CALCIUM UPTAKE BY RAT KIDNEY MITOCHONDRIA
CALCIUM UPTAKE BY RAT KIDNEY MITOCHONDRIA
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DOI：
10.1073/pnas.47.11.1744
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发表时间：
1961-01-01
影响因子：
11.1
通讯作者：
ENGSTROM, GW
中科院分区：
文献类型：
--
作者：
DELUCA, HF;ENGSTROM, GW
方法。-将体重为70 - 80 gm的Holtzman品系的年轻雄性大鼠饲养在吊丝笼中，并随意给予食物和水。除了用不含维生素的酪蛋白加0.2%的L-胱氨酸代替蛋白质来源的白色外，所喂的饲料基本上是Steenbock和Herting的足够钙和磷的饲料。在每个实验中，大鼠被分成两个相等的组。一组没有接受维生素D，而在另一组中，每只大鼠每三天接受75 IU的结晶钙化醇棉籽油。维生素D缺乏组的生长迟缓和血清钙降低很明显。4.在大鼠分别进食至少21天后，对缺乏维生素D和补充维生素D的大鼠进行头部重击，随后断头处死。快速取出肾脏，并在冰冷的等渗蔗糖中冷却。在0 ℃下，用配有Teflon研杵的Potter-Elvehjem匀浆器制备10%的肾脏匀浆。将10 ml匀浆在10 ml 0.34 M蔗糖上分层，并以600 X g离心10 min以除去细胞核、碎片和未破碎的细胞。小心地除去上层，并以8.000 X g离心10分钟以沉淀线粒体。用0.25 M蔗糖洗涤该制剂一次，最后悬浮于5 ml等渗蔗糖中。除非另有说明，将0.5ml该悬浮液在30 ℃下在含有0.08 μ mol细胞色素c、6 μ mol ATP、45 μ mol琥珀酸盐或15 μ mol谷氨酸盐、10 μ mol MgCl 2、0.9 μ mol含Ca 4的CaCl 2和40 μ mol Tris缓冲液（pH7.4）或40 μ mol磷酸盐缓冲液（pH7.4）的混合物中孵育。40 μ mol KCl和300 μ mol蔗糖，最终体积为3.2 ml。将混合物在Dubnoff型振荡器中以空气作为气相孵育。取出反应混合物的等分试样，冷却至0 ℃，并立即以20，000 × g离心7 - 10分钟。倾析上清液，必要时，通过改良的乙二胺四乙酸盐（EDTA）滴定法5分析钙或计数放射性。为了测量沉淀物的放射性，将离心杯排干并用薄纸擦干;将沉淀物溶解在稀硝酸中并消化，或将其溶解在0.2%十二烷基硫酸钠中，铺在不锈钢板上，并计数为无限薄的样品。用薄端窗自动计数装置在盖革区测量放射性。通过Nesslerization常规分析每种线粒体制备物的样品的氮。6制备乙二胺四乙酸盐（EDTA）制剂时，将肾脏在含有0.001 M EDTA钠的0.25 M蔗糖中匀浆。分离如前所述，不同之处在于线粒体用含EDTA的0.25 M蔗糖洗涤一次，然后在不含EDTA的蔗糖中洗涤。将制剂悬浮在不含EDTA的等渗蔗糖中，并如前所述使用。
Methods.-Young male rats of the Holtzman strain weighing 70-80 gm were housed irn hanging wire cages and given food and water ad libitum. The diet fed was essentially the adequate calcium and phosphorus diet of Steenbock and Herting'4 except that vitamin-free casein plus 0.2 per cent L-cystine replaced egg white as the protein source. In each experiment, the rats were divided into two equal groups. One group received no vitamin D, while in the remaining group each rat received 75 iu of crystalline calciferol in cottonseed oil every three days. Retarded growth and reduced serum calcium were evident in the vitamin-D deficient group.'4 After the rats had been on their respective diets for at least 21 days, pairs of vitamin-D deficient and supplemented rats were killed by asharp blow on the head followed by decapitation. The kidneys were removed quickly and chilled in ice-cold isotonic sucrose. A 10 per cent homogenate of the kidneys was prepared at 00C with a Potter-Elvehjem homogenizer fitted with a Teflon pestle. Ten ml of homogenate was layered over 10 ml of 0.34 M sucrose and centrifuged at 600 X g for 10 min to remove nuclei, debris, and unbroken cells. The upper layer was removed carefully and centrifuged at 8.000 X g for 10 min to sediment the mitochondria. This preparation was washed once with 0.25 M sucrose and finally suspended in 5 ml of the isotonic sucrose. Unless otherwise indi-cated, 0.5 ml of this suspensionwas incubated at 300C in a mixture which contained 0.08, umoles cytochrome c; 6, umoles ATP; 45, umoles succinate or 15, umoles glutamate; 10 jumoles MgCl2; 0.9, umoles CaC12 containing Ca4; and either 40 Mumoles Tris buffer, pH 7.4, or 40, umoles phosphate buffer, pH 7.4; 40, umoles KC1 and 300, umoles sucrose in a final volume of 3.2 ml. Mixtures were incubated in a Dubnoff-type shaker with air as the gasphase. Aliquots of the reaction mixture were removed, chilled to 00C and immediately centrifuged at 20,000 X g for 7-10 minutes. The supernatant was decanted and, when desired, analyzed for calcium by a modification of an ethylenediaminetetraacetate (EDTA) titration method'5 or counted for radioactivity. To measure the radioactivity of the precipitate, the centrifuge cup was drained and wiped dry with tissue paper; the pellet was dissolved in dilute nitric acid and digested or it was dissolved in 0.2 per cent sodium lauryl sulfate, plated on stainless steel planchets, and counted as infinitely thin samples. Measurements of radioactivitywere made in the Geiger region with a thin end-window automatic counting device. A sample of each mitochondrial preparation was analyzed routinely for nitrogen by Nesslerization.'6When ethylenediaminetetraacetate (EDTA) preparations were made, kidneys were homogenized in 0.25 M sucrose containing 0.001 M sodium EDTA. The isolation was as described previously except that the mitochondria were washed once with the 0.25 M sucrose containing EDTA and then washed in sucrose free of EDTA. The preparations were suspended in isotonic sucrose free of EDTA andused as described previously.