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PubMed1978

Ribonuclease H from rat liver. I. Partial purification and characterization of nuclear ribonuclease H1.

A ribonuclease H, an enzyme that specifically degrades the RNA moiety of RNA-DNA hybrid, has been partially purified from rat liver nuclei and characterized. Neither native or denatured DNA, nor single or double-stranded synthetic polyribonucleotides were degraded by the enzyme. The enzyme possesses a molecular weight of about 36,000 and requires alkaline pH, magnesium ions, and ammonium sulphate for maximum activity. The enzyme acts on the hybrid as an endonuclease, resulting in oligonucleotides with 3'-hydroxyl termini. The properties of this enzyme were distinct from those of the rat liver cytosol enzyme reported by Roewekamp and Sekeris in many respects, such as molecular weight, optimal pH and requirements for divalent cations. Preliminary experiments suggest that the nuclear enzyme is localized in the nucleoplasm and nucleoli. These results indicate that multiple forms of ribonuclease H exist in different regions of rat liver cells.

Animals↗
PubMed1978

Ribonuclease H from rat liver. II. Partial purification and characterization of cytosol ribonuclease H1.

We have detected in rat liver cytosol three enzymes (termed C-1, C-2, and C-3) which cleaved the RNA moiety of RNA-DNA hybrid. These enzymes were separated from each other by DEAE-Sephadex and Sephadex G-200 chromatography. C-1 and C-2 specifically act on the RNA moiety of RNA-DNA hybrid, while C-3 degrades single-stranded RNA as well as the RNA of the hybrid. The molecular weights of C-1, C-2, and C-3 are about 110,000, 35,000 and 110,000 daltons, respectively, and their activities are absolutely dependent on divalent cations such as Mg2+ and Mn2+. Cleavage by C-1 and C-2 is endonucleolytic, producing mostly oligonucleotides and a small amount of mononucleotides which possess 3'-hydroxyl termini. It seems likely that C-2 is originally present in the nucleus and is released into cytosol because of its loose binding to the nuclear components. As for biochemical properties, C-1 is very similar to the cytosol ribonuclease H initially reported by Roewekamp and Sekeris, and C-2 is very similar to the nuclear ribonuclease H reported by us in the preceding paper.

Animals↗
PubMed1994

Ribonuclease E provides substrates for ribonuclease P-dependent processing of a polycistronic mRNA.

The polycistronic mRNA of the histidine operon is subject to a processing event that generates a rather stable transcript encompassing the five distal cistrons. The molecular mechanisms by which such a transcript is produced were investigated in Escherichia coli strains carrying mutations in several genes for exo- and endonucleases. The experimental approach made use of S1 nuclease protection assays on in vivo synthesized transcripts, site-directed mutagenesis and construction of chimeric plasmids, dissection of the processing reaction by RNA mobility retardation experiments, and in vitro RNA degradation assays with cellular extracts. We have found that processing requires (1) a functional endonuclease E; (2) target site(s) for this activity in the RNA region upstream of the 5' end of the processed transcript that can be substituted by another well-characterized rne-dependent cleavage site; (3) efficient translation initiation of the first cistron immediately downstream of the 5' end; and (4) a functional endonuclease P that seems to act on the processing products generated by ribonuclease E. This is the first evidence that ribonuclease P, an essential ribozyme required for the biosynthesis of tRNA, may also be involved in the segmental stabilization of a mRNA.

Amino Acid Sequence↗
PubMed1976

The role of lysine-41 in ribonuclease A studied by proton-magnetic-resonance spectroscopy of guanidinated ribonuclease A.

Ribonuclease A has been guanidinated at the lysine residues and the nona-guanidinated and deca-guanidinated (fully substituted) products separated. In confirmation of an earlier report by Glick and Barnard (1970), it has been shown by chemical procedures that the former derivative is not reacted at lysine-41. Guanidination of lysine-41 to produce the fully substituted product causes loss of enzymic activity without any apparent change of conformation, as tested by conformational comparisons (using proton magnetic resonance spectroscopy) including (a) difference spectroscopy, evidence for the involvement of lysine-41 in a catalytic role in the enzyme. Dimethylation of lysine-41 of nona-guanidinated ribonuclease A produces sharp proton resonances which shifts as the dimethylamino group is titrated and allow the determination of an apparent pK of 8.8 for unsubstituted lysine-41.

