Wittrup K D, Bailey J E
Department of Chemical Engineering, California Institute of Technology, Pasadena 91125.
Cytometry. 1988 Jul;9(4):394-404. doi: 10.1002/cyto.990090418.
A novel assay of single-cell exogenous beta-galactosidase activity in Saccharomyces cerevisiae has been developed. Intracellular fluorescence due to the hydrolysis of resorufin-beta-D-galactopyranoside attains a steady state between production of resorufin and its subsequent leakage from the cell. The cells are permeabilized with Triton X-100, and the assay is performed at 0 degrees C. These conditions were chosen to minimize intercellular fluorescence communication. Free resorufin in the extracellular space is bound by bovine serum albumin to prevent its uptake by cells. Two regimes of fluorescence accumulation are observed, one limited by the rate of diffusion of substrate into the cell, and one limited by the rate of enzymatic cleavage of the substrate. A quantitative correlation between fluorescence and beta-galactosidase activity is obtained under optimized assay conditions.
已开发出一种用于测定酿酒酵母单细胞中外源β-半乳糖苷酶活性的新方法。由于试卤灵-β-D-吡喃半乳糖苷水解产生的细胞内荧光在试卤灵产生及其随后从细胞中泄漏之间达到稳定状态。用Triton X-100使细胞通透，并在0℃下进行测定。选择这些条件以尽量减少细胞间荧光通讯。细胞外空间中的游离试卤灵与牛血清白蛋白结合，以防止其被细胞摄取。观察到两种荧光积累模式，一种受底物扩散到细胞中的速率限制，另一种受底物酶促裂解的速率限制。在优化的测定条件下，荧光与β-半乳糖苷酶活性之间获得了定量相关性。
