Ribonuclease removal

For ribonuclease, removal by pepsin proteolysis of only a tetrapeptide sequence at the C-terminus (Asp-Ala-Ser-Val) lead to an inactive enzyme and unstable structure (Anfinsen 1956 Taniuchi, 1970). When this shortened enzyme, the so-called des(121-124) ribonuclease or pepsin inactivated ribonuclease (PIR), is reduced, it cannot reoxidize to 3deld the native pairing of disulfide bonds (Taniuchi, 1970). Removal of six C-terminal residues to form RNase 1-118 also yields a structureless and inactive enzyme (Lin, 1970 Andria and Taniuchi, 1978). The importance of the C-terminal end to the folding of these two proteins was also emphasized by the data reported in Chapter 9. The information contained in the C-terminal sequence of the two nucleases appears to be crucial for their refolding. It was proposed that the polypeptide chain of nuclease and ribonuclease cannot achieve the native structure, during biosynthesis, until the termination of the polypeptide chain. For RNase even the N-terminal end seems to be important for folding. After removal by proteolytic action of subtilisin of the first 20 amino adds (Richards, 1958), the RNase S protein is unstable and cannot refold correctly when disulfide bridges are reduced. 

After the virus has attached to CD4 and chemokine receptors, another viral glycoprotein (gp41) assists with viral fusion to the cell and internalization of the viral contents. The viral contents include single-stranded RNA, an RNA-dependent DNA polymerase (also known as reverse transcriptase), and other enzymes. Using the single-stranded viral RNA as a template, reverse transcriptase synthesizes a complementary strand of DNA. The single-stranded viral RNA is removed from the newly formed DNA strand by ribonuclease H, and reverse transcriptase completes the synthesis of double-stranded DNA. The... 

The methionine 29 is on the outside of ribonuclease-S and simple absorbed [PtClJ2- or [PtC en] should have been rapidly removed from this site. We therefore believe that the platinum complex has reacted... 

Privalov et al (1989) studied the unfolded forms of several globular proteins [ribonuclease A, hen egg white lysozyme, apomyoglobin (apoMb), cytochrome c, and staphylococcal nuclease]. Unfolding was induced by 6 M Gdm-HCl at 10°C, heating to 80°C, or by low pH at 10°C with cross-links cleaved (reduction and carboxamidomethylation or removal of heme). The unfolded forms showed CD spectra (Fig. 27)... 

Final alcohol precipitation not only allows for removal of the phenol and any remaining non-covalently bound hydrocarbon but also concentrates the DNA. Ribonuclease treatment removes any contaminating RNA. Additional purification by cesium chloride centrifugation (35) is also often performed. This is particularly suited to small quantities of DNA. Hydroxyapatite chromatography is also effective in separating RNA, proteins, and DNA (36.37). 

U. Narang, M.H. Rahman, J.H.Wang, P.N. Prasad, and F.V. Bright, Removal of ribonucleases from solution using an inhibitor-based sol-gel-derived biogel. Anal. Chem. 67, 1935-1939 (1995). 

The tissue or cell sample is firstly homogenized in a buffer containing a detergent such as Triton X-100 and sodium deodecyl sulphate (SDS), which disrupts the cell and dissociates DNA-protein complexes. Protein and RNA are then removed by sequential incubations with a proteolytic enzyme (usually proteinase K) and ribonuclease. Finally the DNA is extracted into ethanol. Ethanol only precipitates long chain nucleic acids and so leaves the single nucleotides from RNA digestion in the aqueous layer. 

Prokaryotic and eukaryotic tRNAs are also made from longer precursor molecules. These must have an intervening sequence (intron) removed, and the 5 - and 3 -ends of the molecule are trimmed by ribonuclease. A 3 -CCA sequence is added and bases at specific positions are modified, producing "unusual" bases. 

A bacterial peptidase splits a 20-residue fragment containing His 12 from the N-terminal end of RNase A. This "S-peptide" can be recombined with the rest of the molecule, which is inactive, to give a functional enzyme called ribonuclease S. In a similar way, residues 119-124 of RNase A can be removed by digestion with carboxypeptidase to give an inactive protein which lacks His 119. In this case, a synthetic peptide with the sequence of residues 111 -124 of RNase A forms a complex with the shortened enzyme restoring full activity.755... 

RNA does not normally precipitate like DNA, but it could still be a minor contaminant. RNA may be degraded during the procedure by treatment with ribonuclease after the first or second deproteinization step. Removal of RNA sometimes makes it possible to denature more protein using chloroform-isoamyl alcohol. If DNA in a highly purified state is required, several deproteinization and alcohol precipitation steps may be carried out. It is estimated that up to 50% of the cellular DNA is isolated by this procedure. The average yield is 1 to 2 mg per gram of wet packed cells. 

Ribonuclease Ti is fairly resistant to proteases. The threonine residue at the carboxyl terminal of the enzyme can be removed by carboxy-peptidase A without loss of activity (67). Leucine aminopeptidase does not release amino acids from the amino terminal (68). Ribonuclease Ti is not inactivated by trypsin or chymotrypsin in the presence of 0.2 M phosphate (69), which probably binds the enzyme and protects it from inactivation (67). Treatment of the enzyme with trypsin in the absence of phosphate inactivates it (67). Ribonuclease Tj is hydrolyzed by pepsin with progressive loss of activity (69). 

In view of the high stability of the enzyme most samples have been prepared by the procedure described by Kunitz (16) and modified by McDonald (17) to remove all traces of proteolytic activity. During this procedure the minced bovine pancreas is exposed to 0.25 N sulfuric acid, ammonium sulfate precipitation, 10 min at 95°-100° and pH 3, and, finally, reprecipitation. The product can be crystallized it was also shown later to contain a number of components all with ribonuclease activity. A practical summary of all details is given by Kunitz and McDonald (18). 

Perturbation of the spectrum of RNase-A by dioxane was almost exactly one-half that of Ox-RNase or of that expected for 6 free tyrosyl residues (303). This solvent was not included in the series reported by Herskovits and Laskowski (301). The results appear to indicate a 3 3 division but could, of course, also fit the 2 2 2 division if the appropriate characteristics are attributed to the free, partially accessible, and buried groups. Ribonuclease-S also appears to have 3 buried tyrosyl residues and S-protein only 2 (304). Presumably Tyr 25 is normalized when S-peptide, and thus the Asp 14 interaction, is removed. 

The first detailed proposal for the mechanism of action of ribonuclease was put forward by Mathias and Rabin and their colleagues (514) An original diagram from their paper is shown in Fig. 28 BIB, 516). It bears a remarkable similarity to the geometry of the active site as defined by the X-ray studies and shown in Fig. 23. For Step 1 the mechanism proposes (1) removal of the proton on the 2 -OH by an imidazole residue in the base form, (2) protonation of the 5 0 of the leaving nucleoside by the other imidazole in the acid form, and (3) attack by the 2 alkoxide on the phosphorus atom to yield the cyclic phosphate. Hydrolysis or alcoholysis of the cyclic phosphate requires the reverse of each of these steps. At the start of step 1, one histidine is in the acidic form and one in the basic form. At the start of step 2 the roles of the two histidine residues are reversed. 

---