Phosphodiesterases RNase

The sole biochemical function of 2 -5A (and hence 2 -5 A synthetase) appears to be as an activator of a dormant endo-RNase, which is expressed constitutively in the cell. This RNase, known as RNase L or RNase F, cleaves all types of single-stranded RNA (ssRNA). This inhibits production of both viral and cellular proteins, thus paralyzing viral replication. Presumably, cellular destruction of the invading ssRNA will be accompanied by destruction of any additional viral components. Removal of dsRNA would facilitate deactivation of the endo-RNase, allowing translation of cellular mRNA to resume. A 2-5 phosphodiesterase represents a third enzymatic... 

Ribonucleases (RNases) may be defined as phosphodiesterases that attack the internucleotide bonds in ribonucleic acid (RNA) and its products but not those in deoxyribonucleic acid (DNA) or simple phos-phodiesters such as bis-p-nitrophenyl phosphate. 

On treatment of guanosine 2, 3 -monophosphate with ethanol, propanol, or glycerol in the presence of RNase Ni, the corresponding 3 -guanylyl esters are formed, in modest yield. In the last case, subsequent digestion with snake venom phosphodiesterase affords a mixture of d- and L-glycerol 3-phosphates in 1 2 ratio. 

In vivo, cleavage of P-0 bonds are performed by enzymes such as phosphatases, phosphodiesterases, phosphohydrolases, nucleases, DNases and RNases (see Section 13.1.1). In vitro, cleavage of a P- O bond is often a trivial synthetic step. Even for an easy step, enzymes attract increasing attention. The enzymatic reactions are preferred when regio- or stereoselectivity is required, and when the substrates are temperature or pH sensitive. Many phosphate analogs have been tested as substrates of enzymes that hydrolyze phosphoryl groups. These analogs are often accepted as substrates for the enzymes, and such reactions could be synthetically valuable. Typical examples are presented in the tables. 

Cyclic phosphate diesters can be hydrolyzed selectively with RNases and phosphodiesterases to give the corresponding phosphate monoesters (Table 13-10, entries 1 and 2). 

Mechanisms Interferons are glycoproteins produced in human leukocytes (IFN-a), fibroblasts (IFN-(3), and immune cells (IFN-y)- They exert multiple actions that affect viral RNA and DNA synthesis. Interferons induce the formation of enzymes, including a protein kinase that phosphorylates a factor which blocks peptide chain initiation, a phosphodiesterase that degrades terminal nucleotides of tRNA. and enzymes that activate RNase. 

Fig. 2A-D. Effect of RNase A on the average chain length of poly(ADPR) syn-thetized by the mRNP poly(ADPR) polymerase. The reaction was carried out as described in [4] with addition of variable amounts of RNase A A control B 1.5 jug ml" C 5 Mg ml" D 20 Mg ml". The reaction product was analyzed by thin layer chromatography on PEI cellulose after digestion by snake venom phosphodiesterase. Average chain length A 3.9, B4.2,C4.5,D4.7...

In order to confirm that the acid-insoluble radioactive material synthesised during the ADPRT assay is in fact (ADP-ribose) , this material was digested with a number of hydrolytic enzymes. RNase A, DNase I, Micrococcal nuclease and proteinase K failed to degrade this material. However, snake venome phosphodiesterase degraded 96% of the acid-insoluble radioactive material (Table 1). This strongly suggests that the incorporated radioactive material was (ADP-ribose) . 

At 2.5 mmolar concentration, thymidine, nicotinamide and 3-aminobenzamide were efficient inhibitors of the enzymatic activity, the apparent Ki with these inhibitors were 55 pM, 139 pM and 23 pM respectively in the case of plasmacytoma. When free mRNPs were incubated with snake venom phosphodiesterase or poly(ADP-ribose) glycohydrolase, but not with RNAses, a release of the labelled ADP-ribose in the incubation medium occurred (25-28). 

The specificity of snake venom phosphodiesterase, unlike that of RNase, is not concerned with the nature of the bases. Therefore, it can release one by one all nucleotides found in the polynucleotide chain. However, in contrast to pancreatic RNase, the snake venom phosphodiesterase acts at the other end of the phosphodiester bond, and 5 -mononucleotides are produced. 

Phosphoramidates. Phosphoramidates are another commercially available DNA analogue where the 3 -oxygen of the phosphodiester center is replaced with an amino or aminoalkyl group (Fig. 13) (49). Phosphoramidates exhibit complete resistance to nuclease PI and snake venom phosphodiesterase and a moderate improvement in affinity for DNA and RNA targets (ATm per monomer = 4°C for 2 -fluoro N3 -P5 phosphoramidates) (57). However, they exhibit no RNase H activity (49). 

Viral RNA, RF-RNA and 1 % were exposed to RI-RNA formaldehyde (Bishop and Koch, 1967, 1969), to UV (Koch, Quintrell and Bishop, 1967), to heat (80° C) in phosphate buffered saline (Koch, unpublished), to 0.5% diethylpyrocarbonate at 0° C (Breindl and Koch, I972) or to RNase A and exonucleases (RNase II, venom phosphodiesterase, spleen phosphodiesterase) (Mittelstaedt and Koch, 1974). 

Ribonucleases (RNases) are enzymes that specifically hydrolyse the phosphodiester bonds in ribonucleic acid (RNA). They are distinct from general nucleases which digest both RNA and deoxyribonucleic acid (DNA), from deoxyribonucleases (DNases) which digest exclusively DNA, and from phosphodiesterases... 

---