Ribonuclease activity assays

Cytokine profiling has also been measured as a function of changes in cytokine mRNA expression using either reverse transcription polymerase chain reaction (RT-PCR) [87, 91-93] or ribonuclease protection assay (RPA) [94-97], Measurement of cytokine transcripts by RT-PCR revealed that prolonged exposure to TMA induced increased levels of IL-4 mRNA expression compared with treatment with DNCB [87,92-93]. However, expression of the type 1 cytokine IFN-y by DNCB-activated LNC was variable and failed to discriminate between contact and respiratory allergens [87,91,93). A similar profile was observed for freshly isolated tissue analyzed by RPA. This somewhat less... 

A wide variety of procedures have been used for assaying ribonuclease activity. Some measure only step 1 or step 2, some measure both, some use high molecular weight substrates, some low, some substrates are... 

Further investigations focused on evaluating the three steps of IFN-y-stimulated class II expression signal transduction and transcription factor activation, CIITA expression, and class II transcription in CMV-infected cells. Ribonuclease protection assays (RPA) and RT-PCR experiments reveal that neither MHC class II nor CIITA RNA is upregulated in response to IFN-y treatment in HCMV-infected ECs at 72h after infection (Miller et al. 1998). Moreover, electrophoretic mobility shift assays (EMSA) demonstrate that STAT-1 homodimers are not... 

It has been reported that a ribonuclease is induced when extracts of interferon-treated non-infected cell extracts are incubated with dsRNA and ATP (2j). Since dsRNA is present in interferon-treated mengo virus-infected cells the induction of a dsRNA dependent ribonuclease might occur. Kinetic assays for ribonuclease activity showed a marked increase in ribonuclease activity of the P-20 from interf eron-treatment, mengo virus-infected cells. Poly C, Poly U and poly A were also degraded by the enzyme, whereas (5h) replicative form of mengo virus was not degraded. Extracts from interferon-treated non-infected cells did not show enhanced ribonuclease activity compared with non-infected control cells. 

Tripathy, D. R. Dinda, A. K. Dasgupta, S. A simple assay for the ribonuclease activity of ribonucleases in the presence of ethidium bromide. Anal Biochem. 2013,437,126-129. 

Lee, J. Choi, S. Improved fluorometric assay method for ribonuclease activity. J. Biochem. Mol Biol 1997,30, 258-261. 

Cell walls, total membrane-bound components, and ribosomes were separated and assayed for cellulase activity to study the subcellular localization of the enzymes as follows. Segments (approx. 5 g fresh wt) were ground in two volumes of extraction medium containing 0.4M sucrose (ribonuclease-free), 5mM Mg acetate, lOmM Tris-HCl (pH 7.5 at 22°C), 20mM KC1 and 5mM / -mercaptoethanol. The brei was filtered and the filtrate centrifuged at 500 Xg for 20 min. The post-500 Xg supernatant was fractionated essentially as previously described (28). Aliquots (7 mL) of the supernatant were layered on a discontinuous gradient composed of 2 mL 70% (w/v) sucrose and 3 mL 15% (w/v) sucrose both in lOmM Tris-HCl (pH 7.5 at 22°C), lOmM KC1, 2.5mM Mg acetate and ImM / -mercaptoethanol. The tubes were centrifuged at... 

A simple procedure described for the detection of encarbohydrate moiety of ribonuclease B has a-D-mannopyranosyl residues as terminal non-reducing groups, but such residues are lost as part of an oligosaccharide on action of the endo -o-l-acet-amido-2-deoxyglucanase, agarose immobilized concanavalin A was used to separate radioactive substrate (absorbed) from radioactive product (unadsorbed). The assay was used to follow the preparation of the enzyme from Streptomyces plicatus. 

Pardee and co-workers (225,225a,227,227a) have investigated the activity of 9 bacterial enzymes after infection of E. coli with various T phages, i.e., apyrase, ribonuclease, deoxyribonuclease, alkaline and acid proteases, pyruvic oxidase, formic dehydrogenase, serine deaminase, and catalase. The difficulties of assay of labile enzymes in the broken cell systems are indicated by the early report of an increase in apyrase activity (225) which later was attributed to a technical defect in the... 

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