2 SPECIES OF LYSOSOMAL ORGANELLES IN CULTURED HUMAN-FIBROBLASTS
2 SPECIES OF LYSOSOMAL ORGANELLES IN CULTURED HUMAN-FIBROBLASTS
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DOI：
10.1016/0092-8674(79)90302-7
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发表时间：
1979-01-01
期刊：
影响因子：
64.5
通讯作者：
NEUFELD, EF
中科院分区：
文献类型：
--
作者：
ROME, LH;GARVIN, AJ;NEUFELD, EF
培养的二倍体人皮肤成纤维细胞通过最大限度地回收含有酸性水解酶的颗粒的程序进行分级。通过受控的胰蛋白酶消化使细胞分离，在低压下通过N2空化破坏细胞，并在18，000 × 10 - 6下分级分离。g在胶体二氧化硅的自生梯度上。该程序分离了2种可视为溶酶体的颗粒。密度较大的一个（峰密度1.11）显然没有其他污染物，但浮力较大的一个（峰密度1.085）与其他细胞器（包括线粒体、高尔基体、内质网和质膜）的峰一起或靠近峰沉积。这2个颗粒群含有大致相等比例的酸性水解酶（磷酸酶、6种糖苷酶和4种组织蛋白酶），显示出潜伏期，在正常细胞和艾杜糖苷酶缺陷细胞中具有相似的35 S-粘多糖周转，并且是α-葡萄糖苷酶的受体。L-艾杜糖醛酸酶，先前显示通过受体介导的内吞作用获得。完整成纤维细胞的酸性磷酸酶染色显示残留体分散在整个细胞质中，在细胞核附近，有一个明显的小管网络和相关的突起和旋钮样扩大。在薄切片和厚切片中，这些都是连续的，好像形成了一个类似于GERL [高尔基体，内质网，溶酶体]的三维网络。分离组分的超微结构研究表明，致密的溶酶体峰由小的圆形或椭圆形酸性磷酸酶阳性小体组成。更多的浮力峰包含非溶酶体细胞器预测的生化标志物，小酸性磷酸酶阳性机构和大型多囊泡结构，其中酸性磷酸酶被定位在一个矩阵周围显然空囊泡。这些大结构可以表示GERL的片段。致密和浮力溶酶体细胞器可能主要来自残留体和GERL网络，分别。
Cultured diploid human skin fibroblasts were fractionated by a procedure that maximizes recovery of particles containing acid hydrolases. The cells were detached by controlled trypsinization, disrupted by N2 cavitation at low pressure and fractionated at 18,000 .times. g on a self-generating gradient of colloidal silica. This procedure separated 2 species of particles that could be considered lysosomal. The denser one (peak density 1.11) was apparently free of other contaminants, but the more buoyant one (peak density 1.085) sedimented with or close to the peaks of other organelles, including mitochondria, Golgi, endoplasmic reticulum and plasma membranes. The 2 populations of particles contained acid hydrolases (phosphatase, 6 glycosidases and 4 cathepsins) in roughly equal proportions, displayed latency, had similar turnover of 35S-mucopolysaccharide in normal and in iduronidase-deficient cells, and were recipients of .alpha.-L-iduronidase, previously shown to be acquired by receptor-mediated endocytosis. Acid phosphatase staining of the intact fibroblasts showed residual bodies scattered throughout the cytoplasm and, near the nucleus, a prominent network of tubules and associated dilatations and knob-like enlargements. In both thin and thick sections, these appeared continuous, as if forming a 3-dimensional network similar to the network described as GERL [Golgi, endoplasmic reticulum, lysosomes]. Ultrastructural studies of the isolated fractions showed the denser lysosomal peak to be composed of small round or oblong acid phosphatase-positive bodies. The more buoyant peak contained the nonlysosomal organelles predicted from the biochemical markers, small acid phosphatase-positive bodies and large multivesiculated structures in which acid phosphastase was localized in a matrix surrounding apparently empty vesicles. These large structures may represent fragments of GERL. The dense and buoyant lysosomal organelles may originate primarily from residual bodies and from the GERL network, respectively.