Isolation of plasma membranes from purified mouse spermatogenic cells.
Isolation of plasma membranes from purified mouse spermatogenic cells.
复制标题
从纯化的小鼠生精细胞中分离质膜。
作者：
C. Millette;D. A. O’Brien;C. Moulding
用其他作者改进的方法，从纯化的粗线精细胞、圆形精细胞和成年小鼠睾丸残体中制备了质膜。用电子显微镜、凝集素结合和酶测定对分离膜进行了检查。超微结构观察显示直径0.4 ~ 1.7微米的光滑单位膜囊泡。显微镜下未检测到细胞核、线粒体或溶酶体的污染。放射性标记凝集素结合实验[125I-RCAI， 125i -绿豌豆凝集素]表明，细胞表面标记与形态学上鉴定为质膜的物质相分离。根据凝集素数据，估计膜的总回收率平均为33%。亚细胞标记物的生化分析显示，没有检测到DNA，只有1.2%的细胞总RNA与膜分离。多种酶分析表明细胞质酶、高尔基体或线粒体的污染很小。5′-核苷酸酶（E.C. 3.1.3.5）的测定表明，该酶不是发育小鼠生精细胞膜的主要成分。相反，支持细胞是这种酶在成年精小管中最重要的来源。对纯化的生殖细胞分离膜进行聚丙烯酰胺凝胶分析，发现粗线精母细胞和圆形精母细胞膜的蛋白质组成有显著差异。从均匀的生精细胞群体中制备高纯度的质膜，有助于对发育中的男性生殖细胞特异性的细胞表面抗原进行生化表征。
Plasma membranes have been prepared from purified pachytene spermatocytes, round spermatids and residual bodies of the adult mouse testis using procedures modified from other authors'. Isolated membranes have been examined using electron microscopy, lectin binding and enzymic assays. Ultrastructural observation reveals smooth unit-membrane vesicles from 0.4-1.7 micrometer diameter. No contamination by nuclei, mitochondria or lysosomes is detected microscopically. Radiolabelled lectin-binding experiments [125I-RCAI, 125I-green pea lectin] indicate that cell surface label cofractionates with material identified morphologically as plasma membrane. Estimates of total recovery of membrane, based upn the lectin data, average 33%. Biochemical analysis of subcellular markers reveal that no detectable DNA and only 1.2% of the total cellular RNA cofractionate with membranes. A variety of enzyme assays suggests little contamination by cytosol enzymes, Golgi material or mitochondria. Assays of 5'-nucleotidase (E.C. 3.1.3.5) indicate that this enzyme is not a major component of developing mouse spermatogenic cell membranes. Instead, Sertoli cells represent the most important source of this enzyme in the adult seminiferous tubule. Polyacrylamide gel analysis of membranes isolated from purified germ cells reveals significant differences in the protein compositions of pachytene spermatocyte and round spermatid membranes. The preparation of highly purified plasma membranes from homogeneous populations of spermatogenic cells should facilitate the biochemical characterization of cell surface antigens specific to developing male germ cells.