s) - sucrose gradient , density gradient , cell fractionation , differential centrifugation , fractionation , sucrose , centrifugation , skeletal muscle , human muscle , chemistry , chromatography , biochemistry , biology , anatomy , enzyme , physics , quantum mechanics
. The principle organelle marker enzymes and various adenosine triphosphatase (ATPase) activities were studied in human skeletal muscle. The reproducibility of each assay was established under optimal and linear assay conditions. Whole homogenates of normal human quadriceps muscle were fractionated by centrifugation on a continuous sucrose density gradient. Gradient fractions were assayed for organelle marker enzymes and frequency‐density histograms were constructed for each enzyme. Good resolution of the principal organelles was obtained. Adenosine triphosphatase (ATPase) was assayed under conditions of maximal stimulation by Ca 2+ , or Mg 2+ or Na 2+ , K + + Mg 2+ . The distribution of these activities was compared with those of the organelle marker enzymes. Both Ca 2+ ‐ATPase and Mg 2+ ‐ATPase were distributed to both the mitochondrial and myofibrillar fractions but could be distinguished by the inhibition of mitochondrial ATPase with sodium azide. The distribution of Na + , K + ‐activated, Mg 2+ ‐dependent ATPase (Na + , K + ATPase) activity suggested a sarcolemmal localization. The results of electron microscopy of gradient fractions were consistent with the organelle content of the fractions as determined by enzymic analyses. These studies provide reference information for the subsequent investigation of organelle pathology of human muscle disorders.