Microassays for a set of enzymes in individual small marine copepods

Abstract

Fluorogenic assays for a set of five hydrolytic enzymes involved in digestion and food utilization (alanine and arginine aminopeptidase, lipase/ esterase, chitobiase, and beta-glucosidase) were optimized to measure activities of these enzymes in the same extracts of individual small North Sea copepods. The enzyme activities of Acartia clausi, Centropages typicus, Corycaeus anglicus, Paracalanus parvus, and Temora longicornis showed distinct species specific activity patterns, but also high intra-specific variability. Protein, lipids, carbon and nitrogen (C, N) were determined with micro-scale assays in individual copepods or in batches of 10 to 50 animals. Water soluble protein contents ranged from 16% to 38%, and lipid contents from 2.4% to 5.5% of dry mass. The molar C/N ratios were between 4.1 and 4.5. The presented microassays provide suitable tools for studying physiological reactions of copepods and other small pelagic crustaceans in response to variable environmental conditions.

Physiological response of adult Antarctic krill, Euphausia superba, to long-term starvation

“…Total esterases (carboxylic ester hydrolases; EC 3.1.1.) were measured as described by Knotz et al (2006), and activity of lipases (glycerol ester hydrolases; EC 3.1.1.) was determined according to Prim et al (2003).…”

Digestive enzymes of two brachyuran and two anomuran land crabs from Christmas Island, Indian Ocean

“…lipase, esterase and N-acetyl-β-d-glucosaminidase activities were measured in digestive fluid using the respective substrates 4-methylumbelliferyl oleate (75164, Fluka), 4-methylumbelliferyl butyrate (19362, Sigma) and 4-methylumbelliferyl N-acetyl-β-d-glucosaminide (M2133 Sigma) as per the method of Knotz et al (2006) ( Table 2). These assays measure the continuous release of (10 mg ml −1 ) linton and greenaway (2004) the fluorescent molecule 4-β-methylumbelliferone (MUF) from the hydrolysis of the substrates.…”

NanoDrop fluorometry adopted for microassays of proteasomal enzyme activities

“…This may lead to a better scientific exploration of rare drugs. Similarly, reducing the assay volume may also allow enzymatic analysis within small medical biopsy samples, cell culture suspensions, and very small organisms and the parts thereof [8,9].…”

“…A small amount of extract (50-100 ll) is more than sufficient to run all three proteasomal enzyme assays, including controls and effectors, and to quantify protein as well. Moreover, the method enables fluorogenic enzyme assays to be conducted on small individual animals, which might be important in environmental, ecophysiological, or ecotoxicological studies [8].…”

“…This may lead to a better scientific exploration of rare drugs. Similarly, reducing the assay volume may also allow enzymatic analysis within small medical biopsy samples, cell culture suspensions, and very small organisms and the parts thereof [8,9].In our laboratory, we have developed NanoDrop fluorometry for routine measurements of enzyme activities within small samples. For example, activities of digestive enzymes have been measured in small marine invertebrates, and proteolytic enzymes have been measured in the muscle tissues from individual lobster larvae.…”