Binding Sites↗
PubMed1983

The active site of ribonuclease A from the crystallographic studies of ribonuclease-A-inhibitor complexes.

The results of X-ray analyses of three ribonuclease-A-nucleotide complexes, at 2.3 A, are reported. A modified purine mononucleotide, 8-oxo-guanosine 2'-phosphate in a syn conformation, binds at the pyrimidine-binding site of the catalytic cleft. Solvent molecules are expelled from the active site due to inhibitor binding. The positions of the side-chains of the active-site residues Gln-11, His-12 and Thr-45 are well defined and do not alter on inhibitor binding. The mobility of Lys-41 is greatly reduced in the protein-nucleotide complexes and the terminal amino group interacts directly with the 2'-phosphate group of the nucleotides. In the complex of the enzyme with a modified pyrimidine, cytidine-N(3)-oxide 2'-phosphate, His-119 is stabilised in the minor site of the native protein [Borkakoti, N., Moss, D.S. and Palmer, R.A. (1982) Acta Crystallogr. B38, 2210-2217], while in the protein-purine derivative the imidazole group is located in the major site. Inhibitor binding induces movements in the side-chains of Lys-7 and Lys-66 which also modify the conformation of the active-site cleft of ribonuclease A.

Amino Acids↗
PubMed1971

Mutants of Escherichia coli lacking endonuclease I, ribonuclease I, or ribonuclease II.

To isolate mutants of Escherichia coli K-12 lacking endonuclease I activity (end), a method has been developed which detects, by differential methyl green staining, undegraded deoxyribonucleic acid (DNA) in colonies previously incubated in toluene. This procedure allows isolation of mutant strains in which DNA degradation is reduced. For half of these strains, this defect has been correlated with deficiencies of endonuclease I, ribonuclease I (rns), or ribonuclease II (rne) activities. The enzymatic deficiencies of the other strains remain unknown. An rne mutation is cotransducible with serA (which is located at 56 min on the genetic map). Most end mutations, called endA, are also cotransducible with serA and are located between serA and strA. One end mutation, called endB, is located between purE and trp (i.e., between 13 and 25 min on the genetic map).

Cell-Free System↗
PubMed2000

Effect of bovine seminal ribonuclease and bovine pancreatic ribonuclease A on bovine oocyte maturation.

Bovine seminal ribonuclease (BS-RNase) contains the MxM (noncovalent dimer) and M=M (free monomer) in constant ratio. The aim of this work was to evaluate the effect of BS-RNase, its monomer and dimer forms, and also various mutants of this enzyme on meiotic completion in cattle oocytes. It was found that BS-RNase has irreversible effects on the meiotic maturation of bovine oocytes in vitro, particularly on the completion of meiosis. The effect of BS-RNase is dose-dependent. In medium supplemented with 1 microg/ml, the results were comparable with those of the control (70% MII oocytes after 24 hr of culture). Whereas 5 microg/ml reduced the number of MII oocytes to 50%, 10 and 25 microg/ml arrested this process completely. The MxM form and RNase A at 5 microg/ml inhibited the maturation rate by 71 and 48%, respectively, but a less significant effect was observed for the M=M form, or the carboxymethylated monomers MCM31 and MCM32 (21%, 16%, and 42% MII oocytes, respectively, in comparison with control). These data demonstrate that bovine ribonucleases can have variable detrimental effects on the maturation of bovine oocyte. J. Exp. Zool. 287:394-399, 2000.

Animals↗
PubMedDate not supplied

[Brownian dynamics simulation of the association of Bacillus amyloliquefaciens ribonuclease (barnase) and Bacillus intermedius ribonuclease (binase) with barstar].

A comparative study of the association of two ribonucleases, barnase and binase, with the polypeptide inhibitor barstar has been performed by the Brownian dynamics simulation method. It was shown that the method adequately reproduced the dependence of the association rate on pH and ionic strength of solution and the influence of mutations of some ribonuclease amino acids. Two types of energetically favorable complexes of binase-barstar encounter were determined. In the type I complex, the amino acids of binase active center take part in the complex formation. In the second complex, the active center is free. It was supposed that the temporary binding of barstar into complex of type II is competitive relative to the inhibition reaction. This can partially explain the decrease in the rate of binase inhibition as compared with the corresponding reaction of barnase.

