Ribonuclease assay

Marsh et al. (1983) have shown that SFV can still infect BHK-21 cells. Moreover, the release of nucleocapsid into the cytoplasm (as measured by the ribonuclease assay) proceeds, albeit 40% more slowly than at 37 C. Thin-section electron microscopy confirms that SFV particles are not transferred to lysosomes at this temperature. 

Crook EM, Mathias AP, Rabin BR. Spectrophotometric assay of bovine pancreatic ribonuclease by the use of cytidine 2, 3 -phosphate. Biochem. J. 1960 74 234-238. 

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... 

Ribonuclease Nj is most stable in neutral or acid media at 37°. It is resistant even to heating at 80° for 2 min in acidic media of pH 2-4, and is fairly unstable in alkaline medium like RNase Ti. It is not as stable as RNase Ti in higher salt concentrations and exhaustive dilution with water makes the enzyme unstable. This enzyme needs a protective protein such as gelatin or bovine serum albumin for assay. 

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... 

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... 

Surry DD, McAllister G, Meneses-Lorente G, et al. High-throughput ribonuclease protection assay for the determination of CYP3A mRNA induction in cultured rat hepatocytes. Xenobiotica 1999 29 827-838. 

With the existence of this new cyclodextrin lock, it was again important to select a key to fit it and to serve as substrate. For this we wanted a cyclic phosphate ester that this cyclodextrin bisimidazole could hydrolyze. The enzyme ribonuclease hydrolyzes cyclic phosphates as the second step in the hydrolysis of RNA, and cyclic phosphates are used as assay substrates for the enzyme. The advantage to us of such a substrate was that the geometry of the transition state would be relatively well-defined, so that it should be possible to design congruence between the catalyst and the transition state. Molecular model building indicated that a possible substrate was the cyclic phosphate derived from 4-f-butylcatechol (VIII). Indeed, the cyclodextrin bisimidazole (VII) is a catalyst for the cleavage of cyclic phosphate (VIII) (14). 

Choi WS, Machida CA, Ronnekleiv OK. 1995. Distribution of dopamine Dl, D2, and D5 receptor mRNAs in the monkey brain Ribonuclease protection assay analysis. Brain Res Mol Brain Res 31 86-94. 

Richtand NM, Kelsoe JR, Segal DS, Kuczenski R (1995) Regional quantification of Dl, D2, and D3 dopamine receptor mRNA in rat brain using a ribonuclease protection assay. Mol Brain Res 33 97-103. 

Also, HPLC methods with electrochemical or fluorescent detection are used (H19, M3). In proteins, dityrosine can be estimated by immunochemical methods employing dityrosine-specific antibodies (K5). Measurements of o,o -dityrosine and o-tyrosine levels in rat urine express dityrosine contents in skeletal muscle proteins, and have been proposed as the noninvasive oxidative stress test in vivo. One should be aware, however, that A-formylkynurenine, also formed in protein oxidation, has similar fluorescence properties as dityrosine (excitation 325 nm, emission at 400-450 nm) (G29). Also, oxidation of mellitin when excited at 325 nm produces an increase in fluorescence at 400—450 nm, despite the fact that mellitin does not contain tyrosine. Oxidation of noncontaining Trp residues ribonuclease A and bovine pancreatic trypsin inhibitor with "OH produces loss of tyrosine residues with no increase in fluorescence at 410 nm (S51). There are also methods measuring the increased hydrophobicity of oxidized proteins. Assays are carried out measuring protein binding of a fluorescent probe, 8-anilino-l-naphthalene-sulfonic acid (ANS). Increase in probe binding reflects increased surface hydrophobicity (C7). 

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