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PubMed1976

RNase sensitivity of an anterior morphogenetic determinant in an insect egg (Smittia sp., Chironomidae, Diptera).

In chironomid midges, the development of head and thorax in the embryo requires the function of cytoplasmic determinants localized near the anterior pole of the egg. Experimental inactivation of these determinants causes a dramatic switch in the developmental program of the embryo. Instead of the normal segment pattern, the aberrant pattern "double abdomen" is formed. Head, thorax, and anterior abdominal segments are then replaced by an additional set of posterior abdominal segments joined in mirror image symmetry to the original abdomen. Such double abdomens have been produced, with a maximum yield of 29%, by application of RNase (ribonuclease I, ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) to the anterior pole region of the egg. This was achieved by microinjection or puncturing the eggs during submersion in RNase. Control experiments with inactive RNase S fragments reliably proved that double abdomen formation resulted from RNase activity. Neither application of other enzymes to the anterior pole region nor application of RNase to other egg regions produced double abdomens in significant yields. The effects of RNase concentration and stage of development were determined. The data from these and earlier experiments are compatible with the idea that stored cytoplasmic RNP particles act as anterior determinants. Similarities to genetically caused switches in developmental pathways (homeotic mutations) are discussed.

Age Factors↗
PubMed1979

Serum RNase in the diagnosis of pancreatic carcinoma.

Serum RNase (RNase I; ribonuclease 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) activity (mean +/- SD) with polycytidine as substrate was determined in normal individuals (24.9 +/- 3.0 units/ml) and in patients with pancreatic cancer (37.3 +/- 14.8), pancreatitis (38.5 +/- 12.6), nonpancreatic diseases (48.7 +/- 14.8), or renal failure (175.8 +/- 92.8). Patients with pancreatic cancer could not be distinguished from those with pancreatitis or with nonpancreatic disease, although the RNase activities in all of these differed from the activity in normal individuals. The serum RNase activities of four patients with resectable "curable") pancreatic carcinoma and two others with advanced pancreatic cancer without obstructive jaundice were normal. After total pancreatectomy, serum RNase activity remained in the high-normal range. The data presented here and data in the literature show that serum RNase cannot be of primarily pancreatic origin. The present study also demonstrates that measurement of its activity is not useful in early detection of pancreatic cancer.

Creatinine↗
PubMed1981

Identification of the dnaQ gene product and location of the structural gene for RNase H of Escherichia coli by cloning of the genes.

By in vitro recombination we have constructed hybrid plasmids capable of complementing a conditional lethal mutator mutation, dnaQ49, in Escherichia coli K12. The dnaQ+ plasmids consist of a full-length pBR322 DNA and a 1.5-kilobase DNA fragment derived from the E. coli chromosome. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the 1.5-kilobase insert codes for two proteins, one whose molecular weight is 25,000 [the 25-kilodalton (kDal) protein] and the other whose molecular weight is 21,000 (the 21-kDal protein). Because insertion of gamma delta sequence into the dnaQ gene of the plasmid resulted in disappearance of the 25-kDal protein, it was concluded that the 25-kDal protein is the dnaQ gene product. The 21-kDal protein was identified as RNase H on the basis of the following evidence. (i) Cells harboring the dnaQ+ plasmids, with or without the gamma delta insertion in the dnaQ gene, had a 5- to 7-fold higher level of RNase H activity than cells harboring pBR322. (ii) After induction of cells that are lysogenized with dnaQ+-transducing lambda phages, RNase H activity increased considerably. A similar high level of RNase H activity was observed with transducing phages whose dnaQ function was inactivated by insertion of a transposon, Tn3, into the gene, (iii) The plasmid-encoded RNase H, labeled with [35S]methionine, was purified in a manner essentially similar to that of the chromosome-encoded enzyme. These results suggest that the dnaQ gene and the structural gene for RNase H, termed gene rnh, are closely linked and located at 5 min on the linkage map.

Bacterial Proteins↗
PubMed1988

Yeast RNase P: catalytic activity and substrate binding are separate functions.

