Extracellular phosphatase activity of natural plankton studied with ELF97 phosphate: fluorescence quantification and labelling kinetics.
ELF(R)97 phosphate (ELFP) is a phosphatase substrate which produces ELF(R)97 alcohol (ELFA), a fluorescent water-insoluble product, upon hydrolysis. We studied the kinetics of ELFA precipitation in freshwater samples at levels of total plankton and single phytoplankton cells, and tested the suitability of ELFP for measurement of surface-bound algal extracellular phosphatases. Samples from acidic Plesné Lake (pH approximately 5; high phosphatase activity) and eutrophic Rímov reservoir (pH approximately 7-10; moderate phosphatase activity) were incubated with ELFP for 5-300 min, fixed with HgCl2 and filtered through polycarbonate filters. Relative fluorescence of filter-retained ELFA precipitates was quantified with image analysis. Time-courses of ELFA formation exhibited lag periods followed by finite periods of linear increase. In Plesné Lake, lag-times were shorter (1-18 min) and rates of increase in ELFA fluorescence higher (by approximately 2 orders of magnitude) than in Rímov reservoir (lag-times 30-200 min). Similar patterns of ELFA formation kinetics were also observed in Plesné Lake samples in cuvette spectrofluorometer measurements (which failed in Rímov reservoir). Linear regression of seasonal data on rates of increase in ELFA fluorescence from image cytometry and spectrofluorometry (r2 = 0.65, n = 10) allowed for calibration of image cytometry in terms of amount of cell-associated ELFA. Preliminary measurements of extracellular phosphatase activities of several algae resulted in rates (10-2260 fmol cell-1 h-1) which are comparable to data reported in the literature for algal cultures.
Nedoma
Jirí
J
Hydrobiological Institute, Academy of Sciences of the Czech Republic, Na sádkách 7, CZ-370 05 Ceské Budejovice, Czech Republic. nedoma@hbu.cas.cz
Strojsová
Alena
A
Vrba
Jaroslav
J
Komárková
Jaroslava
J
Simek
Karel
K
D018153:Czech Republic; D005456:Fluorescent Dyes / Q000378:metabolism*; D005618:Fresh Water; D007091:Image Processing, Computer-Assisted; D009943:Organophosphorus Compounds / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010839:Phytoplankton / Q000201:enzymology*; D010933:Plankton / Q000201:enzymology*; D011799:Quinazolines / Q000378:metabolism*; D052999:Quinazolinones; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D009943:Organophosphorus Compounds; D011799:Quinazolines; D052999:Quinazolinones; C079177:2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone; D010744:Phosphoric Monoester Hydrolases