Direct Measurement of Acetylesterase in Living Protist Cells

Abstract

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 in Bacillus 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 10-5 M. Eserine (10-5 M) and Paraoxon (10-7 M) inhibited B. megaterium enzyme. Sodium acetate at 10-2 M did not inhibit bacterial enzyme. The implications of these findings on the location and expression of esterase activity in living cells are discussed.Esterases have been identified in bacteria (3, 33, 36), in bacterial spores (25,26,34), in fungi (5, 22, 23), in algae (27), and in protozoa (1,11, 19). The enzymes were identified in cell extracts by gel electrophoresis, kinetic assays, and cytochemical localization by light and electron microscopy. The use of a multitude of substrates makes it difficult to compare the enzyme activities of various organisms.Rotman and Papermaster (29) demonstrated the uptake and enzymatic hydrolysis of fluorescein esters by living animal cells. The released fluorescein was concentrated inside the cell, rendering it visible by fluorescence microscopy. This phenomenon was termed fluorochromasia. An eyepiece photometer was used to measure the evolved fluorescein, which followed Michaelis-Menten kinetics. The adjective fluorogenic was applied to this process. Fluorescein diacetate (FDA) was I Presented at the 17th annual meeting of the Canadian Society of Microbiologists, Hamilton, Ontario, June 1967. found to be the best fluorogenic substrate. The same substrate was used by Roberts and Rosenkrantz (26) to demonstrate acetylesterase (EC 3.1.1.6.) activity in Bacillus coagulans cells and spores by a fluorogenic assay, but no report of fluorochromasia was made.This communication is concemed with the search f...

Fluorescein Diacetate Hydrolysis as a Measure of Total Microbial Activity in Soil and Litter

“…Earlier studies have shown that all fungi investigated (10,14), most bacteria (8, 10), and some protozoa and algae (10) exhibit FDA hydrolytic activity. FDA has also been used in studies of the enzymatic activities of mammalian cells (12).…”

“…For all determinations of FDA hydrolytic activity, the sample (soil, straw, bacterial or fungal suspension) was simultaneously added with FDA (final concentration, 10 ,ug/ml) to sterile 60 mM sodium phosphate buffer, pH 7.6, and the mixture was incubated at 24°C on a rotary shaker. The buffering capacity was sufficient to keep the pH at 7.6 for the duration of the experiments.…”

ALGAL ASSEMBLAGES IN ANTARCTIC PACK ICE AND IN ICE‐EDGE PLANKTON1

“…Freshly collected material was examined during the cruise using epifluorescent microscopy. Several ice samples were exarnined using fluorescein diacetate as an indicator of cell viability (Medzon and Brady 1969).…”

Fluorescein diacetate hydrolysis as a measure of microbial activity in aquatic systems: Application to activated sludges

“…This measure involves reactions that agree with most of the expected properties for such methods: -sites where reactions occur along metabolic pathways are, in most cases, well known (2); -as a consequence of their connection with the electron transport system, the reactions are correlated with the respiration rate (3,4), even under anaerobic conditions. Hydrolysis of fluorescein diacetate (FDA) was first applied to environmental studies for enumeration of procaryotic and eucaryotic microorganisms in soil (5,6). It has also been used to study microbial metabolic activity in soil (7,8).…”