RNase, pancreatic

FIGURE 11.32 All example of nuclease specificity The specificity of RNA hydrolysis by bovine pancreatic RNase. This RNase cleaves h at 3 -pyriinidines, yielding oligonncleoddes with pyrimidine 3 -P04 ends. 

Hydrolysis of RNA by alkali or pancreatic RNase leads initially to fragments which terminate in 2, 3 -cyclic phosphodiesters. Micrococcal nuclease, on the other hand, gives rise to fragments terminating in 3 -phos-phomonoester groups which facilitate their isolation, and this enzymic hydrolysis has been used to prepare 3 -ribodinucleotides. ... 

Rait VK, Xu L, O Leary TJ, et al. Modeling formalin fixation and antigen retrieval with bovine pancreatic RNase A II. Interrelationship of cross-linking, immunore-activity, and heat treatment. Lab. Invest. 2004 84 300-306. 

Figure 15.9 The results of capture ELISA on native RNase A and formalin-treated RNase A. Right panel, native RNase A (curve 1) and unfractionated formalin-treated RNase A (curve 2). Left panel, individual fractions of formalin-treated RNase A monomer (curve 3), dimmer (curve 4), trimer (curve 5), tetramer (curve 6), and a mixture of oligomers with >5 cross-linked proteins (curve 7). The ELISA plate wells were coated with monoclonal antibody against bovine pancreatic RNase A (1 pg/mL) overnight at 4°C and then blocked with bovine serum albumin. The wells were incubated for lh at 37°C in the presence of various concentrations of antigen in lOOpL of PBS. After washing, each plate well received a 1 4000 dilution of horseradish peroxidase conjugated rabbit polyclonal anti-RNase A antibody followed by incubation at ambient temperature for lh. After washing, detection was achieved using a mixture of 2,2,-azino-di-(3-ethylbenzthiazoline-6-sulphonate) and hydrogen peroxide. Absorbance was monitored at 405 nm. See Rait etal.11 for details.

Affinity chromatography was carried out on columns prepared with lightly carboxymethylated chitin, which is known to be a poor substrate for lysozyme. Both native lysozyme and regenerated 13-105 were bound to the column at pH 7 and eluted at pH 3. As controls, the basic proteins cytochrome c and pancreatic RNase A, as well as concanavalin A and a-amylase, were not bound from the same solvent at pH 7. These findings constitute a third line of evidence for formation of native-like structure in regenerated 13-105. 

An example of enzyme depletion is the ribonuclease inhibitor isolated from human placenta by Blackburn, Wilson Moore. This protein forms a 1 1 complex with bovine pancreatic RNase A and is a noncompetitive... 

Pearson and Johns.81,138 Irradiated poly U was degraded by treatment with the enzyme pancreatic RNase. Three major photoproducts were isolated from the hydrolysis products and identified as Up, DpUp,... 

Ribonuclease EC 3.1.27.5 Hydrolysis of RNA Milk is a very rich source similar to pancreatic RNase... 

Bovine pancreatic RNase A is a member of a homologous superfamily. In addition, there is a separate family of guanine-specific microbial RNases that have evolved to have a similar active site.192,193 Ribonuclease T1 from Aspergillus oryzae and the 110-residue bamase from Bacillus amyloliquefaciens of Mr 12 392 (see Chapter 19) are the best known examples. One of the histidine residues is replaced by a glutamate in these enzymes. The microbial enzymes are much more amenable to study by protein engineering. 

Most typical RNases such as pancreatic RNase (RNase A), described in the chapter by Richards and Wyckoff, and RNases Ti, T2, Nl( and U2, which will be described in this chapter, are characterized as follows ... 

Ribonuclease U2 is a novel enzyme found in the culture broth of Ustilago sphaerogena (7, 106). Ribonuclease U2 splits, practically specifically, the phosphodiester bonds of purine nucleotides in RNA with the intermediary formation of purine nucleoside 2, 3 -cyclic phosphates, indicating the specificity is complementary to that of pancreatic RNase A (106). Like RNase N, RNase U2 very slowly hydrolyzes the intermediate, nucleoside 2, 3 -cyclic phosphate, to 3 -nucleotides (80, 106). Thus, RNase U2 is a useful tool, not only for the analysis of nucleotide sequences of RNA (90, 92, 107, 108) but also for the synthesis of various oligonucleotides containing adenylyl or guanylyl residue (30) (T. Koike, T. Uchida, and F. Egami, unpublished). 

A good source of uncommon bases is tRNA. It provides substrates for studying the effect of base on the rate of hydrolysis. Baev et al. (62) showed that V2-dimethylguanylyl-(3 -5 )-cytidine-3 phosphate (G2m-pCp) was hydrolyzed much slower than the usual GpCp. Venkstern (63) reported that Tp was hydrolyzed very slowly. Naylor et al. (64) found that Cp was hydrolyzed with half the rate of CpU. The same group of workers introduced (64, 65) a chemical block on uridine and pseudo-uridine residues by reacting RNA with l-cyclohexyl-3-(2-morpho-liny]-(4)-ethyl)-carbodiimide metho-p-toluene sulfonate. The modification of the uridine residues completely blocked the action of venom exonuclease and also blocked the action of pancreatic RNase. 

It should be pointed out that the successful purification of spleen exonuclease (11) was greatly helped by use of a DNA hydrolyzate produced by spleen acid DNase as the substrate, since the synthetic substrates are nonspecific, and RNA core (the water-undialyzable ribooligonucleo-tides obtained by exhaustive digestion of RNA with pancreatic RNase) is also hydrolyzed by both acid and basic spleen ribonucleases (38, 39). Spleen exonuclease is unable to hydrolyze cyclic phosphates (14). 

The carbohydrate moiety of RNA is D-ribose with the / -D-ribofurano-side ring. The 2 - and 3 -OH groups are cis to each other and easily form the cyclic phosphate intermediate. Although the 2 -deoxynucleotides bind to the enzyme, they only serve as inhibitors. The 2 -OH group is mandatory for the catalytic activity of pancreatic RNase. 

Surface immunoglobulin receptor of human B-cell lymphoma Tumor suppressor protein p53 mAb 5.5 against the ligand-binding site ot the nicotinic acetylcholine receptor Bovine pancreatic RNase fragment concanavalin A... 

Following such treatment, the cultured cells in monolayer are washed with phosphate-buffered saline and extracted in 4 ml 0.5% Triton X-100 in saline/EDTA (100 mM NaCl, 10 mM EDTA, pH 8.0) for 2 min at room temperature. This releases most of the cytoplasmic material whilst the nuclei remain attached to the culture dish. 0.5% sodium dodecyl sulphate and 40 //g/ml pancreatic RNAse (preincubated at 80°C for 10 min, to inactivate DNAse) in saline/EDTA (above) is then added and the mixture incubated for 20 min at 37°C. One volume of chloroform/isoamyl alcohol (20 5, v/v) is then added and the phases mixed gently. The aqueous phase is separated by centrifugation and extracted again with chloroform/isoamyl alcohol. DNA is precipitated from the aqueous phase with 2 vol 95% ethanol and resuspended in 0.01 M Tris, pH 7.5. Alkaline sucrose density gradients (5-20%) are prepared in 0.1 M NaCl, 0.1 M NaOH with a final volume of 4.1 ml. Samples of DNA (max 3 fig) are layered on the top of these gradients and spun at 32 000 rpm at 20°C in a SW.50.1 Beckman rotor for 120 min. Fractions are collected and the [3H]DNA precipitated... 

Probably not all proline residues are important for protein folding. Evidence for nonessential prolines came from a comparison of several homologous pancreatic RNases (Krebs et al., 1983, 1985) and cytochromes c (Babul et ai, 1978 Nall, 1990) that differ in the number of proline residues. Such prolines could be nonessential because they do not interfere with folding, or, alternatively, because they remain nativelike as regards isomeric state, after unfolding. 

Fig. 6. Acceleration of refolding of different proteins as a function of PPI activity. The acceleration factor is given as the ratio hlk of the observed rate constants for folding in the presence of PPI, k, and in the absence of PPI, ki,. The PPI activity is given as /f/ml. The K values are defined as described in footnote b to Table I. The following protein concentrations were used in the refolding experiments ( ) 2 /iM immunoglobulin light chain, ( ) 11 juAf porcine pancreatic RNase, and ( ) 17 fiM S-protein fragment of bovine RNase A. The final conditions for refolding were 0.25 M urea (0.30 M urea for porcine RNase), 0.1 Af Tris-HCl, pH 8.0, 10°C. Based on data from Lang el at. (1987).

Ribonuclease A (RNase) Dissolve 10 mg of pancreatic RNase A per mL of 20 mM sodium acetate (pH 5.0). Place in boiling water for 10 min. Store aliquots at -20°C. For a working solution dilute 1 100 in 2x SSC. Hybridization Mixture (HM) ... 

Table 5. Inhibition of reverse-transcriptase activity of FL-virions by distamycin derivatives in the absence of exogenous template. Virions containing Triton were preincubated at room temp, for 25 min with 50 jUg/ml of pancreatic RNase Chandra et al.6 ...

Virions containing Nonidet P-40 were preincubated at room temp. 25 min with 50 Mg/ml of pancreatic RNase. 

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