Inhibition of RNase

We have examined the metal dependence (Mn + and Mg +) of E.coli RNase H activity. Mn2+-dependent activity requires much less metal for activation than does Mg2+-activity and is inhibited upon the further addition of Mn +. Using electron paramagnetic resonance (EPR), we have measured two distinct Mn + binding constants, consistent with the concentration requirements for activation and inhibition in vitro. Our data are most consistent with a single-divalent metal catalyzed reaction which can be attenuated (inhibited) upon binding a second metal. We discuss a possible mechanism for metal activation and inhibition of RNase H in light of previous mutagenesis and structural studies. 

Mn " -dependence of E.coli RNase HI The Mn2+-dependence of E.coli RNase HI catalysis was determined using a soluble assay that monitors acid-solubility of radiolabelled RNA in an RNA DNA hybrid (25). This Mn2+-dependence shows activation at low concentrations of Mn2+, followed by inhibition at higher Mn2 + concentrations (Figure 1). The optimum activity is achieved in 5 pM MnCl2, and is 30 % of the maximum activity in MgCl2 (data not shown). Maximum inhibition at 1 mM MnCl2 is 20-fold inhibited relative to activity at 5 pM MnC. Activation and inhibition of RNase H was indistinguishable from RNase HI (data not shown). 

Step 1 is the primary one that is catalyzed by RNAses. It is a fairly straightforward reaction and therefore is amenable to analysis by standard procedures [59]. RNase is also susceptible to inhibition by substances such as 2 -CMP. In our study [40], we used ITC to determine the binding affinity and thermodynamic parameters associated with the reversible inhibition of RNAse-A by 2 -CMP at body temperature (37 °C) and in a more physiologically relevant (i.e., multi-ion)... 

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). 

Recently, extractions using cesium trifluoroacetate (CsTFA) have been recommanded. This salt is expensive but is more effective than CsCl in deproteinization and inhibition of RNases. RNA can be pelleted or banded by adjustement of the density. CsTFA is far from essential to purify total RNA for sequencing. 

However, there are a number of other miscellaneous biological roles played by this complex. The [Co(NH3)6]3+ ion has been shown to inhibit the hammerhead ribozyme by displacing a Mn2+ ion from the active site.576 However, [Co(NH3)6]3+ does not inhibit ribonuclease H (RNase),577 topoisomerase I,578 or hairpin ribozyme,579 which require activation by Mg2+ ions. The conclusions from these studies were that an outer sphere complex formation between the enzyme and Mgaq2+ is occuring rather than specific coordination of the divalent ion to the protein. These results are in contrast to DNase I inhibition by the same hexaammine complex. Inhibition of glucose-induced insulin secretion from pancreatic cells by [Co(NH3)6]3+ has been found.580 Intracellular injection of [Co(NH3)6]3+ into a neurone has been found to cause characteristic changes to the structure of its mitochondria, and this offers a simple technique to label neuronal profiles for examination of their ultrastructures.581... 

Considerable DNase but no RNase activity results if Ca-+ is replaced by Sr-+, while Fe-+ and Cu J+ cause minimal activation (3, 40). A number of heavy metal cations inhibit DNase and RNase activities competitively with Ca-+ Hg-+, Zn2+, and Cd-+ are the most potent of these (3). Studies with synthetic substrates, to be discussed below, indicate that Ca2+ is not only required for the proper binding of substrates but also that it is required for the subsequent independent hydrolytic process. Although several divalent cations can substitute for Ca2t in the binding function, as evidenced by their competitive inhibition of enzymic activity (3) and their ability to promote nucleotide binding (62), the catalytic role of Ca2+ appears to be unique. 