Bacterial Proteins↗
PubMed1991

Studies on the interaction of ribonuclease inhibitor with pancreatic ribonuclease involving differential labeling of cysteinyl residues.

Ribonuclease inhibitor (RI) is a protein that forms a very tight complex with ribonucleases (RNases) of the pancreatic type. RI contains 30 thiol groups, some of which are important for the enzyme-inhibitor interaction. To examine which thiols are affected by the binding of RNase, differential labeling experiments were performed. Reaction of porcine RI with the cysteine-specific labeling reagent 4-N,N-dimethylaminoazobenzene-4'-iodoacetamido-2'-sulfonic acid resulted in labeling of an average of 7.4 of the 30 cysteinyl residues. Binding of bovine pancreatic RNase A caused a 3.2-fold reduction in the extent of modification. Peptide mapping showed that in free RI, Cys-57, -371, and -404 were labeled to the greatest extent (yield, 0.4-0.6 mol/mol). RNase A did not protect Cys-57 against modification, whereas the labeling of Cys-371 and -404 was reduced by more than 90%. A second group of residues was labeled to a lesser extent in free RI (yield, 0.04-0.2 mol/mol). Within this group 11 residues were protected by RNase A by more than 90%, 2 were not affected at all, and 7 were protected between 10 and 90%. Seven cysteinyl residues in RI that were protected in the RI.RNase A complex were no longer protected in the RI.S-protein complex. These residues were mainly present in the N-terminal region of RI. However, when the S-peptide was included to yield the RI.RNase S complex, the same pattern of labeling was obtained as with the RI.RNase A complex. Addition of the S-peptide alone had no effect on the labeling. The implications of these observations with respect to RNase binding areas of RI are discussed in relation to the results obtained from the analysis of active RI molecules that contain deletions.

Amino Acid Sequence↗
PubMed1980

[Serum ribonuclease activity in gynecologic neoplasms. The behavior of serum ribonuclease in ovarian cancer under cytostatic therapy].

Ribonuclease activity in the serum of patients with histologically confirmed ovarian carcinoma is significantly higher than in serum of patients with benign tumors of the ovary and that of healthy controls. In 847 samples collected during a period of 20 months the course of the ribonuclease activity of 41 patients receiving cytostatic treatment was determined. The ovarian carcinoma of all 41 patients was histologically ascertained. Three typical examples are presented.

Female↗
PubMed1989

Primary structure of a ribonuclease from porcine liver, a new member of the ribonuclease superfamily.

In most tissues, ribonucleases (RNases) are found in a latent form complexed with ribonuclease inhibitor (RI). To examine whether these so-called cytoplasmic RNases belong to the same superfamily as pancreatic RNases, we have purified from porcine liver two such RNases (PL1 and PL3) and examined their primary structures. It was found that RNase PL1 belonged to the same family as human RNase Us [Beintema et al. (1988) Biochemistry 27, 4530-4538] and bovine RNase K2 [Irie et al. (1988) J. Biochem. (Tokyo) 104, 289-296]. RNase PL3 was found to be a hitherto structurally uncharacterized type of RNase. Its polypeptide chain of 119 amino acid residues was N-terminally blocked with pyroglutamic acid, and its sequence differed at 63 positions with that of the pancreatic enzyme. All residues important for catalysis and substrate binding have been conserved. Comparison of the primary structure of RNase PL3 with that of its bovine counterpart (RNase BL4; M. Irie, personal communication) revealed an unusual conservation for this class of enzymes; the 2 enzymes were identical at 112 positions. Moreover, comparison of the amino acid compositions of these RNases with that of a human colon carcinoma-derived RNase, RNase HT-29 [Shapiro et al. (1986) Biochemistry 25, 7255-7264], suggested that these three proteins are orthologous gene products. The structural characteristics of RNases PL1 and PL3 were typical of secreted RNases, and this observation questions the proposed cytoplasmic origin of these RI-associated enzymes.

Amino Acid Sequence↗
PubMed1992

Revisiting the action of bovine ribonuclease A and pancreatic-type ribonucleases on double-stranded RNA.

