Exogenous alkaline phosphatase activity of algal cells determined by fluorimetric and flow cytometric detection of soluble enzyme products (4-methyl-umbelliferone, fluorescein)

Phosphate acquisition in algae is an important process in ecosystem development. To explore exogenous alkaline phosphatase activity, a laboratory culture of Chlamydomonas reinhardtii Dangeard was investigated by fluorometric and cytometric techniques. Two fluorogenic substrates, 4-methyl-umbelliferone-phosphate (MUP) and 3,6-fluorescein-diphosphate, were applied to examine induction of phosphorus regeneration as well as enzyme dynamics in P-starved cells. Fluorometric analysis revealed the absence of constitutive or secretory phosphatases but traced the induction of surface-bound exogenous phosphatases with a cellular K-m of 52 mu M MUP. In cytometric assays, single-cell phosphate acquisition was examined. Exogenous phosphatase activity was detectable from cell halos and recorded continuously as the slope on fluorescence increase and cellular steady state of fluorochrome production. An experimental time course on P-starvation indicated the induction of a phosphatase system after 4 days. The use of flow cytometry in combination with specific fluorogenic substrates is a valuable tool for fine-tuned single-cell analysis of phosphatase activity in algal communities.