RNase inhibition inhibitors

Heparin, which inhibits RNase A, does not inhibit RNase Ti, nor does a natural RNase A inhibitor from rat liver (20), or Aspergillus orysae nuclease inhibitor (21). 

Very few inhibitors specifically target HIV-1 RNase H activity. Illimaquinone, a natural marine product, was shown to preferentially inhibit the HIV-1 RNase H activity [113,114]. However, this compound appears to react with a sulfhydryl group in the polymerase domain and not with RNase H itself. It may be possible to use the available information on structural and biochemi-... 

The effects of inhibitors and activators on RNases Ti, N1 Ui, U2, and T2 are summarized in Table IV (5, 7, 8), showing the characteristics of each RNase. Ribonuclease Ti is strongly inhibited by 10 3M Zn2+ RNases and T2 are inhibited about half but RNases N and U2 are not inhibited. Ribonuclease T, is also strongly inhibited by 10 3M Ag+, while all other RNases are inhibited about half. But 10 3 M Cu2+ strongly inhibits RNase T2 and only about half inhibits the other RNases. All five RNases are not inhibited by 10 2 M ethylenediaminetetraacetate (EDTA) and 10-3 M ICH2COOH. No metallic cofactor is required for their action. 

There may be many proteins that inhibit ribonucleases. Clear evidence has been presented that there is at least one such substance in rat liver that is a powerful and specific inhibitor of the pancreatic enzyme, but is inactive against RNases obtained from plants or bacteriophage (R14, S17). 

Phosphorothioate oligonucleotides have been reported to be competitive inhibitors for HIV-reverse transcriptase and inhibit RT-as-sociated RNase H activity (175, 176). They have been reported to bind to the cell surface protein CD4 and to protein kinase C (PKC) (177). Various viral polymerases have also been shown to be inhibited by phosphorothioates (140). Additionally, we have shown potent, non-sequence-specific inhibition of RNA splicing by phosphorothioates (105). 

Le Dinh T, Freneaux E, Labbe G, Letteron P, Degott C, Geneve J, Berson A, Larrey D, Pessayre D (1988) Amineptine, a tricyclic antidepressant, inhibits the mitochondrial oxidation of fatty acids and produces microvesicular steatosis of the liver in mice. J Pharmacol Exp Ther 247 745-750 Le Roy F, Bisbal C, Silhol M, Martinand C, Lebleu B, Salehzada T (2001) The 2-5A/RNase L/RNase inhibitor (RLl) pathway regulates mitochondrial mRNAs stability in interferon-a-treated H9 cells. J Biol Chem 276 48473 8482 Le Roy F, Silhol M, Salehzada T, Bisbal C (2007) Regulation of mitochondrial mRNA stability by RNase L is translation-dependent and controls IFNalpha-induced apoptosis. Cell Death Differ 14 1406-1413... 

In addition to acting as an inhibitor of dynein and (Na, K)-ATPase, vanadium is also a potent inhibitor of RNase (57) and alkaline and acid phosphatases (58.59). This suggests that vanadium generally tends to inhibit enzymes of phosphate metabolism. However, according to Gibbons et al. (53), the mechanism of Inhibition is not the same in each enzyme. The Inhibition of RNase and alkaline phosphatase is greater by oxyvanadium (IV) than by vanadium (V). 

Successful RNA preparation depends on the inhibition of both endogenous RNase activity liberated on cell lysis and contamination of preparations by exogenous RNases. In this method treatment of equipment and solutions with diethyl pyrocarbonate (DEPC), a strong inhibitor of RNases, is used to prevent degradation of samples. In addition working quickly and keeping preparations on ice whenever possible will help to minimize problems with RNase activity. 

In most cases, the respective protein purification buffer (NOT the storage buffer) will be most appropriate for RNA-binding reactions. However, the concentrations of some components of these buffers, such as Mg + and NaCl, may need to be varied so as to determine optimal binding conditions. Also, inclusion of compounds such as heparin sulfate can minimize nonspecific binding of RNA by the target protein. Finally, an appropriate quantity of commercially available RNase inhibitor (up to 10 U of the human placenta RNase inhibitor from New England Biolabs or an equivalent activity of a comparable inhibitor) may be needed to inhibit trace amounts of ribonu-clease contamination. 

Centrifuge (at 12,000g) at 4°C for 15 mm, pour off the supernatant, wash the pellet with 100 )xL of cold ethanol, 75% (v/v), recentnfuge and aspirate the supernatant, dry the pellet under a vacuum, and dissolve it in 100 xL of TE buffer or in DEPC-treated water for 30 mm at 37°C 1 iL of RNase-inhibitor can be added to inhibit possible contaminating RNase. May be stored at -70°C for long time periods before use or at -20°C for daily use (see Note 2)... 

One issue that came from the work of Kienberger et al. was that the lack of use of RNAse inhibitor will allow partial degradation of RNA by endogenous enzymes or contaminants. It was claimed that where RNA immobilisation on mica was dense, the tight packing inhibited the action of the RNAse, so little degradation was observed [143]. 

PRI is also an inhibitor (K <0.1 nM) of both the angiogenic and ribonucleolytic activities of angiogenin, a blood vessel-inducing protein (20). PRI may therefore be called a ribonuclease/angiogenin inhibitor (RAI). Note that PRI does not inhibit some other RNases such as RNase Tl, nuclease SI, and RNase H. 

Pyrophosphate (Na-PPj) at a 0.5 mM concentration reduces the RTase activity to 50%. Among PP analogs, phosphonoformate (or foscarnet) is the strongest inhibitor to AMV RTase (8,20). The AMV RNase H activity is unaffected by the phosphonoformate. Na-PPj has also been reported to have no inhibitory effect on RNase H activity (21). The mode of RTase inhibition by phosphonoformate is noncompetitive (K = 5 to 100 pM) with respect to and depending on nucleotide substrates. The structurally related phosphonoacetate is not a RTase inhibitor. 

Phosphonoformate, a PPj analog, is a strong inhibitor of MoLV RTase (IC50 = 8—250 fiM) (10). The inhibition is noncompetitive with respect to nucleotide substrates (Kj == 4.7 /zM). Phosphonoformate inhibits neither the RNase H activity of AMV RTase nor that of MoLV RTase. 

Reverse transcriptase (RT) plays a critical role in the early steps of the life of human immunodeficiency virus (HIV) (304), and for over a decade has been one of the major targets of AIDS therapy. Polycitone A (280) was found to be a potent general inhibitor of retroviral reverse transcriptases and cellular DNA polymerases (305). Polycitone A exhibited potent inhibitory capacity of both RNA- and DNA-directed DNA polymerases. It inhibits retroviral reverse transcriptases (RTs) of human immunodeficiency virus type 1 (HIV), murine leukemia virus (MLV) and mouse mammary tumor virus (MMTV)] as efficiently as cellular DNA polymerases of both DNA polymerases a and p and the prokaryotic Klenow fragment of Escherichia coli DNA polymerase I. The mode and mechanism of inhibition of the DNA-polymerase activity associated with HIV-1 RT by polycitone A (280) have been studied. The results suggest that the inhibitory capacity of the DNA polymerase activity is independent of the template-primer used. The RNase H function is hardly affected by this inhibitor. Polycitone A has been shown to interfere with DNA primer extension, as well as with the formation of the RT-DNA complex. Steady-state kinetic studies demonstrate that this inhibitor can be considered as an allosteric inhibitor of HIV-1 RT. The target site on the enzyme may be also spatially related to the... 

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