Isolation, purification and partial characterization of chloragocytes from the earthworm species Lumbricus terrestris
Isolation, purification and partial characterization of chloragocytes from the earthworm species Lumbricus terrestris
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DOI：
10.1023/a:1006882207581
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发表时间：
1998-08-01
影响因子：
4.3
通讯作者：
Nadeau, D
中科院分区：
文献类型：
--
作者：
Affar, EB;Dufour, M;Nadeau, D
从蚯蚓中分离出绿原细胞。经过机械解离和Percoll沉降后，获得了高度纯化的活绿红细胞级分。分离的绿红细胞积累了活性染料中性红，并减少了四唑染料MTT，从而表明细胞的完整性。飞行时间流式细胞仪分析显示，在30-33 μ m的大小范围内的大型和高度颗粒化的细胞的主要群体。水解酶的测定结果表明，β-D-N-乙酰氨基葡萄糖苷酶和酸性磷酸酶表现出最高的活动（146.6和24.9 mU/mg蛋白质，分别），可能表明这2个水解酶的主要作用，在绿细胞的生理功能。相比之下，其他酸性水解酶如β-D-半乳糖苷酶和β-D-葡糖醛酸糖苷酶的比活性分别比氨基葡糖苷酶低26倍和182倍。膜结合碱性磷酸酶的比活性与其酸性对应物（分别为18.9和24.9 mU/mg蛋白质）相当，这种活性水平可能显示出在绿原细胞中重要的跨膜活性。细胞质和线粒体酶异柠檬酸脱氢酶的活性水平与仅细胞质酶乳酸脱氢酶的活性水平相当（分别为6.6和8.1 mIU/mg蛋白质）。当L.在Percoll上分离了陆生绿细胞匀浆，结果表明水解酶和淀粉酶主要与Percoll层上残留的较轻物质有关。尽管如此，在富集的氯小体组分中检测到酸性磷酸酶和β-半乳糖苷酶的总回收活性的显著比例（15-25%）支持了一些氯小体可能是“溶酶体样”细胞器的观点。
Chloragocytes were isolated from the earthworm species Lumbricus terrestris. After mechanical dissociation and sedimentation through Percoll, a highly purified fraction of viable chloragocytes was obtained. The isolated chloragocytes accumulated the vital dye neutral red and reduced the tetrazolium dye MTT, thereby indicating cellular integrity. Time of flight flow cytometric analyses revealed a main population of large and highly granulated cells in the 30-33 mu m size range. Hydrolase measurements showed that beta-D-N-acetyl-glucosaminidase and acid phosphatase exhibited the highest activities (146.6 and 24.9 mU/mg of protein, respectively), possibly indicating a major role for these 2 hydrolases in the physiological function of chloragocytes. In contrast, other acid hydrolases such as beta-D-galactosidase and beta-D-glucuronidase had specific activities of respectively 26 and 182 times lower than the glucosaminidase. The specific activity of the membrane-bound alkaline phosphatase was comparable to that of its acid counterpart (18.9 vs. 24.9 mU/mg of protein, respectively) and this level of activity may show an important trans-membrane activity in chloragocytes. The cytoplasmic and mitochondrial enzyme isocitrate dehydrogenase had a level of activity comparable to that of the exclusively cytoplasmic enzyme lactate dehydrogenase (6.6 vs. 8.1 mIU/mg of protein, respectively). When L. terrestris chloragocyte homogenates were separated on Percoll, results showed that hydrolases and dehydrogenases were mainly associated with the lighter materials that remained above the Percoll layer. Nonetheless, the detection of significant proportions (15-25%) of the total recovered activity of acid phosphatase and beta-galactosidase in the enriched chloragosome fraction supports the notion that some chloragosomes may be 'lysosome-like' organelles.