Ribonuclease A RNase

A prototype assay for human IgG has been developed by Ullman and Maggio (1980), with a detectability of IQ- M IgG (15 ng/ml). A serious problem with RNase is its ubiquitous nature, causing frequent interference and limiting its practical utility. 

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The squaraine probe 9g was tested for its sensitivity to trace the formation of protein-lipid complexes [57]. The binding of dye 9g to model membranes composed of zwitter-ionic lipid phosphatidylcholine (PC) and its mixtures with anionic lipid cardiolipin (CL) in different molar ratios was found to be controlled mainly by hydrophobic interactions. Lysozyme (Lz) and ribonuclease A (RNase) influenced the association of 9g with lipid vesicles. The magnitude of this effect was much higher... 

We found that solutions of hen egg white lysozyme, bovine ribonuclease A (RNase A), or a 1 2 mol ratio of bovine carbonic anhydrase lysozyme formed opaque gels within 2 min when mixed with an equal volume of 20% NBF.25,26 Multi-protein tissue surrogates comprised of 50% w/v lysozyme and up to four additional proteins have also been formed (Fowler et al., unpublished results). After overnight fixation, the surrogates were firm and sliced easily with a razor blade for sampling. To determine the optimal... 

An example of this effect is provided by ribonuclease A (RNase A). At pH 8 and 37°, the rate of deamidation of Asn67 was more than 30-fold lower in the native than in the unfolded protein [111]. Deamidation of the native RNase A was also ca. 30-fold slower than of an octapeptide whose sequence is similar to that of the deamidation site, although the reaction mechanisms were similar [108][123],... 

Ribonuclease A (RNase A) was selected as the target enzyme for solid-phase synthesis because its sequence was known (Scheme S), 22 25 and an X-ray structure had been deduced. 24 Importantly, it had been shown that this 124-residue protein could be reduced and unfolded and then reoxidized to re-form the four disulfide bonds with recovery of full enzymatic activity. 25 ... 

Ribonuclease A (RNase A) is dissolved at 1 mg/ml in water, and stored at -20°C. To inactivate the possible contaminated DNase completely, RNase A solution is sometimes boiled once when prepared. 

Ribonucleases are a widely distributed family of en-zymes that hydrolyze RNA by cutting the P—O ester bond attached to a ribose 5 carbon (fig. 8.12). A good representative of the family is the pancreatic enzyme ribonuclease A (RNase A), which is specific for a pyrimidine base (uracil or cytosine) on the 3 side of the phosphate bond that is cleaved. When the amino acid sequence of bovine RNase A was determined in 1960 by Stanford Moore and William Stein, it was the first enzyme and only the second protein to be sequenced. RNase A thus played an important role in the development of ideas about enzymatic catalysis. It was one of the first enzymes to have its three-dimensional structure elucidated by x-ray diffraction and was also the first to be synthesized completely from its amino acids. The synthetic protein proved to be enzymatically indistinguishable from the native enzyme. 

MATERIALS. Ribonuclease A (RNase A) (bovine pancreas), cytochrome C (Cyt. C) (horse heart), lysozyme (chicken egg white), myoglobin (sperm whale), P-lactoglobulin A... 

Fig. 2. Temperature dependence of the partial specific heat capacity for pancreatic ribonuclease A (RNase), hen egg-white lysozyme (Lys), sperm whale myoglobin (Mb), and catalase from Thermus thermophilus (CTT). The flattened curves are for RNase and Lys with disrupted disulfide cross-links and for apomyoglobin, when polypeptide chains have a random coil conformation without noticeable residual structure (Privalov et al., 1988).

Emulsion activity (EA) of ribonuclease A (RNase A, 10 mg mL 1) has been shown to be enhanced by glycation with glucose-6-phosphate (G6P), using capillary electrophoresis to follow the changes in RNase A.506 A cluster of about 20 peaks formed and migrated more slowly than RNase A monomer. The area of the cluster increased with temperature and with G6P concentration. EA decreased with the time of incubation in the absence of G6P. In its presence, EA reached a maximum with time of incubation, being highest for 60 mM G6P after 96 h at 30 °C and 18-24 h at 40 °C. 

Two potentially cytotoxic proteins were isolated in this manner, bovine pancreatic ribonuclease A (RNase A) and a restriction enzyme from Haemophilus parainfluenzae (Hpa ) (55). A naturally occurring cysteine residue close to the... 

The reaction of H atoms with bovine pancreatic ribonuclease A (RNAse A) has been studied by steady-state /-radiolysis of lipid vesicle suspensions containing RNAse A. The inactivation of RNAse A caused by interaction of H atoms with protein involved selective attack on methionine residues and was connected with release of diffusible thiyl radicals [reaction (36)] ... 

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) ... 

Bovine pancreatic ribonuclease A (RNase A RNA depolymerase EC 3.1.27.5) is a distributive endoribonuclease that catalyzes the cleavage of the P-O5 bond of RNA on the 3 side of pyrimidine residues. RNase A binds to polymeric substrates (Imura et al., 1965 Irie et al., 1984 Moussaoui et al., 1995), but the mechanism by which RNase A locates a pyrimidine residue within a polymeric substrate is not known. 

The previously discussed enzymes may all be used for solid-phase AM assays. For homogeneous AM-type EIA, lysozyme, malate dehy-rogenase (MDase), glucose-6-phosphate dehydrogenase (GPDase), and ribonuclease A (RNase A), are used in addition to BGase. 

DSC studies of thermal denaturation studies of ribonuclease A (RNase A) showed a decrease in T values in ILs with various cations and anions as follows [11] ... 

In 1970, Eckstein and co-workers reported the first stereochemical study of an enzyme-catalyzed hydrolysis of a phosphate ester, the hydrolysis of the endo isomer of uridine 2, 3 -cyclic phosphorothioate (enrfo-cyclic UMPS) (72) by ribonuclease A (RNase A) 13). The hydrolysis of RNA catalyzed either by base or by RNase A proceeds by a two-step mechanism in which the 2 -hydroxyl group of a nucleotide unit within an RNA molecule acts as a nucleophile on the 3 -phosphodiester bond to displace the 5 -hydroxyl group of the neighboring nucleoside to form a 2, 3 -cyciic phosphate intermediate. RNase A then catalyzes the hydrolysis of this cyclic phosphate, mimicked by Eckstein s endo-cyclic UMPS, to yield the ultimate 3 -mononucleotide product. 

Breslow and co-workers have studied the mechanism of imidazole-catalyzed hydrolysis of both cyclic phosphate esters and of RNA (67-72). These studies are directed toward a more detailed understanding of the mechanism of the hydrolysis of RNA catalyzed by ribonuclease A (RNase A). In particular, recent studies by Breslow and co-workers have addressed the interesting and enzymologically pertinent question of the origin of the bell-shaped dependence of hydrolytic rate constant on pH in both enzymic and nonenzymic reactions. 

Figure 8.4 Ribonuclease A. (a) Hydrolytic reaction catalyzed by ribonuclease A (RNAse A), note that hydrolytic cleavage occurs at the 3 -s1de of a pyrimidine nucleoside (b) cartoon display structure of RNAse A (bovine pancreatic) (side view) (pdb 7rsa).

The first model is based on the ability of Au(m) to alter the response of T-cells to ribonuclease-A (RNase). RNase injected into mice normally generates, via pathway 1, the peptide composed of amino acids 74-88 in the protein sequence, which is considered to be immunodominant and stimulates a T-cell response. 

Onconase (ONC), a protein (Mr ll.SkDa 104 aa) from the oocytes and early embryos of the Northern leopard frog, Rana pipiens, which shares 30% sequence identity with ribonuclease A (RNase A). This unstable protein shows also a similar three-dimensional structure as RNase A. The active site of ONC is located in the cleft of its kidney shape, and contains the catalytic triad (His °, Lys and His ) that is characteristic of the RNase A superfamily. ONC is currently in clinical trials for the treatment of cancer [E. Notomista et al.. Biochemistry 2000, 39, 8711 J. E. Lee, R. T. Raines, Biochemistry 2003, 42, 11443]. 

We will illustrate the coupling of protein and water dynamics using results of MD simulations of several systems, including native and MG states of human a-lactalbumin (HaLA) in aqueous solution [5], ribonuclease A (RNase) in dry and hydrated powders and glycerol solution [6,7], maltose-binding protein (MBP) in a hydrated powder [8], and bacteriorhodopsin (BR) in purple membrane (PM) stacks [9,10]. Our choice of specific systems has generally been made based on the availability of experimental data to which the simulation results can be closely compared. We have accordingly set up the systems so that the simulations are very similar to the experiments, both in terms of sample composition and thermodynamic state points (i.e., temperature and pressure). 

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