Burón M I, Navas P, García-Herdugo G, Morré D J
Departamento de Biología Celular, Facultad de Ciencias, Universidad de Cordóba/Spain.
Eur J Cell Biol. 1987 Oct;44(2):176-86.
Aqueous two-phase partition and preparative free-flow electrophoresis were used in series to isolate the plasma membranes of amphibian epidermis. Fractions obtained by two-phase partition were 40-fold enriched in a K+-stimulated, ouabain-inhibited, p-nitrophenylphosphatase relative to the total homogenate and based on morphology were representative isolates of all epidermal cells together. Small mucosal granules and mucin aggregates were the primary contaminants. Based on activities of marker enzymes, contents of mitochondria, Golgi apparatus and endoplasmic reticulum were low (0.15 that of total homogenate) or absent. When plasma membranes isolated by aqueous two-phase partition were subjected to preparative free-flow electrophoresis, they were distributed toward the anode in a series of fractions of increasing net negative charge, sialic acid content and specific activity of the K+-stimulated, ouabain-inhibited, p-nitrophenylphosphatase reminiscent of the activity gradient from base to apex for frog epidermis observed from cytochemical investigations. The most electronegative fractions nearest the anode and to the left of the main protein peak were enriched in both sulfate groups and thick membranes of the stratum corneum. A fraction migrating less toward the anode and to the right of the main protein peak contained hemidesmosomes together with the lowest enrichments of sialic acid, sulfate and the phosphatase. The results suggest that the plasma membranes isolated from mixed cell populations, such as those encountered in epidermal homogenates, may be resolved by free-flow electrophoresis according to cell type of origin following activity gradients present in the original tissue. Additionally, the findings provide independent biochemical confirmation of a base-to-apex gradient of transport (ATPase) activity associated with the plasma membranes of cells of the different strata of the amphibian epidermis.
采用水相两相分配和制备型自由流电泳联用的方法分离两栖类表皮的质膜。相对于总匀浆，通过两相分配获得的组分中，一种受钾离子刺激、对哇巴因敏感的对硝基苯磷酸酶的含量富集了40倍，并且基于形态学分析，这些组分代表了所有表皮细胞的分离物。小的黏膜颗粒和黏蛋白聚集体是主要的污染物。基于标记酶的活性，线粒体、高尔基体和内质网的含量较低（为总匀浆的0.15倍）或不存在。当对通过水相两相分配分离得到的质膜进行制备型自由流电泳时，它们在一系列净负电荷、唾液酸含量以及受钾离子刺激、对哇巴因敏感的对硝基苯磷酸酶的比活性不断增加的组分中向阳极移动，这让人联想到从细胞化学研究中观察到的青蛙表皮从基部到顶端的活性梯度。最靠近阳极且在主要蛋白质峰左侧的最具电负性的组分中，硫酸基团和角质层的厚膜都有所富集。一个向阳极迁移较少且在主要蛋白质峰右侧的组分含有半桥粒，同时唾液酸、硫酸盐和磷酸酶的富集程度最低。结果表明，从混合细胞群体（如表皮匀浆中遇到的细胞群体）中分离得到的质膜，可能会根据原始组织中存在的活性梯度，通过自由流电泳按照细胞来源类型进行分离。此外，这些发现为两栖类表皮不同层细胞的质膜相关的从基部到顶端的转运（ATP酶）活性梯度提供了独立的生化证实。