During tRNA biosynthesis the 5'-leader sequences in precursor tRNAs are removed by the ribonucleoprotein RNase P, an enzyme whose RNA moiety is required for activity. To clarify some aspects of the enzyme mechanism, we examined substrate binding and product formation with mutant precursor tRNAs. Mutations G-1----A or U-2----C in the Schizosaccharomyces pombe sup3-e tRNASer, which cause mispairing at or near the top of the acceptor stem, prevent the removal of the 5'-leader sequences by Saccharomyces cerevisiae RNase P. Equilibrium binding studies involving specific gel retardation of RNase P-precursor tRNA complexes showed that complexes with wild-type and A-1 and C-2 mutant precursor tRNAs had very similar dissociation constants (average Kd for sup3 = 1.5 +/- 0.2 nM). Thus, the 5'-terminal nucleotides of mature tRNA, on the 3' proximal side of the RNase P cleavage site, affect the enzyme's catalytic function but not substrate binding. The catalytic integrity of the RNA component of RNase P is not essential for binding of tRNA precursors, as demonstrated by gel retardation of micrococcal nuclease-inactivated enzyme. This suggests a possible role for the protein component of the enzyme in substrate binding. Upon restoration of base pairing to the acceptor stem in the A-1 or C-2 mutants, we found that, in addition to a requirement for pairing at these positions, conservation of the wild-type first and second nucleotides of the tRNA was necessary to obtain maximal cleavage by RNase P. This indicates a distinct sequence preference of this enzyme.

Base Sequence↗
PubMed1990

Site-specific excision from RNA by RNase H and mixed-phosphate-backbone oligodeoxynucleotides.

Oligodeoxynucleotides containing phosphodiester or modified internucleoside linkages were investigated with respect to their ability to be acted on by ribonuclease H activities present in a HeLa cell nuclear extract after hybridization with complementary sequences in RNA. Oligodeoxynucleotides complementary to nucleotides 2-14 of human U1 small nuclear RNA were investigated. Extensive cleavage of U1 RNA was observed with the unmodified oligodeoxynucleotide and with the phosphorothioate analogue but not with U1-complementary oligodeoxynucleotides containing methylphosphonate, phosphoro-N-morpholidate, or phosphoro-N-butylamidate internucleoside linkages. Additional experiments using a 514-nucleotide-long RNA substrate demonstrated the capacity of complementary phosphodiester- and phosphorothioate-linked oligodeoxynucleotides (but not ones containing methylphosphonate, phosphoro-N-morpholidate, or phosphoro-N-butylamidate linkages) to serve as RNase H targets when hybridized to an internal RNA site. Detailed comparisons revealed phosphodiester-linked oligodeoxynucleotides to be more efficient than the comparable phosphorothioate-linked oligomers with respect to RNase H action. Various pentadecamer oligodeoxynucleotides complementary to the 514-nucleotide-long test RNA and containing 2-6 consecutive phosphodiester- or phosphorothioate-linked nucleotides flanked by RNase H-resistant methylphosphonate linkages afforded precise "site-directed" RNase H excision within the DNA.RNA hybrid. These results serve to assort modified oligodeoxynucleotide-containing hybrids into RNase H-sensitive and -resistant classes and also provide clues as to how RNase H makes contact with the DNA strand in a DNA.RNA hybrid.

Base Sequence↗
PubMed1997

Mutations in HIV reverse transcriptase which alter RNase H activity and decrease strand transfer efficiency are suppressed by HIV nucleocapsid protein.

Structural studies of authentic HIV reverse transcriptase (RT) suggest a role for the p51 carboxyl terminus in forming an active RNase H conformation [Rodgers, D. W., Gamblin, S. J., Harris, B. A., Ray, S., Culp, J. S., Hellmig, B., Woolf, D. J., Debouck, C. & Harrison, S. C. (1995) Proc. Natl. Acad. Sci. USA 92, 1222-1226]. We have purified mutant RT heterodimers containing deletion of 5, 9, or 13 amino acids from the p51 carboxyl terminus. These "selectively deleted" heterodimers have been analyzed for changes in RNA-dependent DNA polymerase activity, RNase H activity, and the ability to catalyze DNA strand transfer. As deletions extended into the p51 subunit, a decrease in the stability of the RT-DNA complex was apparent. The largest effect was observed for p66/p51Delta13 RT, which showed a 3-fold decrease relative to wild-type RT. RNase H activity was measured by digestion of the RNA in a 5' 32P-labeled RNA/DNA hybrid. Deletion of 5 or 9 amino acids from p51 had little effect on synthesis-dependent and synthesis-independent RNase H activities. In contrast, deletion of 13 amino acids from p51 increased the length of the hydrolysis products of both RNase H activities by 8-10 bp, thus changing the spatial relationship between the polymerase and RNase H active sites from a distance of 17-18 bp to 26-27 bp. The Delta13 derivative was also incapable of efficient DNA strand transfer. This defect in strand transfer could be suppressed by the 71-amino acid form of HIV nucleocapsid protein (NC) but not by the 55-amino acid form (NC55) or by equine infectious anemia virus NC. These results provide evidence for the existence of a specific complex between RT and NC and are discussed in terms of the role of this complex in proviral DNA synthesis.