A summary of the transition temperatures of RNase in the presence of a variety of electrolytes is given in Table XX 361, 362). The effects of the usual salts follow the Hofmeister series. The remarkable difference in denaturing power of the various guanidinium salts is quite general. The sulfate actually tends to stabilize slightly while the thiocyanate is a more powerful denaturant than the chloride. Irreversible denaturation is markedly inhibited by spermine 363). There is no clear correlation between stabilizing effects and the complex inhibition curves obtained by Wold (see Section VI,E,2). 

The sugar configuration about the T, 2, 3, and 4 positions can be changed by synthesis. A variety of pyrimidine nucleoside cyclic phosphates have been made. Ukita et al. (425) prepared -D-lyxo-uridine 2 3 -cyclic phosphate (Fig. 21a). The configuration about the 2 and 3 positions is inverted and the two OH groups are now cis to the base rather than trans as in the D-ribose series. No hydrolysis at all of this compound was observed in the presence of RNase. However, both the cyclic phosphate and the free 2 (3 )-nucleotides inhibit the enzyme. The... 

The variable activity of RNase toward different RNA preparations has been tracked down in part to the variable metal content of the substrates [see Wojnar and Roth (4-76), and earlier references quoted]. Takahashi et al. (477) have reported that Mg2+, Ca2+, and Mn2+ have little or no effect on step 1 or step 2 activity when these are assayed with low molecular weight substrates. However, Ca2+ and Mg2+ do interact with RNA and they inhibit the RNase-catalyzed reaction at pH 7 because of this interaction with substrate (478). Eichhorn et al. (479) found activation by Mg2+ and various transition metals at pH 5. In any event it is clear that in general each metal can be expected to show different effects as a function of pH, ionic strength, specific buffer effects, etc. A substantial correlation of much of the data has been made by Alger (480) who studied RNA and C > p substrates over wide ranges of metal concentration. Activation appeared to involve predominantly metal-substrate interactions while inhibition occurred with direct enzyme-metal interaction. 

Antisense DNA or RNA RNA2 Cytoplasm Translation arrest, RNase H activation, inhibition of splicing, disruption of RNA structure Clinical use3 clinical trials... 

Chen, H. and Gold, L. (1994) Selection of high-affinity RNA ligands to reverse transcriptase inhibition of cDNA synthesis and RNase H activity. Biochemistry, 33, 8746-8756. 

Illimaquinone, such as avarone and avarol isolated from a Red Sea sponge, i.e., Smenospongia, has been reported to inhibit the RNase H activity associated with the HIV-1 reverse transcriptase at a concentration of 5 to 10 pg/rnl, whereas it was not active against the RNA-dependent DNA polymerase (RDDP) and DNA-dependent DNA polymerase (DDDP) activities of the enzyme at a concentration of 50 pg/rnl. 

Cys202 [208]. Human immunodeficiency virus (HIV) is the primary cause of acquired immunodeficiency syndrome (AIDS). In an effort to find new drugs preventing the growth of HIV, Masao et al developed an in vitro assay method of RNase H activity associated with reverse transcriptase (RT) from HIV-1. Some 1,4-naphthoquinones moderately inhibited RNase H activity [209]. Several natural occurring naphtoquinones have showed antiretroviral activity [210-211],... 

Fig. 9. Inhibition of endogenous RNase-sensitive FLV-DNA-polymerase activity by tilorone and congeners...

Most antisense experiments made with LNA have been focused mechanistically on mRNA inhibition by RNase H recmitment (7). Expression studies in a wide variety of human epithelial and cancer cell lines are made and the overall picture of these experiments is that LNA antisense oligonucleotides are very potent with IC50 values for mRNA inhibition obtained frequently in the low to sub nanomolar range—and actually more potent than competing oligonucleotide analogs (7). 

Didierjean J, Isel C, Querre F, Mouscadet J-F, Aubertin A-M, Valnot J-Y, Piettre SR, Marquet, R. Inhibition of human immunodeficiency virus type 1 reverse transcriptase, RNase H, and integrase activities by hydroytropolones. Antimicrob. Agents Chemother. 2005 49 4884-4894. 

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