A single‐cell assay of β‐galactosidase activity in Saccharomyces cerevisiae

Open Access
Publisher Website
Google Scholar
Abstract
A novel assay of single‐cell exogenous β‐galactosidase activity in Saccharomyces cerevisiae has been developed. Intracellular fluorescence due to the hydrolysis of resorufin‐β‐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°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 β‐galactosidase activity is obtained under optimized assay conditions.

This publication has 23 references indexed in Scilit: