NanoDrop fluorometry adopted for microassays of proteasomal enzyme activities

Abstract

a b s t r a c tNanoDrop spectrophotometry and NanoDrop fluorospectrometry are used almost exclusively to determine the concentrations of nucleic acids and proteins. We propose that NanoDrop fluorospectrometry can also be applied for measuring enzyme activities using fluorogenic substrates such as the proteolytic activities of the 26S proteasome. Because the NanoDrop ND-3300 device requires only 2 ll of sample, the amount of sample extract, substrate, and cofactors used for an enzyme assay can be significantly reduced. In this report, we present exemplary microassays for proteasomal activities (chymotrypsin-, trypsin-, and PGPH [peptidyl-glutamyl peptide hydrolase]-like sites) in extracts of isolated hemocytes from a marine crab, Cancer pagurus (Crustaceae).Ó 2011 Elsevier Inc. All rights reserved.The use of highly sensitive fluorogenic substrates is becoming increasingly relevant not only in analytical and clinical enzymology but also in various other fields of life sciences, including biochemical, physiological, and biomedical research. Particularly important applications include the design of artificial substrates for use as a tool in cytology and pathology [1,2]. In this respect, the discovery of the intracellular ubiquitin-mediated proteolytic pathway and the controlled degradation of waste proteins by the multi-enzyme complex called 26S proteasome [3,4] has opened another important field for fluorometric enzyme assays [5][6][7].Fluorogenic substrates as well as enzyme effectors may be quite expensive or available in limited amounts; the latter is particularly relevant if their isolation from natural sources is laborious or they are synthesized in small amounts for specific applications. Therefore, it would be ideal if the amount of substrate used per assay could be decreased through a reduction in assay volume. 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. In this report, we describe in detail a method for measuring 26S proteasomal activities in small amounts of hemocyte extracts from crustaceans. We also demonstrate the sensitivity of this method and describe its potential applications within various fields of the life sciences.Because the NanoDrop device (ND-3300, version 2.7.0, PEQLAB Biotechnologie) requires only 1-2 ll of reaction mixture for fluorescent measurement [10], the total volume of the assay was reduced significantly.The reproducibility and linearity of the fluorescence signals were determined with the fluorophore 7-amino-4-methyl coumarin (AMC, 1 Fluka, product no. 08440). This ...

Proteasome properties of hemocytes differ between the whiteleg shrimp Penaeus vannamei and the brown shrimp Crangon crangon (Crustacea, Decapoda)

“…Besides methodological reports (Götze and Saborowski 2011;Götze et al 2013), almost no information is available about the catalytic properties of crustacean hemocyte proteasomes. However, it is crucial to understand such a cellular key process controlling several essential pathways (e.g., cell cycle, apoptosis, or differentiation) as this forms the basis for the investigation and interpretation of cellular stress responses or pathogenmediated reactions.…”

“…All assays were carried out in a NanoDrop device (Thermo Scientific, ND3300) as described previously (Götze and Saborowski 2011). In brief, the total reaction mixture of 25 μL contained 17.5 μL buffer (50 mmol L −1 Tris·HCl pH 7.5 at 30°C), 5 μL enzyme extract yielding 10 μg protein on average, 1.25 μL of substrate solution, and either 1.25 μL dimethyl sulfoxide (DMSO) or 1.25 μL of the inhibitor epoxomicin.…”

“…Epoxomicin (PeptaNova, 4381), gliotoxin (Applichem, APPA7665), lactacystin (Cayman chemical company, Cas 133343-34-7), and MG132 (Sigma, C2211) were dissolved in DMSO, aliquoted, and stored at −20°C. The final concentrations of inhibitors in the assays were epoxomicin 50 mmol L −1 (Götze and Saborowski 2011), lactacystin 1 mmol L −1 , gliotoxin 100 mmol L −1 (Kroll et al 1999), and MG132 25 mmol L −1 (Luker et al 2003). Enzymatic assays were performed as described above (n = 3).…”

“…For example, proteasome-mediated degradation was reported to be involved in the molting process of adult and juvenile lobsters (Mykles 1999;Götze and Saborowski 2011) or in the process of learning and memory of crabs (Merlo and Romano 2007). Furthermore, it has been found that shrimp, infected with the white spot syndrome virus (WSSV), may elicit an immune response directed toward the UPS to prevent the deleterious damage caused by this virus (Jarrousse et al 1999).…”

Proteasomal activities in the claw muscle tissue of European lobster, Homarus gammarus, during larval development

“…Enzyme micro-assays with the NanoDrop device Fluorometric enzyme micro-assays were carried out with a NanoDrop device as described by Götze and Saborowski (2011). The instrument requires only 2 ll of reaction mixture.…”