CHARACTERIZATION OF DOG CARDIAC MICROSOMES - USE OF ZONAL CENTRIFUGATION TO FRACTIONATE FRAGMENTED SARCOPLASMIC RETICULUM, (NA+ + K+)-ACTIVATED ATPASE AND MITOCHONDRIAL FRAGMENTS
CHARACTERIZATION OF DOG CARDIAC MICROSOMES - USE OF ZONAL CENTRIFUGATION TO FRACTIONATE FRAGMENTED SARCOPLASMIC RETICULUM, (NA+ + K+)-ACTIVATED ATPASE AND MITOCHONDRIAL FRAGMENTS
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DOI：
10.1016/0005-2728(70)90113-1
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发表时间：
1970-01-01
期刊：
影响因子：
--
通讯作者：
POLASCIK, MA
中科院分区：
文献类型：
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作者：
KATZ, AM;REPKE, DI;POLASCIK, MA
心脏微粒体是一种异质组分，除了被认为起源于肌浆网的标志物外，还含有线粒体、质膜和溶酶体酶。该馏分的确切组成取决于制备方法，因为心室肌的长时间均质化增加了微粒体蛋白的产量和线粒体污染物的比例。在仅用蔗糖制成的密度梯度上对心脏微粒体进行超离心，在分离碎裂的肌浆网方面价值有限。由于微粒体的聚集，在蔗糖中的带状超浓缩允许材料的分离，仅轻微增强肌浆网标记物的活性。在LiBr的存在下，在抑制这种盐对所研究的活性的破坏作用的条件下使用，微粒体部分的聚集减少，密度梯度分级分离更有效。用0.5M LiBr区带离心法制备的肌浆网碎片，其线粒体酶含量低于原微粒体的15倍，而Ca 2+摄取速率和程度分别比原微粒体粗品提高2倍和4倍。肌浆网标记物集中在含有约100 μ g/ml的梯度区域中。5%的原始蛋白质，不对应于明显的蛋白质峰。
Cardiac microsomes represent a heterogeneous fraction which contains mitochondrial, plasma membrane and lysosomal enzymes in addition to markers believed to originate in the sarcoplasmic reticulum. The exact composition of this fraction depends on the method of preparation in that prolonged homogenization of ventricular myocardium increases both the yield of microsomal protein and the proportion of the mitochondrial contaminant. Ultracentrifugation of cardiac microsomes on density gradients made with sucrose alone is of limited value in isolating fragmented sarcoplasmic reticulum. Because of aggregation of the microsomes, zonal ultracentrifugation in sucrose permits isolation of material with only slight enhancement in the activity of markers for the sarcoplasmic reticulum. In the presence of LiBr, used under conditions which inhibit the damaging effects of this salt on the activities studied, aggregation of the microsomal fraction is reduced and density gradient fractionation is more effective. The fragmented sarcoplasmic reticulum prepared by zonal centrifugation in 0.5 M LiBr contains less than 1 5 the level of mitochondrial enzymes found in the original microsomes while the rate of Ca 2+ uptake is enhanced 2-fold and the extent of Ca 2+ uptake is enhanced 4-fold over that in the crude microsomal fraction. The sarcoplasmic reticulum markers were concentrated in a region of the gradient containing approx. 5% of the original protein that did not correspond to an obvious protein peak.