Energy-dependent calcium transport in endoplasmic reticulum of adipocytes.
Energy-dependent calcium transport in endoplasmic reticulum of adipocytes.
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脂肪细胞内质网中能量依赖性钙转运。
DOI：
10.1016/s0021-9258(17)32961-7
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发表时间：
1976
期刊：
影响因子：
--
通讯作者：
L. Jarett
中科院分区：
文献类型：
--
作者：
D. Bruns;J. McDonald;L. Jarett
分离的大鼠脂肪细胞的内质网具有主动积累钙的能力。使用总细胞匀浆的 20,000 X g 上清液（S1 级分）来表征钙吸收。在测试之前，从 S1 级分中分离出的内质网囊泡为 160,000 X g 微粒体颗粒，表明其积累钙的能力很小。通过形态学外观、通过使用钙吸收的选择性抑制剂以及通过积累的钙的高速沉降，S1级分中的钙吸收被定位于内质网囊泡。摄取依赖于 MgATP 和温度，并由用作囊泡内捕获剂的草酸盐维持。在所有测试的钙浓度（3 至 100 μM）下，吸收与时间呈线性关系至少 30 分钟，并且最佳 pH 值约为 7.0。巯基抑制剂对氯汞苯磺酸盐对钙吸收产生剂量依赖性抑制，总抑制量为 0.07 mumol/mg 蛋白质。钌红和叠氮化钠在浓度（分别为5μM和10mM）下抑制小于5％的吸收，完全阻断从相同细胞中分离的线粒体对钙的吸收。钙吸收的 Km 为 10 μM 总钙，相当于测定系统中大约 3.6 μM 离子钙。在所用测定条件下，24 度时最大摄取速度为 5.0 nmol（微粒体蛋白毫克）-1 (min)-1，Q10 为 1.8。 S1 级分中内质网囊泡的摄取活性表现出明显的时间和温度依赖性不稳定性，这可能部分解释了分离的微粒体级分中缺乏摄取。这种能量依赖性钙吸收系统似乎对细胞内钙的调节具有重要的生理意义。
The endoplasmic reticulum from isolated rat adipocytes has the ability to actively accumulate calcium. The calcium uptake was characterized using the 20,000 X g supernatant (S1 fraction) of total cellular homogenate. Endoplasmic reticulum vesicles isolated from the S1 fraction as a 160,000 X g microsomal pellet prior to testing demonstrated little ability to accumulate calcium. The calcium uptake in the S1 fraction was localized to the endoplasmic reticulum vesicles by morphologic appearance, by the use of selective inhibitors of calcium uptake, and by high speed sedimentation of the accumulated calcium. The uptake was MgATP- and temperature-dependent and was sustained by the oxalate used as the intravesicular trapping agent. Uptake was linear with time for at least 30 min at all calcium concentrations tested (3 to 100 muM) and exhibited a pH optimum of approximately 7.0. The sulfhydryl inhibitor p-chloromercuribenzene sulfonate produced a dose-dependent inhibition of calcium uptake with total inhibition at 0.07 mumol/mg protein. Ruthenium red and sodium azide inhibited less than 5% of the uptake at concentrations (5 muM and 10 mM, respectively) which completely blocked calcium uptake by mitochondria isolated from the same cells. The Km for calcium uptake was 10 muM total calcium which corresponded to approximately 3.6 muM ionized calcium in the assay system. The maximum velocity of the uptake was 5.0 nmol (mg of microsomal protein)-1 (min)-1 at 24 degrees under the assay conditions used and exhibited a Q10 of 1.8. The uptake activity of the endoplasmic reticulum vesicles in the S1 fraction exhibited a marked time- and temperature-dependent lability which might account in part for the lack of uptake in the isolated microsomal fraction. This energy-dependent calcium uptake system would appear to be of physiologic importance to the regulation of intracellular calcium.