STUDIES ON MITOCHONDRIAL STRUCTURE AND FUNCTION IN PHYSARIUM-POLYCEPHALUM .1. FINE-STRUCTURE, CYTOCHEMISTRY, AND H-3-URIDINE AUTORADIOGRAPHY OF A CENTRAL BODY IN MITOCHONDRIA
STUDIES ON MITOCHONDRIAL STRUCTURE AND FUNCTION IN PHYSARIUM-POLYCEPHALUM .1. FINE-STRUCTURE, CYTOCHEMISTRY, AND H-3-URIDINE AUTORADIOGRAPHY OF A CENTRAL BODY IN MITOCHONDRIA
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DOI：
10.1016/0014-4827(73)90079-7
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发表时间：
1973-01-01
影响因子：
3.7
通讯作者：
KUROIWA, T
中科院分区：
文献类型：
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作者：
KUROIWA, T
材料和方法在这些实验中使用了第二次有丝分裂（MII）后的表面疟原虫，并使用丹尼尔和Rusch（14）报道并由Guttes和Guttes（15）评论的方法的修改进行培养。将G2期期间直径为4cm的三个疟原虫从皮氏培养皿中的滤纸刮入30 ml含有0.25M蔗糖、0.01M CaCl 2、0.01M Tris缓冲液（pH 7.0，含HCl）和0.1%（wt/vol）Triton X-100的对照培养基中（16）。将悬浮液在50-ml Waring搅拌杯中以3，000 rpm均质化30 s。用移液管除去泡沫。均聚物通过重力通过两种厚度的具有31 μ m孔的尼龙网（NBC Ind.，日本）。将10 ml匀浆在蔗糖浓度为0.5 - 1.5M的不连续蔗糖密度梯度上分层，两个相邻蔗糖溶液之间的差异为0.25M。将试管在0 ℃下以1，500 rpm（300 g）在具有Kokusan H-103离心机的摇摆桶转子中离心30分钟。线粒体在0.75和1.00 M的蔗糖层之间的边界带状。然后收集线粒体级分，并将线粒体以3，000 rpm沉降10分钟。通过重悬于10 ml 0.25 M蔗糖溶液中洗涤线粒体一次，然后再离心。将沉淀重悬于0.5ml蒸馏水中。分离的线粒体用0.2%天青B水溶液染色，并在光学显微镜下检查。在分离线粒体或线粒体沉淀之前，通过先前使用的程序（11）制备疟原虫的切片。根据先前描述的程序制备分散的线粒体制剂（17）。将散布在空气-水表面的线粒体置于碳包被的网格上，在0.2%戊二醛的pH6.8磷酸盐缓冲液中4 ℃固定5 min，在2%（wt/vol）磷钨酸或0.02%（wt/vol）醋酸双氧铀中室温染色10 min，对散布在网格上的线粒体进行酶标。将网格从固定剂转移到pH6.8的乙酸盐缓冲液中，在该缓冲液中漂洗，并在34 ℃下在以下中提取：（a）1 mg DNA酶（Sigma Chemical Co.，密苏里州圣路易斯，纯牛肉胰脏）/升。0 ml含有0.003 M乙酸镁的0.1 M乙酸盐缓冲液，pH 5.5，持续20分钟，（B）1 mg RNA酶（Sigma Chemical Co.，五次结晶，
MATERIALS AND METHODSSurface plasmodia after the second mitosis (Mll) were used in these experiments and were cultivated using a modification of the methods reported by Daniel and Rusch (14) and reviewed by Guttes and Guttes (15). Three plasmodia 4 cm in diameter during G2 phase were scraped from filter paper in a Petri dish into 30 ml of the control medium containing 0.25 M sucrose, 0.01 M CaC12, 0.01 M Tris buffer (pH 7.0 with HCI), and 0.1%(wt/vol) Triton X-100 (16). The suspension was homogenized for 30 s at 3,000 rpm in a 50-ml Waring blender cup. The foam was removed with a pipette. The homogehate was filtered by gravity through two thicknesses of nylon meshes with pores of 31# m (NBC Ind., Japan). 10 ml of the homogenate was layered over a discontinuous sucrose density gradient with sucrose concentrations ranging from 0.5 to 1.5 M, the difference between two adjacent sucrose solutions being 0.25 M. The tubes were centrifuged at 0 C for 30 min at 1,500 rpm (300 g) i1 a swinging bucket rotor with a Kokusan H-103 centrifuge. The mitochondria banded at the border between the 0.75 and 1.00 M sucrose layers. The mitochondrial fraction was then collected and the mitochondria were sedimented for 10 min at 3,000 rpm. The mitochondria were washed once by resuspending in 10 ml of 0.25 M sucrose solution followed by recentrifugation. The pellet was resuspended in 0.5 ml of distilled water. Isolated mitochondria were stained with a 0.2% aqueous solution of azure B and examined under the light microscope. Preparation of ultrathin sections of the plasmodia before isolation of the mitochondria or of the mitochondrial pellet was done by the procedure employed previously (1 I). Spread mitochondria preparations were made according to the procedures described previously (17). The mitochondria spread on the air-water surface were picked up on a carbon-coated grid, fixed in 0.2% glutaraldehyde in pH 6.8 phosphate buffer for 5 min at 4 C, and stained in 2%(wt/vol) phosphotungstic acid or in 0.02%(wt/vol) uranyl acetate for 10 min at room temperature.Enzymatic digestions were done on the mitochondria spread on the grid. The grids were transferred from fixative to acetate buffer at pH 6.8, rinsed in this buffer, and extracted at 34 C in:(a) 1 mg DNase (Sigma Chemical Co., St. Louis, Mo., electrophoretically pure, beef pancreas)/l. 0 ml 0.1 M acetate buffer containing 0.003 M magnesium acetate at pH 5.5 for 20 min,(b) 1 mg RNase (Sigma Chemical Co., five times crystallized,