Nucleases, inhibition

The mature red blood cell cannot synthesize protein. Reticulocytes are active in protein synthesis. Once reticulocytes enter the circulation, they lose their intracellular organelles (ribosomes, mitochondria, etc) within about 24 hours, becoming young red blood cells and concomitandy losing their ability to synthesize protein. Extracts of rabbit reticulocytes (obtained by injecting rabbits with a chemical—phenylhydrazine—that causes a severe hemolytic anemia, so that the red cells are almost completely replaced by reticulocytes) are widely used as an in vitro system for synthesizing proteins. Endogenous mRNAs present in these reticulocytes are destroyed by use of a nuclease, whose activity can be inhibited by addition of Ca +. The system is then pro-... 

ARCAs are incorporated into RNA exclusively in the correct orientation to an extent that is similar to the standard cap (see previously), which makes them potentially useful compounds in terms of increasing translational efficiency when incorporated into RNA. Similarly, they should be effective for inhibiting protein synthesis as free analogs. To test the influence of the ARCAs on protein synthesis in vitro, we use the microccocal nuclease treated rabbit reticulocyte lysate system (RRL system) optimized for cap-dependent translation (Cai et al., 1999). Highly cap-dependent translation is achieved at 100 mM potassium acetate and 1.4 mM magnesium chloride. 

Deoxyribonucleic acid footprinting studies have shown that HMG domains A and B inhibit cleavage by nucleases over a 12- to 15-base-pair region centered around the platinum adduct (81). The HMG proteins can modulate cisplatin cytotoxicity by inhibition of the excinuclease-mediated removal of Pt-d(GpG) adducts from DNA (82). However, this hypothesis has been questioned because there is no evidence for cellular protein shielding of Pt-d(GpG) adducts from repair enzymes (83). 

It should be noted that the unfolding kinetics can sometimes involve quite complex unfolding schemes of different substates in equilibrium with the native state. Staphylococcal nuclease is an example of such behavior, known to unfold with three different substates that exhibit an equilibrium that does not appear to shift with temperature.49 Irreversible aggregation processes of proteins have been known to involve first- or second-order reactions.132141 The mechanism of recombinant human interferon-y aggregation is an example where thermodynamic and kinetic aspects of the reaction provided a powerful tool for understanding the pathway of instability and permitted a rationale for screening excipients that inhibited the process.141... 

Huang JC, Zamble DB, Reardon JT, Lippard SJ, Sancar A (1994) HMG-domain proteins specifically inhibit the repair of the major DNA adduct of the anticancer-drug cisplatin by human excision nuclease. Froc Natl Acad Sd USA 91 10394-10398... 

Restriction endonucleases require the presence of Mg2+ for activity. The quench buffer contains EDTA, which complexes transition metal ions in the solution. The metal ions are no longer available for binding by the nuclease molecules and enzyme activity is inhibited. 

We have studied crystals of both the uninhibited nuclease and the nuclease-pdTp-Ca2+ complex and treated them as independent structures. The crystals of uninhibited nuclease, hereafter referred to as Type I, have unit cell parameters a = b = 47.75 A, c = 63.5 A the ternary complex (inhibited nuclease) crystals, hereafter called Type II, have unit cell parameters a=b = 48.3A, c = 63.3A the space group of both is P4i (31). We shall show views of the structure of the nuclease at three levels of resolution these structures were determined as follows ... 

Fig. 2. (a) Front view of the conformation of the peptide chain of the inhibited nuclease at high resolution (Type II crystals), (b) Right side view of the chain conformation at high resolution. The curlicue at the upper left center represents the inhibitor pdTp. 

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). 

Type I (uninhibited) crystals. In a rather marked contrast, Fig. 6b shows the same view of the 4-A electron density map of the Type II (inhibited) crystals. A mass of electron density now appears in the pocket, and there also appears to be some added density at the left side of the perimeter of the pocket itself. It should perhaps be reemphasized here that these structures of the Type I and Type II crystals were determined completely independently using one set of heavy atoms and unsubstituted data for the uninhibited nuclease and another entirely different combination for the nuclease-inhibitor complex. However, the fact that heavy-atom positions appear very distinctly when intensities from Type I crystals are combined with Type II phases (and vice versa) indicates that the nuclease undergoes no gross conformational changes with pdTp and Ca2+ binding (31) thus, the kind of comparison presented... 

From differences between the spectral (29) and fluorescence (SO) properties of the inhibited and uninhibited nuclease and from differences in the number of groups susceptible to acetylation (32) in the two forms of the enzyme, Cuatrecasas et al. (S3) concluded that tyrosyl residues were involved in binding pdTp. This led them to some very interesting studies of the specific modification of certain tyrosyl residues with tetranitromethane and of the properties of these modified forms of the nuclease which we will discuss below in the context of this paper. Briefly, the pattern of relative reactivity was found to be... 

It is easy to see why Tyr 85 is unreactive in the inhibited nuclease it is tightly hydrogen bonded to the 3 -phosphate of the pdTp. In harmony... 

In the high resolution structure, Tyr 91 and 93 are indeed buried deep in the hydrophobic area between the back wall of the pocket and the rear edge of the nuclease molecule. Tyrosine 27, which is more resistant to nitration in the inhibited nuclease, is probably hydrogen bonded to the carboxylate of Glu 10, partially shielded by the methylene chain of Lys 28, and, most importantly, at the rear upper right... 

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). 

Two functions can be suggested, both inferred from its preference for binding to single-stranded DNA. It could protect single-stranded DNA from nucleases, or it could facilitate unwinding by inhibiting rewinding. 

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