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Digestive enzyme activity as a quantitative measure of protistan grazing: the acid lysozyme assay for bacterivory

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  • We propose quantification of the activity of digestive enzymes as a novel way to estimate rates of protist grazing in natural waters. Our first application of this approach was determination of protistan bacterivory by assaying the activity of lysozyme at acid pH. Lysozyme specifically degrades peptidoglycan, a major structural component of prokaryotic cell walls. The basis of the method is determination of lysozyme activity present in protistan food vacuoles by using a fluorochrome-linked artificial substrate, 4-methylumbelhferyl β-D-N,N',N"-triacetylchitotriose (MUF-[GlcNAc]₃ as an analogue of peptidoglycan. Measurement of rate of MUF cleavage from the substrate in sonicated samples at acid pH (4.5) distinguishes activity of digestive enzymes present in protistan food vacuoles from extracellular or intracytoplasmic lysozyme activity. Acid lysozyme activity was calibrated against rate of bacterivory estimated using the fluorescently labeled bacteria (FLB) uptake method. Results from the 2 methods were significantly correlated (r² = 0.98) for both cultures of bacterivorous protists and for estuarine and nearshore seawater samples, over a wide range of rates of bacterivory (10³ to 10^6 bacteria /(ml h)) The relation between the 2 variables determined from water samples taken in open North Pacific gyre water had a higher slope compared to that of the other samples. The advantages of the acid lysozyme activity method are that it does not require in vivo incubations, manipulation of live samples, or microscopy, as do other current methods of estimating bacterivory, and that a large number of discrete samples can be quickly processed. Separate calibration of the assay, using alternate measures of bacterivory, is recommended for individual applications.
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  • Gonzalez, J. M., Sherr, E. B., & Sherr, B. F. (1993). Digestive enzyme activity as a quantitative measure of protistan grazing: the acid lysozyme assay for bacterivory. Marine Ecology Progress Series, 100, 197-206.
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  • 100
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