A quantitative assay of beta-galactosidase activity in single cells of Saccharomyces cerevisiae has been developed using a fluorogenic substrate and flow cytometry [reported in Wittrup and Bailey, Cytometry, 9, 394 (1988)]. The beta-galactosidase activity is expressed in yeast from the Escherichia coli lacZ gene under the control of the yeast GAL10 promoter, and is used as a marker for multicopy plasmid content. A nonfluorescent fluorogenic substrate is enzymatically cleaved by intracellular beta-galactosidase to form a fluorescent product. The accumulation of fluorescent product in single cells was found to depend on bulk substrate concentration and single-cell enzyme activity in a fashion that could not be described by a Michaelis-Menten kinetic rate form. It has been demonstrated that diffusion limitation rather than enzyme activity can determine the level of single-cell fluorescence under certain assay conditions, and a mathematical model has been formulated which accounts for substrate and product diffusion. Guided by the mathematical model, the assay conditions were modified to allow measurement of single-cell enzyme activity rather than diffusion rates.
显示更多 [+] 显示较少 [-]
AGRIS - 国际农业科技情报系统
Share
页码
v.525-532(5)
其它主题
Actividad enzimatica; Modelos matematicos; Activite enzymatique; Modele mathematique
语言
英语
注释
references. AVAILABILITY: US (DNAL 381 J8224).
类型
Journal Article; Journal Part
来源
Biotechnology-and-bioengineering (USA). (5 Mar 1990). v. 35(5) p. 525-532.
团体作者
University of Illinois, Urbana, IL
Fertilizantes de Costa Rica, San Jose (Costa Rica).
2012-11-15
AGRIS AP