Nucleases Ribonucleases

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)... 

If polyribonucleotides are treated simultaneously with methoxylamine and bisulphite, cytidine residues are converted into 5,6-dihydro-7V4-methoxycytidine-6-sulphonate,154 and uridine into 5,6-dihydrouridine-6-sulphonate.155 Treatment with dilute ammonia regenerates the uridine residues, leaving the dihydrocytidine derivatives unaffected. When only the cytidine residues have been derivatized, pancreatic ribonuclease becomes uridyl ribonuclease, since it is unable to cleave the chain on the 3 -side of the modified cytidine.154 This allows the isolation of blocks of modified cytidine residues. T2 ribonuclease may also be used. Alternatively, a ribonuclease from Physarum polycephalum has been found to hydrolyse CpX links very slowly, allowing the isolation of cytidine blocks.156 If both uridine and cytidine residues are modified, T2 ribonuclease acts as puryl ribonuclease, allowing the isolation of cumulative blocks of pyrimidines.155 This ability to alter the specificity of nuclease cleavage is a useful tool in sequence analysis. 

Studies of proteolytic fragments of staphylococcal nuclease (Tan-iuchi and Anfinsen, 1969) and RNase A (Taniuchi, 1970) seemed to support this view. Taniuchi (1970), in summary remarks, said Thus, the minimum information of the specific folding of a protein requiring almost the entire amino acid sequence is observed with both staph-yloccocal nuclease and bovine pancreatic ribonuclease. ... 

Pancreatic ribonuclease Staphylococcal nuclease Peroxidases Glutathione peroxidase Cytochrome c peroxidase Oxygen carriers Myoglobin, hemoglobin Myohemerythrin, hemerythrin Hormone-binding proteins Uteroglobin Pre albumin Lectins... 

Nucleic acids are broken down into their components by nucleases from the pancreas and small intestine (ribonucleases and deoxyribonucleases). Further breakdown yields the nucleobases (purine and pyrimidine derivatives), pentoses (ribose and deoxyribose). 

A potent enzyme inhibitor (abbreviated DEP) that acts by ethoxyformylation of proteins, usually at histidine residues. DEP is an irreversible inhibitor of ribonuclease, and rinsing glassware with a 0.1% (weight/volume) DEP solution is recommended to render glassware nuclease-free. Aqueous solutions must be freshly prepared for maximal effectiveness, because DEP will hydrolyze in 6-12 hours at neutral pH. 

Selected entries from Methods in Enzymology [vol, page(s)] Inhibitory properties, 68, 212 inactivator, of ribonucleases, 65, 681 lipase modification, 64, 390 nuclease inactivation, 79, 63 ribonuclease inactivation, 79, 52, 112-113, 267. 

Ribonuclease II [EC 3.1.13.1], also called exoribo-nuclease II, catalyzes the exonucleolytic cleavage of the polynucleic acid, preferring single-stranded RNA, in the 3 - to 5 -direction to yield 5 -phosphomononucleotides. The enzyme processes 3 -terminal extra-nucleotides of monomeric tRNA precursors, following the action of ribonuclease P. Similar enzymes include RNase Q, RNase BN, RNase PHI, and RNase Y. Ribonuclease T2 [EC 3.1.27.1] is also known as ribonuclease II. 

This enzyme [EC 3.1.26.6], also known as endoribo-nuclease IV and poly(A)-specific ribonuclease, catalyzes the endonucleolytic cleavage of poly(A) to fragments terminated by 3 -hydroxyl and 5 -phosphate groups. Oligonucleotides are formed with an average chain length of ten. 

Crouch RJ, Dirksen ML, Ribonuclease H. In Linn SM, Roberts RJ, eds. Nuclease. Plainview, New York Cold Spring Harbor Laboratory Press, 1982 211-241. 

The specificity of RNase A for a pyrimidine on the 3 side of the phosphodiester bond that is cleaved is evidently ensured by the pair of hydrogen bonds from 0-2 of the pyrimidine to the backbone NH of Thr 45 and a second from the N-4 proton to the side chain OH of the same threonine (Fig. 12-25). Other nucleases, such as ribonuclease T2,762 with different specificities also make use of hydrogen bonding of the base at the 3 side of the cleavage point with backbone amide groupings. 

RNS, ribonuclease LZM, lysozyme SNS, staphylococcal nuclease LZ4, T4 lysozyme PAP, papain TLS, thermolysin, TRX, thioiedoxin FLN, flavodoxin ADH, alcohol dehydrogenase coenzyme domain AKN, adenyl kinase MDG, malate dehydrogenase TIM, triosephosphate isomerase SUB, subtilisin CPA, carboxypeptidase LDH, lactate dehydrogenase PGK, phosphoglycerate kinase GPD, glyceraldehyde 3-phosphate dehydrogenase, HKN, hexokinase. 

Nucleic acid extracted from purified virus using phenol or dodecyl sulfate is easily destioyed by the homologous nucleases present m normal sera or tissues. DNA is destroyed by the enzyme deoxyribonuclease RNA by ribonucleases, This provides one means of identifying the type of nucleic acid. The intact virus is not affected by these enzymes. 

The five N-terminal residues and the six or seven C-terminal residues cannot be seen in the high resolution electron density map, and the loop referred to above, formed by residues 44 to 53, appears at only one-third to one-half the amplitude of the well-resolved parts of the map. The lack of clarity in these three regions might possibly result from poor phasing or some other crystallographic factor, but we consider it more likely that these predominantly hydrophilic sections of the peptide project in a disordered way into the solvent. In this connection, it is interesting that in the presence of Ca2+ and pdTp trypsin cleaves inhibited nuclease at only two points between residues 5 and 6 and between residues 48 and 49 (36-38) which are at the very extremity of the loop. It also seems relevant that ribonuclease S also shows lack of clarity at the ends of the peptide chains and in the region of a relatively exposed loop (56). 

Studies on microbial RNases began in 1924 when Noguchi found ribonucleic acid degrading enzymes in Takadiastase (134). Since then extensive studies have been carried out on RNA degrading enzymes. It is rather surprising that guanyloribonuclease so widely distributed in microorganisms was found only in 1957. This is because earlier studies did not consider base specificity. Even quite recently studies on nucleases or ribonucleases do not consider base specificity or do not separate nuclease mixtures from each other thus, information available on microbial RNases is still scant. 

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