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Characterization of proteolytic enzyme activities in macroalgae

Proteases are essential components of cells and participate in processes ranging from photoacclimation and nutrient acquisition to development and stress responses. Virtually nothing is known about this diverse group of enzymes in macroalgae. Methods to measure proteases developed for phytoplankton (caseinolysis, leucine aminopeptidase (LAP) activity and casein zymograms) have been modified to allow use with spectrophotometers as well as spectrofluorometers, so making them accessible to a wider number of users. Applying the methods to a wide range of macroalgae from intertidal zones in south-western Spain and Northern Ireland detected proteases in all species examined. Protease activities differed among species but there was little systematic variation with taxonomic group. Moreover, similar species in Spain and Northern Ireland often showed differences. With the exception of LAP activities in brown algae, protease activities were one to two orders of magnitude greater in macroalgae than those previously measured in phytoplankton, when expressed per unit protein. However, scaling of activities to protein was complicated by significant interferences. Copper-based protein assays gave erroneously high results when applied to the brown algae, and partial purification of proteins by trichloroacetic acid precipitation did not overcome the problem. The higher levels of protease activity suggest that proteolysis plays a more significant role in multicellular than in unicellular algae. Proteases were characterized in terms of activities at different pH and the effect of protease inhibitors. In common with microalgae but in contrast to findings for animals and higher plants, proteases tended to show higher activities in the neutral and alkaline ranges. Many commonly used protease inhibitors (e.g. leupeptin and phenylmethylsulphonyl fluoride) had relatively little effect, which may explain why biochemical work with macroalgal species has traditionally been difficult. Our results suggest that macroalgal proteases are easily measurable but highly variable. A major source of variability that has not been assessed is differing environmental conditions. If this is correct, measurements of proteolytic enzymes may provide a valuable tool for examining biologically relevant changes in environments. Controlled laboratory experiments and seasonal monitoring are the next logical steps towards this goal.