O'Beirne G B, Williams D C
Department of Biochemistry, Trinity College, Dublin, Ireland.
Eur J Biochem. 1988 Aug 1;175(2):413-21. doi: 10.1111/j.1432-1033.1988.tb14211.x.
In rat kidney high-affinity binding sites for [3H]Ro-5-4864 and [3H]PK-11195 with the properties of the peripheral-type acceptor were found enriched in mitochondrial (M) and light-mitochondrial-lysosomal (L) fractions on differential centrifugation. When the combined M and L fractions were subjected to sucrose density gradient centrifugation, these binding sites were found enriched at a density of 1.155 g/ml coincident with a population of light mitochondria, whereas a population of heavier mitochondria (rho = 1.175 g/ml) had few or no binding sites. Transmission electron microscopy showed that whereas the heavier mitochondria appeared highly pure and intact, the lighter mitochondria appeared less intact and to be contaminated with vesicular structures. After fractionation of the light mitochondria and vesicles by centrifugation, both fractions showed the same ratio of [3H]Ro-4864 binding sites to monoamine oxidase activity consistent with the vesicles being of mitochondrial outer-membrane origin. Digitonin pre-treatment had no effect on the density of acceptor-rich fractions on sucrose density gradient centrifugation. However, pretreatment with succinate/iodophenylnitrophenylphenyltetrazolium (INT) perturbed equally the density of acceptor-rich fractions and mitochondrial marker enzymes. When mitochondrial fractions were subjected to sonication prior to density gradient centrifugation the binding sites were now found highly enriched in a much lighter fraction coincident with the monoamine oxidase activity and thus consistent with being outer-membrane vesicles. When a mitochondrial fraction was subjected to hypotonic treatment before assay no evidence for activation/unmasking of binding sites was found. The hypotonic treatment did not release any inhibitor of the binding sites. These results are consistent with the peripheral benzodiazepine acceptor having an outer-membrane location on a sub-population of rat kidney mitochondria. Those mitochondria showing high levels of the acceptor are either light mitochondria or appear more susceptible to osmotic damage than those mitochondria in which the acceptor is absent or at low levels.
在大鼠肾脏中，通过差速离心发现，具有外周型受体特性的[3H]Ro-5-4864和[3H]PK-11195高亲和力结合位点在线粒体（M）和轻线粒体-溶酶体（L）组分中富集。当将合并的M和L组分进行蔗糖密度梯度离心时，发现这些结合位点在密度为1.155 g/ml处富集，与一群轻线粒体一致，而一群较重的线粒体（ρ = 1.175 g/ml）几乎没有或没有结合位点。透射电子显微镜显示，较重的线粒体看起来高度纯净且完整，而较轻的线粒体看起来不太完整且被囊泡结构污染。通过离心对轻线粒体和囊泡进行分级分离后，两个组分显示出相同的[3H]Ro-4864结合位点与单胺氧化酶活性的比例，这与囊泡起源于线粒体外膜一致。洋地黄皂苷预处理对蔗糖密度梯度离心时富含受体组分的密度没有影响。然而，琥珀酸盐/碘苯基硝基苯基苯基四氮唑（INT）预处理同样扰乱了富含受体组分的密度和线粒体标记酶。当线粒体组分在密度梯度离心前进行超声处理时，现在发现结合位点高度富集在一个与单胺氧化酶活性一致的更轻的组分中，因此与外膜囊泡一致。当线粒体组分在测定前进行低渗处理时，未发现结合位点激活/暴露的证据。低渗处理未释放任何结合位点的抑制剂。这些结果与外周苯二氮䓬受体位于大鼠肾脏线粒体亚群的外膜位置一致。那些显示高受体水平的线粒体要么是轻线粒体，要么比那些缺乏或低水平受体的线粒体更容易受到渗透损伤。
