ANALYTICAL CELL FRACTIONATION OF ISOLATED RABBIT RENAL PROXIMAL TUBULES
ANALYTICAL CELL FRACTIONATION OF ISOLATED RABBIT RENAL PROXIMAL TUBULES
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DOI：
10.1038/ki.1981.106
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发表时间：
1981-01-01
影响因子：
19.6
通讯作者：
TROUET, A
中科院分区：
文献类型：
--
作者：
HJELLE, JT;MORIN, JP;TROUET, A
通过已知可保留肾小管细胞结构和代谢方面的机械程序，从兔皮质中分离出高纯度的近端肾小管。从分离的小管制备的核后上清液在线性蔗糖梯度中进行等密度离心。与质膜相关的酶活性（γ-谷氨酰转肽酶、氨肽酶M、碱性磷酸酶、Na-K-ATP酶和磷酸二酯酶I）表现出尖锐的单峰频率-密度曲线，中值密度接近1.16g/ml，当用洋地黄皂苷处理时，其转变为较重的密度。溶酶体酶、N-乙酰基-β-氨基葡萄糖苷酶、α-甘露糖苷酶和组织蛋白酶B以及过氧化物酶体酶过氧化氢酶在接近1.22g/ml的密度下表现出颗粒相关活性。通过冷冻和解冻对这些颗粒的破坏导致这些活动出现在 .rho 中。 = 1.10 g/ml 梯度区域，其中可溶性胞质酶、磷酸葡萄糖变位酶表现出活性。线粒体典型的细胞色素氧化酶活性在.rho处给出了尖锐的单峰分布。 = 1.18 克/毫升。微粒体葡萄糖-6-磷酸酶和 NADPH:细胞色素 c 还原酶活性的中值密度接近 1.16 g/ml，与洋地黄皂苷孵育后没有变化。半乳糖基转移酶活性在.rho 处呈现倾斜分布。 = 1.16 g/ml，并在洋地黄皂苷后显示出轻微的密度变化。这项对近曲小管细胞成分的酶活性和密度梯度分布的研究为进一步研究这种重要细胞类型对内源性和外源性物质的细胞加工提供了方法。
Proximal tubules were isolated in highly pure form from rabbit cortices by a mechnical procedure known to preserve the structural and metabolic aspects of the tubular cells. Postnuclear supernates prepared from the isolated tubules were subjected to isopycnic centrifugation in linear sucrose gradients. The enzyme activities associated with the plasma membrane (.gamma.-glutamyl transpeptidase, amino-peptidase M, alkaline phosphatase, Na-K-ATPase and phosphodiesterase I) exhibited sharp unimodal frequency-density profiles with a median density near 1.16 g/ml, which shifted to a heavier density when treated with digitonin. The lysosomal enzymes, N-acetyl-.beta.-glucosaminidase, .alpha.-mannosidase, and cathepsin B, and the peroxisomal enzyme catalase exhibited particle-associated activity near a density of 1.22 g/ml. Disruption of these particles by freezing and thawing resulted in these activities appearing in the .rho. = 1.10 g/ml region of the gradient where the soluble cytosolic enzyme, phosphoglucomutase, exhibited activity. Cytochrome oxidase activity typical of mitochondria gave a sharp unimodal profile at .rho. = 1.18 g/ml. Microsomal glucose-6-phosphatase and NADPH:cytochrome c reductase activities gave median densities near 1.16 g/ml, which did not change after incubation with digitonin. Galactosyl transferase activity gave a skewed profile at .rho. = 1.16 g/ml and showed a slight shift to heavier density after digitonin. This study of the enzyme activities and density gradient distribution of the components of the proximal tubule cells provides the methodology for the further study of the cellular processing of endogenous and exogenous substances by this vital cell type.