Abstract
Studies of calcium compartmentalization in smooth muscle organelles have been limited due to the operational methods of isolation of sarcoplasmic reticulum and mitochondria (1, 2). Although these methods give qualitatively excellent results their quantitation in respect to the whole cell is difficult. Since smooth muscle cells can now be isolated before fractionation (3), we employed these newer techniques to examine the distributions of sarcoplasmic reticulum fragments, lysosomes, catalase-containing organelles, mitochondria, and plasma membrane fragments in continuous sucrose gradients. This study confirms that smooth muscle organelles isolated from vascular tissue are heterogeneous and demonstrates that plasma membrane fragments and, to some extent, mitochondria can be separated from the other organelles.
Materials and methods. Hog carotid arteries (6-10 g) were obtained locally3 from freshly slaughtered hogs. Fat and connective tissue were removed and the arteries were cut and placed in ice-cold complete Hank's solution for transport to the laboratory. There they were dissected and isolated cells were obtained by enzymatic digestion (3), omitting the hyaluronidase from the isolation mixture, since equally good results were obtained without its addition.
The washed cells isolated by this procecedure were disrupted in 0.25 M sucrose solution containing 0.02 M KCl with 25 strokes of a Type B pestle in a small Dounce homogenizer. This total homogenate was centrifuged at 600g for 10 min. The postnuclear supernatant (PNS) was removed and saved; the nuclear fraction (N) was re-suspended in 0.25 M sucrose containing 0.02 M KCl.
The postnuclear supernatant (PNS) was adjusted to a sucrose concentration of 22% (w/v) and a volume of 160 ml and used as the light limiting solution in the construction of a 22-65% linear sucrose gradient that was pumped into a Z-60 zonal rotor (Beckman) spinning at 3000 rpm.
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