Kaneski C R, French S A, Brescia M R, Harbour M J, Miller S P
Developmental and Metabolic Neurology Branch, National Institute of Neurological Disordrs and Stroke, National Institutes of Health, Bethesda, MD 20892.
J Lipid Res. 1994 Aug;35(8):1441-51.
With the goal of improving the detection of lysosomal sphingolipid hydrolases within intact cells, we have recently synthesized a new fluorophor, O-[4-(1-imidazolyl)butyl]-2,3-dicyano-1,4-hydroquinonyl beta-D-galactopyranoside (Im-DCH-beta-Gal). In the present study, we evaluated the interaction of Im-DCH-beta-Gal and its tetraacetate derivative, Im-DCH-beta-Gal(OAc)4, with living human fibroblasts. Im-DCH-beta-Gal was shown to be a specific substrate for human lysosomal beta-galactosidase in cell homogenates. Im-DCH-beta-Gal(OAc)4 was taken up and hydrolyzed by normal fibroblasts under physiological culture conditions. Very little hydrolysis of Im-DCH-beta-Gal(OAc)4 was observed in fibroblasts genetically deficient in lysosomal acid beta-galactosidase or in normal cells pretreated with the lysosomal inhibitors chloroquine and ammonium chloride. Analysis of substrate processing by cells indicated that normal and acid beta-galactosidase-deficient cells showed similar rates of uptake and deacetylation of Im-DCH-beta-Gal(OAc)4, with an 80% decrease in the rate of deglycosylation of substrate by beta-galactosidase-deficient fibroblasts. However, under our conditions, the fluorescent product was not well retained by cells. Our results indicate that this novel class of compounds may be useful in measuring lysosomal enzyme function in intact cells and may have application as a fluorescent marker for genetically altered cells.
为了提高在完整细胞内对溶酶体鞘脂水解酶的检测，我们最近合成了一种新的荧光团，即O-[4-(1-咪唑基)丁基]-2,3-二氰基-1,4-对苯二酚基-β-D-吡喃半乳糖苷（Im-DCH-β-Gal）。在本研究中，我们评估了Im-DCH-β-Gal及其四乙酸酯衍生物Im-DCH-β-Gal(OAc)4与活的人成纤维细胞的相互作用。在细胞匀浆中，Im-DCH-β-Gal被证明是人类溶酶体β-半乳糖苷酶的特异性底物。在生理培养条件下，正常成纤维细胞摄取并水解Im-DCH-β-Gal(OAc)4。在溶酶体酸性β-半乳糖苷酶基因缺陷的成纤维细胞或用溶酶体抑制剂氯喹和氯化铵预处理的正常细胞中，几乎未观察到Im-DCH-β-Gal(OAc)4的水解。细胞对底物处理的分析表明，正常细胞和酸性β-半乳糖苷酶缺陷细胞对Im-DCH-β-Gal(OAc)4的摄取和脱乙酰化速率相似，而β-半乳糖苷酶缺陷的成纤维细胞对底物的去糖基化速率降低了80%。然而，在我们的条件下，荧光产物在细胞中保留不佳。我们的结果表明，这类新型化合物可能有助于测量完整细胞中的溶酶体酶功能，并可能作为基因改变细胞的荧光标记物。
