DIRECT MEASUREMENT OF ACETYLESTERASE IN LIVING PROTIST CELLS
DIRECT MEASUREMENT OF ACETYLESTERASE IN LIVING PROTIST CELLS
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DOI：
10.1128/jb.97.1.402-415.1969
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发表时间：
1969-01-01
影响因子：
3.2
通讯作者：
BRADY, ML
中科院分区：
文献类型：
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作者：
MEDZON, EL;BRADY, ML
用荧光乙酰酯酶（醋酸酯水解酶EC 3.1.1.6）底物双醋酸荧光素测定活原生细胞的酶活性。通过荧光显微镜监测荧光色差进行视酶测定。定量荧光测定是在荧光计中测定进化的荧光素。59株细菌中35株荧光染色阳性。8株荧光染色阴性菌株荧光染色阳性。22株黏菌和真菌均为荧光染色阳性。12种不同的藻类中有3种呈荧光阳性。几种未确定的原生动物也呈荧光阳性。六种原生动物中有四种荧光染色阳性。显示乙酰酯酶活性的特别有趣的结构有：真菌生长的菌丝尖端，酵母和原生动物的液泡区，新形成的细菌孢子或未成熟的真菌孢子，巨芽孢杆菌的“中粒体”，以及绿藻的细胞膜和核区。酵母原生质体、细菌原生质体和球质体来源于阳性细胞时呈荧光阳性，来源于阴性细胞时呈荧光阴性。胶囊的持有与乙酰酯酶活性之间没有相关性。在双醋酸荧光素的存在下，对细菌细胞的生存能力没有影响。对氧磷在10−5m时抑制细菌和酵母菌酶。Eserine（10−5m）和Paraoxon（10−7m）抑制了b。megateriumenzyme。乙酸钠在10−2℃时对细菌酶无抑制作用。讨论了这些发现对活细胞中酯酶活性的位置和表达的影响。
The fluorogenic acetylesterase (acetic ester hydrolase EC 3.1.1.6.) substrate, fluorescein diacetate, was used to measure enzyme activity in living protist cells. The visual enzyme assay was done by monitoring fluorochromasia by fluorescent microscopy. Quantitative fluorogenic assays were done by measuring the evolved fluorescein in a fluorometer. Of 59 strains of bacteria, 35 were fluorochromatically positive. Eight of the fluorochromatically negative strains were fluorogenically positive. Of 22 strains of slime molds and fungi, all were fluorochromatically positive. Three out of 12 different algae were fluorochromatically positive. Several unidentified protozoa were also fluorochromatically positive. Four out of six protozoa were fluorochromatically positive. Structures of special interest showing acetylesterase activity were: the growing hyphal tips of fungi, the vacuolated areas of yeast and protozoa, newly formed bacterial spores or immature fungal spores, “mesosome-like” bodies inBacillus megaterium, and the cell membrane and nuclear region of green algae. Yeast protoplasts and bacterial protoplasts and spheroplasts were fluorochromatically positive when derived from positive cells and negative when derived from negative cells. There was no correlation between the possession of a capsule and acetylesterase activity. There was no effect on the viability of bacterial cells incubated in the presence of fluorescein diacetate. Paraoxon inhibited bacterial and yeast enzyme at 10−5m. Eserine (10−5m) and Paraoxon (10−7m) inhibitedB. megateriumenzyme. Sodium acetate at 10−2mdid not inhibit bacterial enzyme. The implications of these findings on the location and expression of esterase activity in living cells are discussed.