Base Sequence↗
PubMed1997

In vitro selection for altered divalent metal specificity in the RNase P RNA.

The ribozyme RNase P absolutely requires divalent metal ions for catalytic function. Multiple Mg2+ ions contribute to the optimal catalytic efficiency of RNase P, and it is likely that the tertiary structure of the ribozyme forms a specific metal-binding pocket for these ions within the active-site. To identify base moieties that contribute to catalytic metal-binding sites, we have used in vitro selection to isolate variants of the Escherichia coli RNase P RNA with altered specificities for divalent metal. RNase P RNA variants with increased activity in Ca2+ were enriched over 18 generations of selection for catalysis in the presence of Ca2+, which is normally disfavored relative to Mg2+. Although a wide spectrum of mutations was found in the generation-18 clones, only a single point mutation was common to all clones: a cytosine-to-uracil transition at position 70 (E. coli numbering) of RNase P. Analysis of the C70U point mutant in a wild-type background confirmed that the identity of the base at position 70 is the sole determinant of Ca2+ selectivity. It is noteworthy that C70 lies within the phylogenetically well conserved J3/4-P4-J2/4 region, previously implicated in Mg2+ binding. Our finding that a single base change is sufficient to alter the metal preference of RNase P is further evidence that the J3/4-P4-J2/4 domain forms a portion of the ribozyme's active site.

Base Sequence↗
PubMed1995

Catalytic cleavage of an RNA target by 2-5A antisense and RNase L.

2-5A antisense (2-5A-AS) molecules are chimeric oligonucleotides that cause 2-5A-dependent RNase (RNase L) to catalyze the selective cleavage of RNA in human cells. These composite nucleic acids consist of a 5'-monophosphorylated, 2',5'-linked oligoadenylate known as 2-5A (an activator of RNase L) covalently attached to antisense 3',5'-oligodeoxyribonucleotides. Here, we characterize the targeted cleavage of the double-stranded RNA-dependent protein kinase (PKR) mRNA by purified, recombinant human RNase L. A 2-5A-AS chimera, which contains complementary sequence to PKR mRNA, and unmodified 2-5A, which causes general RNA decay, were about 20- and 40-fold more active, respectively, than 2-5A-AS chimeras in which the DNA domains are not complementary to sequences in PKR mRNA. Directed cleavage was efficient because each 2-5A-AS chimera targeted many RNA molecules. Moreover, RNase L caused the catalytic cleavage of the RNA target (kcat of approximately 7 s-1). The precise sites of PKR mRNA cleavage caused by 2-5A-AS were mapped, using a primer extension assay, to phosphodiester bonds adjacent to the 3' terminus of the chimera binding site (5' on the RNA target) as well as within the chimera's oligonucleotide binding site itself. The selectivity of this approach is shown to be provided by the antisense arm of the chimera, which places the RNA target in close proximity to the RNase.

Base Sequence↗
PubMed1996

Differential effects of Moloney murine leukemia virus reverse transcriptase mutations on RNase H activity in Mg2+ and Mn2+.

We have previously described the in vitro and in vivo characterization of a panel of mutations affecting the RNase H domain of Moloney murine leukemia virus reverse transcriptase (Blain, S. W., and Goff, S.P. (1993) J. Biol. Chem. 268, 23585-23592; Blain, S. W., and Goff, S. P. (1995) J. Virol. 69, 4440-4452). We were intrigued by a discrepancy between in vitro and in vivo RNase H results for two of the mutants. While delta C and delta 5E appeared to have nearly wild-type RNase H activity in vitro, they were unable to degrade their genomic RNA in vivo and thus were effectively RNase H null mutants in this context. In this present report, we describe the differential effects of these mutations on RNase H activity in vitro in the presence of Mg2+ versus Mn2+: mutants delta C and delta 5E were active in the presence of the less biologically relevant Mn2+ and not in the presence of Mg2+. We also describe three mutants with only partial activity in Mg2+. The presence of the different cations can also affect DNA polymerization and processivity of an RNase H-deficient mutant.

Amino Acid Sequence↗
PubMed1997

ARD-1 cDNA from human cells encodes a site-specific single-strand endoribonuclease that functionally resembles Escherichia coli RNase E.

The human ARD-1 (activator of RNA decay) cDNA sequence can rescue mutations in the Escherichia coli rne gene, which specifies the essential endoribonuclease RNase E, resulting in RNase E-like cleavages in vivo in rne-defective bacteria and in vitro in extracts isolated from these cells (Wang, M., and Cohen, S. N. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10591-10595). Recent studies indicate that the 13.3-kDa protein encoded by ARD-1 cDNA is almost identical to the carboxyl-terminal end of the bovine protein NIPP-1, a nuclear inhibitor of protein phosphatase 1; separate transcripts formed by alternative splicing are proposed to encode the discrete ARD-1 and combined ARD-1/NIPP-1 products (Van Eynde, A., Wera, S., Beullens, M. , Torrekens, S., Van Leuven, F., Stalmans, W., and Bollens, M. (1995) J. Biol. Chem. 270, 28068-28074). Here we show that affinity column-purified protein encoded by human ARD-1 cDNA in E. coli is a site-specific Mg2+-dependent endoribonuclease that binds in vitro to RNase E substrates, cleaves RNA at the same sites as RNase E, and, like RNase E, generates 5' phosphate termini at sites of cleavage. Our results indicate that the ARD-1 peptide can function as a ribonucleolytic analog of E. coli RNase E as well as a domain of the protein phosphatase inhibitor, NIPP-1.

Animals↗
PubMed1998

Matrices of paired substitutions show the effects of tRNA D/T loop sequence on Drosophila RNase P and 3'-tRNase processing.

Drosophila RNase P and 3'-tRNase endonucleolytically process the 5' and 3' ends of tRNA precursors. We examined the processing kinetics of normal substrates and the inhibitory effect of the tRNA product on both processing reactions. The product is not a good RNase P inhibitor, with a KI approximately 7 times greater than the substrate KM of approximately 200 nM and is a better inhibitor of 3'-tRNase, with a KI approximately two times the KM of approximately 80 nM. We generated matrices of substitutions at positions G18/U55 and G19/C56 (two contiguous universally conserved D/T loop base pairs) in Drosophila tRNAHis precursors. More than half the variants display a significant reduction in their ability to be processed by RNase P and 3'-tRNase. Minimal substrates with deleted D and anticodon stems could be processed by RNase P and 3'-tRNase much like full-length substrates, indicating that D/T loop contacts and D arm/enzyme contacts are not required by either enzyme. Selected tRNAs that were poor substrates for one or both enzymes were further analyzed using Michaelis-Menten kinetics and by structure probing. Processing reductions arise principally due to an increase in KM with relatively little change in Vmax, consistent with the remote location of the sequence and structure changes from the processing site for both enzymes. Local changes in variant tRNA susceptibility to RNase T1 and RNase A did not coincide with processing disabilities.

Animals↗
PubMed1999

Properties of cloned and expressed human RNase H1.

We have characterized cloned His-tag human RNase H1. The activity of the enzyme exhibited a bell-shaped response to divalent cations and pH. The optimum conditions for catalysis consisted of 1 mM Mg(2+) and pH 7-8. In the presence of Mg(2+), Mn(2+) was inhibitory. Human RNase H1 shares many enzymatic properties with Escherichia coli RNase H1. The human enzyme cleaves RNA in a DNA-RNA duplex resulting in products with 5'-phosphate and 3'-hydroxy termini, can cleave overhanging single strand RNA adjacent to a DNA-RNA duplex, and is unable to cleave substrates in which either the RNA or DNA strand has 2' modifications at the cleavage site. Human RNase H1 binds selectively to "A-form"-type duplexes with approximately 10-20-fold greater affinity than that observed for E. coli RNase H1. The human enzyme displays a greater initial rate of cleavage of a heteroduplex-containing RNA-phosphorothioate DNA than an RNA-DNA duplex. Unlike the E. coli enzyme, human RNase H1 displays a strong positional preference for cleavage, i.e. it cleaves between 8 and 12 nucleotides from the 5'-RNA-3'-DNA terminus of the duplex. Within the preferred cleavage site, the enzyme displays modest sequence preference with GU being a preferred dinucleotide. The enzyme is inhibited by single-strand phosphorothioate oligonucleotides and displays no evidence of processivity. The minimum RNA-DNA duplex length that supports cleavage is 6 base pairs, and the minimum RNA-DNA "gap size" that supports cleavage is 5 base pairs.

Base Sequence↗
PubMed2001

Biochemical and kinetic analysis of the RNase active sites of the integrase/tyrosine family site-specific DNA recombinases.

In this study, we have used multiple strategies to characterize the mechanisms of the type I and type II RNA cleavage activities harbored by the Flp (pronounced here as "flip") site-specific DNA recombinase (Flp-RNase I and II, respectively). Reactions using half-sites pre-bound by step-arrest mutants of Flp agree with a "shared active site" being responsible for the type I reaction (as is the case with normal DNA recombination). In a "pre-cleaved" type I substrate containing a 3'-phosphotyrosyl bond, the Flp-RNase I activity can be elicited by either wild type Flp or by Flp(Y343F). Kinetic analyses of the type I reaction are consistent with the above observations and support the notion that the DNA recombinase and type I RNase active sites are identical. The type II RNase activity is expressed by Flp(Y343F) in a half-site substrate and is unaffected by the catalytic constitution of a Flp monomer present on a partner half-site. Reaction conditions that proscribe the assembly of a DNA bound Flp dimer have no effect on Flp-RNase II. These biochemical results, together with kinetic data, are consistent with the reaction being performed from a "non-shared active site" contained within a single Flp monomer. The Flp-related recombinase Cre, which utilizes a non-shared recombination active site, exhibits the type I RNA cleavage reaction. So far, we have failed to detect the type II RNase activity in Cre. Despite their differences in active site assembly, Cre functionally mimics Flp in being able to provide two functional active sites from a trimer of Cre bound to a three-armed (Y-shaped) substrate.

Base Sequence↗
PubMed2003

Crystal structure of the tRNA processing enzyme RNase PH from Aquifex aeolicus.

RNase PH is one of the exoribonucleases that catalyze the 3' end processing of tRNA in bacteria. RNase PH removes nucleotides following the CCA sequence of tRNA precursors by phosphorolysis and generates mature tRNAs with amino acid acceptor activity. In this study, we determined the crystal structure of Aquifex aeolicus RNase PH bound with a phosphate, a co-substrate, in the active site at 2.3-A resolution. RNase PH has the typical alpha/beta fold, which forms a hexameric ring structure as a trimer of dimers. This ring structure resembles that of the polynucleotide phosphorylase core domain homotrimer, another phosphorolytic exoribonuclease. Four amino acid residues, Arg-86, Gly-124, Thr-125, and Arg-126, of RNase PH are involved in the phosphate-binding site. Mutational analyses of these residues showed their importance in the phosphorolysis reaction. A docking model with the tRNA acceptor stem suggests how RNase PH accommodates substrate RNAs.

Amino Acid Sequence↗
PubMed2005

Maturation of bacteriophage T4 lagging strand fragments depends on interaction of T4 RNase H with T4 32 protein rather than the T4 gene 45 clamp.

In the bacteriophage T4 DNA replication system, T4 RNase H removes the RNA primers and some adjacent DNA before the lagging strand fragments are ligated. This 5'-nuclease has strong structural and functional similarity to the FEN1 nuclease family. We have shown previously that T4 32 protein binds DNA behind the nuclease and increases its processivity. Here we show that T4 RNase H with a C-terminal deletion (residues 278-305) retains its exonuclease activity but is no longer affected by 32 protein. T4 gene 45 replication clamp stimulates T4 RNase H on nicked or gapped substrates, where it can be loaded behind the nuclease, but does not increase its processivity. An N-terminal deletion (residues 2-10) of a conserved clamp interaction motif eliminates stimulation by the clamp. In the crystal structure of T4 RNase H, the binding sites for the clamp at the N terminus and for 32 protein at the C terminus are located close together, away from the catalytic site of the enzyme. By using mutant T4 RNase H with deletions in the binding site for either the clamp or 32 protein, we show that it is the interaction of T4 RNase H with 32 protein, rather than the clamp, that most affects the maturation of lagging strand fragments in the T4 replication system in vitro and T4 phage production in vivo.

Amino Acid Sequence↗
PubMed2006

Antisense inhibition of RNase P: mechanistic aspects and application to live bacteria.

We explored bacterial RNase P as a drug target using antisense oligomers against the P15 loop region of Escherichia coli RNase P RNA. An RNA 14-mer, or locked nucleic acid (LNA) and peptide nucleic acid (PNA) versions thereof, disrupted local secondary structure in the catalytic core, forming hybrid duplexes over their entire length. Binding of the PNA and LNA 14-mers to RNase P RNA in vitro was essentially irreversible and even resisted denaturing PAGE. Association rates for the RNA, LNA, and PNA 14-mers were approximately 10(5) m(-1) s(-1) with a rate advantage for PNA and were thus rather fast despite the need to disrupt local structure. Conjugates in which the PNA 14-mer was coupled to an invasive peptide via a novel monoglycine linker showed RNase P RNA-specific growth inhibition of E. coli cells. Cell growth could be rescued when expressing a second bacterial RNase P RNA with an unrelated sequence in the target region. We report here for the first time specific and growth-inhibitory drug targeting of RNase P in live bacteria. This is also the first example of a duplex-forming oligomer that invades a structured catalytic RNA and inactivates the RNA by (i) trapping it in a state in which the catalytic core is partially unfolded, (ii) sterically interfering with substrate binding, and (iii) perturbing the coordination of catalytically relevant Mg2+ ions.

Bacillus subtilis↗
PubMed2000

Characterization of RNABP, an RNA binding protein that associates with RNase L.

The 2',5'-oligoadenylate (2-5A)/RNase L pathway is one of several enzymatic pathways induced by interferons (IFN). RNase L is a latent endoribonuclease that is activated on its binding by 2-5A and inhibited by the ribonuclease L inhibitor (RLI). We have shown previously by coimmunoprecipitation that RNase L may be associated with a 90-kDa RNA binding protein (RNABP), identified with a monoclonal antibody (mAb) raised against an RNase L complex purified under native conditions on 2-5A-sepharose. Here we confirm, by gel-filtration and pull-down analysis, the association of RNase L and RNABP, and we demonstrate that this association is significantly increased in the presence of 2-5A. Moreover, we found that RNABP protein levels decrease during terminal differentiation in various cell lines but do not vary during vesicular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV) infection or following IFN-alpha/beta treatment. In this latter case, although total cellular RNABP levels do not vary, the amount of RNABP found in the cytoplasm increases in comparison to that found in the nucleus, indicating a cytoplasmic localization of RNABP after IFN-alpha/beta treatment. Finally, we demonstrate the interaction between RNase L and RNABP in intact cells. Microinjection of an mAb against RNABP into HeLa cells inhibits RNase L antiviral activity and partially inhibits the IFN-alpha/beta-induced antiviral activity.

Adenine Nucleotides↗
PubMed1997

Biochemical evidence for a novel low molecular weight 2-5A-dependent RNase L in chronic fatigue syndrome.

Previous studies from this laboratory have demonstrated a statistically significant dysregulation in several key components of the 2',5'-oligoadenylate (2-5A) synthetase/RNase L and PKR antiviral pathways in chronic fatigue syndrome (CFS) (Suhadolnik et al. Clin Infect Dis 18, S96-104, 1994; Suhadolnik et al. In Vivo 8, 599-604, 1994). Two methodologies have been developed to further examine the upregulated RNase L activity in CFS. First, photoaffinity labeling of extracts of peripheral blood mononuclear cells (PBMC) with the azido 2-5A photoaffinity probe, [32P]pApAp(8-azidoA), followed by immunoprecipitation with a polyclonal antibody against recombinant, human 80-kDa RNase L and analysis under denaturing conditions. A subset of individuals with CFS was identified with only one 2-5A binding protein at 37 kDa, whereas in extracts of PBMC from a second subset of CFS PBMC and from healthy controls, photolabeled/immunoreactive 2-5A binding proteins were detected at 80, 42, and 37 kDa. Second, analytic gel permeation HPLC was completed under native conditions. Extracts of healthy control PBMC revealed 2-5A binding and 2-5A-dependent RNase L enzyme activity at 80 and 42 kDa as determined by hydrolysis of poly(U)-3'-[32P]pCp. A subset of CFS PBMC contained 2-5A binding proteins with 2-5A-dependent RNase L enzyme activity at 80, 42, and 30 kDa. However, a second subset of CFS PBMC contained 2-5A binding and 2-5A-dependent RNase L enzyme activity only at 30 kDa. Evidence is provided indicating that the RNase L enzyme dysfunction in CFS is more complex than previously reported.

2',5'-Oligoadenylate Synthetase↗