Quantitation of β-Galac- tosidase from Yeast Cells Using a Chemilumines- cent Substrate

Studies on the regulation of gene expression have frequently used the βgalactosidase (β-gal) coding sequence (lacZ) from Escherichia coli as a reporter gene. A more recent application for lacZ gene fusions has been to detect and analyze protein-protein interactions using the yeast two-hybrid system (4). The chromogenic β-gal substrate 5bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) is often used for the sensitive but only semiquantitative colorimetric detection of enzyme-producing yeast cell colonies growing on agar plates (1,7). However, for the quantitative determination of β-gal activity from liquid yeast cultures, colorimetric assays using o-nitrophenyl-β-D-galactopyranoside (ONPG; References 6,10 and 12) or chlorophenol red-β-D-galactopyranoside (CPRG; References 3 and 14) as chromogenic substrates are commonly used. These assays offer poor sensitivities with detection limits of approximately 1 ng and 100 pg of enzyme, respectively (3,9). Additionally, they are time-consuming, as it takes up to 4 h from cell extracts to final results. It has been reported that as little as 2 fg of β-gal can be detected with an assay incorporating the chemiluminescent substrate 3-(4-methoxyspiro{1,2dioxetane-3,2′-tricyclo[3,3,1,13,7]decan}-4-yl)phenyl-β-D-galactopyranoside (AMPGD; References 2 and 9). Besides an increase in sensitivity, this assay offers a superior dynamic range. The chemiluminescent signal ranges over four orders of magnitude, while the conventional quantitation methods have dynamic ranges of less than two orders (9). Furthermore, the AMPGD assay is easy to use and much more rapid to perform, leading from extracts to results in less than 1 h. Thus, for the quantitation of β-gal from transfected mammalian cells, chromogenic substrates have been widely replaced by AMPGD. In yeast however, the colorimetric assays are still the only established quantitation methods for β-gal. In our laboratory, a chemiluminometric assay originally developed for the quantitation of β-gal from mammalian cells was modified (2,9) in a way that makes it applicable to yeast cells. It has been optimized for Saccharomyces cerevisiae reporter strains that