A radiometric assay for ganglioside sialidase applied to the determination of the enzyme subcellular location in cultured human fibroblasts.
A radiometric assay for ganglioside sialidase applied to the determination of the enzyme subcellular location in cultured human fibroblasts.
复制标题
神经节苷脂唾液酸酶的放射测定法应用于确定培养的人成纤维细胞中酶的亚细胞位置。
DOI：
10.1016/0003-2697(86)90094-1
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发表时间：
1986
影响因子：
2.9
通讯作者：
G. Tettamanti
中科院分区：
文献类型：
--
作者：
V. Chigorno;G. Cardace;M. Pitto;S. Sonnino;R. Ghidoni;G. Tettamanti
建立了一种测定培养的人成纤维细胞中神经节苷脂唾液酸酶的放射性方法。以高放射性（1.28 Ci/mmol）长链C-3碳氚标记的神经节苷脂GD 1a为底物，通过计算机辅助放射色谱扫描测定释放的和TLC分离的[3 H] GM 1。在提供低且相当可接受的变异系数（4-5%）的实验条件下，该方法的检测限为0.1 nmol释放的GM 1，使用低至10 μg的成纤维细胞匀浆作为蛋白质。采用分析误差较大（变异系数大于10%）的条件时，检测限可降至0.02-0.03 nmol。在75 cm 2塑料烧瓶中培养的人成纤维细胞中神经节苷脂唾液酸酶的含量为5.8 × 2.5（SD）nmol释放的GM 1h − 1 mg蛋白−1。对成纤维细胞匀浆进行的亚组分研究表明，神经节苷脂唾液酸酶主要与富含质膜和细胞内膜的轻膜亚组分有关。该亚组分对人工底物4-甲基伞形酮基-d-N-乙酰神经氨酸几乎没有唾液酸酶活性。一个小的，但可测量的神经节苷脂唾液酸酶活性也存在于富含溶酶体的亚组分，其中含有一个非常高的唾液酸酶活性的上述人工基板。所有这些都支持人成纤维细胞含有具有不同亚细胞位置和底物特异性的唾液酸酶的假设。特别地，作用于神经节苷脂的唾液酸酶似乎具有两个亚细胞位置的位点，一个主要位点在质膜和/或与质膜功能相关的细胞内细胞器水平，一个次要位点在溶酶体中。
A radiometric method for the assay of ganglioside sialidase in cultured human fibroblasts was set up. As substrate, highly radioactive (1.28 Ci/mmol) ganglioside GD1aisotopically tritium-labeled at carbon C-3 of the long chain base was employed; the liberated, and TLC separated [3H]GM1was determined by computer-assisted radiochromatoscanning. Under experimental conditions that provided a low and quite acceptable (4–5%) coefficient of variation, the detection limit of the method was 0.1 nmol of liberated GM1, using as low as 10 μg of fibroblast homogenate as protein. The detection limit could be lowered to 0.02–0.03 nmol, adopting conditions that, however, carried a higher analytical error (coefficient of variation over 10%). The content of ganglioside sialidase in human fibroblasts cultured in 75-cm2plastic flasks was 5.8 ∓ 2.5 (SD) nmol liberated GM1h−1mg protein−1. Subfractionation studies performed on fibroblast homogenate showed that the ganglioside sialidase was mainly associated with the light membrane subfraction that was rich in plasma and intracellular membranes. This subfraction displayed almost no sialidase activity on the artificial substrate 4-methylumbelliferyl-d-N-acetylneuraminic acid. A small but measurable ganglioside sialidase activity was also present in the lysosome-enriched subfraction, which contained a very high sialidase activity on the above artificial substrate. All this supports the hypothesis that human fibroblasts contain sialidases with different subcellular location and substrate specificity. Particularly, the sialidase acting on gangliosides seems to have two sites of subcellular location, a major one at the level of plasma membranes and/or intracellular organelles functionally related with the plasma membranes and a minor one in the lysosomes.
肼解后鞘糖脂唾液酸和 N-乙酰己糖胺残基的特异性 14C 标记。
影响因子：
2.9
作者：
Higashi,H;Basu,S
通讯作者：
Basu,S