A COMBINATION OF METHODS FOR THE PREPARATION OF HIGHLY INTACT MITOPLASTS FROM BEEF-HEART MITOCHONDRIA
A COMBINATION OF METHODS FOR THE PREPARATION OF HIGHLY INTACT MITOPLASTS FROM BEEF-HEART MITOCHONDRIA
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DOI：
10.1016/0003-2697(85)90329-x
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发表时间：
1985-01-01
影响因子：
2.9
通讯作者：
BATRA, PP
中科院分区：
文献类型：
--
作者：
BURNETTE, B;BATRA, PP
与来自其他组织的线粒体相比，牛心线粒体被注意到难以分离。似乎没有一种方法可以在保持内膜完整的同时提供所需的效率。四个变量，单独和所有可能的组合，评价其在分馏的有效性。这些变量为毛地黄皂苷治疗、低渗肿胀、磷脂酶A2预消化和添加高浓度EDTA。通过EM和标记酶测定来评估分级分离的效率。高度完整的有丝分裂体时，得到的线粒体与毛地黄皂苷一起处理强烈的低渗肿胀，或中度低渗肿胀和磷脂酶A2预消化，或EDTA添加和磷脂酶预消化。酶GTP：AMP磷酸转移酶被发现是专门位于基质隔室。
Beef heart mitochondria are noted for difficulty of fractionation compared to mitochondria from other tissues. A method that would give the desired efficiency while leaving the inner membrane intact did not seem to be available. Four variables, alone and in all possible combinations, were evaluated for their effectiveness in fractionation. These variables were treatment with digitonin, hypotonic swelling, phospholipase A2 predigestion and addition of high concentrations of EDTA. The efficiency of fractionation was evaluated by EM and marker enzyme assays. Highly intact mitoplasts were obtained when the mitochondria were treated with digitonin together with intense hypotonic swelling, or moderate hypotonic swelling and phospholipase A2 predigestion, or EDTA addition and phospholipase predigestion. The enzyme GTP:AMP phosphotransferase was found to be located exclusively in the matrix compartment.