CHARACTERIZATION OF UPTAKE AND HYDROLYSIS OF FLUORESCEIN DIACETATE AND CARBOXYFLUORESCEIN DIACETATE BY INTRACELLULAR ESTERASES IN SACCHAROMYCES-CEREVISIAE, WHICH RESULT IN ACCUMULATION OF FLUORESCENT PRODUCT
CHARACTERIZATION OF UPTAKE AND HYDROLYSIS OF FLUORESCEIN DIACETATE AND CARBOXYFLUORESCEIN DIACETATE BY INTRACELLULAR ESTERASES IN SACCHAROMYCES-CEREVISIAE, WHICH RESULT IN ACCUMULATION OF FLUORESCENT PRODUCT
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DOI：
10.1128/aem.61.4.1614-1619.1995
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发表时间：
1995-04-01
影响因子：
4.4
通讯作者：
ABEE, T
中科院分区：
文献类型：
--
作者：
BREEUWER, P;DROCOURT, JL;ABEE, T
流式细胞术是一种快速、灵敏的方法，可用于食品和饮料中微生物的检测。该方法的关键要求是靶细胞的充分荧光染色。研究了荧光素二乙酸酯（FDA）和5-（和6-）羧基荧光素二乙酸酯（cFDA）对酵母菌的染色机理。的prefluorochromes的摄取率增加成正比的浓度，是不饱和的，这表明，运输发生通过被动扩散过程。cFDA的渗透系数为1.3 x 10（-8）m s（-1）。一旦进入细胞，酯被细胞内酯酶水解，其荧光产物积累。细胞提取物中FDA水解（40 ℃）可用一级反应动力学描述，计算出速率常数（K）为0.33 s（-1）。通过Michaelis-Menten动力学描述细胞提取物中cFDA的水解（在40 ℃下），表观V-max和K-m为12.3 nmol。min（-1）。mg蛋白（-1）和0.29 mM。荧光素的蓄积最有可能受到酯酶活性的限制，因为FDA的转运速度快于水解速率。相比之下，羧基荧光素的积累受到cFDA通过细胞包膜的慢得多的转运的限制。一个简单的数学模型来描述荧光染色。FDA和cFDA的酵母细胞的最佳染色的影响进行了讨论。
Flow cytometry is a rapid and sensitive method which may be used for the detection of microorganisms in foods and drinks. A key requirement for this method is a sufficient fluorescence staining of the target cells. The mechanism of staining of the yeast Saccharomyces cerevisiae by fluorescein diacetate (FDA) and 5- (and 6-)carboxyfluorescein diacetate (cFDA) was studied in detail. The uptake rate of the prefluorochromes increased in direct proportion to the concentration and was not saturable, which suggests that transport occurs via a passive diffusion process. The permeability coefficient for cFDA was 1.3 x 10(-8) m s(-1). Once inside the cell, the esters were hydrolyzed by intracellular esterases and their fluorescent products accumulated. FDA hydrolysis (at 40 degrees C) in cell extracts could be described by first-order reaction kinetics, and a rate constant (K) of 0.33 s(-1) was calculated. Hydrolysis of cFDA (at 40 degrees C) in cell extracts was described by Michaelis-Menten kinetics with an apparent V-max and K-m of 12.3 nmol . min(-1). mg of protein(-1) and 0.29 mM, respectively. Accumulation of fluorescein was most likely limited by the esterase activity, since transport of FDA was faster than the hydrolysis rate. In contrast, accumulation of carboxyfluorescein was limited by the much slower transport of cFDA through the cell envelope. A simple mathematical model was developed to describe the fluorescence staining. The implications for optimal staining of yeast cells with FDA and cFDA are discussed.