Single-strand-preferring ribonucleases of the pancreatic type, structurally and/or catalytically similar to bovine RNase A but endowed with a higher protein basicity, are able to degrade double-stranded RNA (dsRNA) or DNA:RNA hybrids under standard assay conditions (0.15 M NaCl, 0.015 M sodium citrate, pH 7), where RNase A is inactive. This enzyme too, however, becomes quite active if assay conditions are slightly modified or its basicity is increased (polyspermine-RNase). In the attempt to review these facts, we have analyzed and discussed the role that in the process have the secondary structure of dsRNA as well as other variables whose influence has come to light in addition to that of the basicity of the enzyme protein, i.e., the ionic strength, the presence of carbohydrates on the RNase molecule, and the structure (monomeric or dimeric) of the enzyme. A possible mechanism by which dsRNAs are attacked by pancreatic-type RNases has been proposed.

Amino Acids↗
PubMed1983

Ribonuclease and ribonuclease inhibitor in the human pancreas.

Several investigators have failed to confirm any specificity of elevated serum ribonuclease (RNase) in the diagnosis of pancreatic cancer. Although RNase had been known to be present in two forms, free and inhibitor-bound, in various tissues of the rat, little was known about it in the human pancreas. The object of this report was to explore the presence of RNase inhibitor in the human pancreas through the assay of both active (= free) and total (= sum of free and inhibitor-bound) RNases. Inhibitor-bound RNase is also referred to as latent RNase. RNase was classified into three types according to pH (acid, neutral, and alkaline RNases) in the pancreatic supernatant fraction. An inhibitor was separated from latent RNase by p-chloromercuribenzoic acid (PCMB), and the latent RNase was changed to an active form. Latent RNase was more active on the alkaline side with a maximum at pH 7.5. Hence, the presence of RNase inhibitor was highly probable in the pancreatic supernatant fraction. RNase inhibitor is most likely a protein, which is bound with both neutral and alkaline RNases. RNase inhibitor may be a cause of nonspecificity and/or low sensitivity of RNase in the serum as a diagnostic marker for pancreatic cancer.

4-Chloromercuribenzenesulfonate↗
PubMed1999

Toward rational design of ribonuclease inhibitors: high-resolution crystal structure of a ribonuclease A complex with a potent 3',5'-pyrophosphate-linked dinucleotide inhibitor.

The crystal structure of ribonuclease A (RNase A) in complex with pdUppA-3'-p [5'-phospho-2'-deoxyuridine-3'-pyrophosphate (P'-->5') adenosine 3'-phosphate] has been determined at 1.7 A resolution. This dinucleotide is the most potent low molecular weight inhibitor of RNase A reported to date (K(i) = 27 nM) and is also effective against two major nonpancreatic RNases: eosinophil-derived neurotoxin and RNase-4; in all cases, tight binding in large part derives from the unusual 3',5'-pyrophosphate internucleotide linkage [Russo, N., and Shapiro, R. (1999) J. Biol. Chem. 274, 14902-14908]. The design of pdUppA-3'-p was based on the crystal structure of RNase A complexed with 5'-diphosphoadenosine 3'-phosphate (ppA-3'-p) [Leonidas, D. D., Shapiro, R., Irons, L. I., Russo, N., and Acharya, K. R. (1997) Biochemistry 36, 5578-5588]. The adenosine of pdUppA-3'-p adopts an atypical syn conformation not observed for standard adenosine nucleotides bound to RNase A. This conformation, which allows extensive interactions with Asn 67, Gln 69, Asn 71, and His 119, is associated with the placement of the 5'-beta-phosphate of the adenylate, rather than alpha-phosphate, at the site where substrate phosphodiester bond cleavage occurs. The contacts of the deoxyuridine 5'-phosphate portion of pdUppA-3'-p appear to be responsible for the 9-fold increased affinity of this compound as compared to ppA-3'-p: the uracil base binds to Thr 45 in the same manner as previous pyrimidine inhibitors, and the terminal 5'-phosphate is positioned to form medium-range Coulombic interactions with Lys 66. The full potential benefit of these added interactions is not realized because of compensatory losses of hydrogen bonds of Lys 7 and Gln 11 with the terminal 3'-phosphate and the adenylate 5'-alpha-phosphate, which were not predicted by modeling. The results reported here have important implications for the design of improved inhibitors of RNase A and for the development of therapeutic agents to control the activities of RNase homologues such as eosinophil-derived neurotoxin and angiogenin that have roles in human pathologies.

Adenosine Diphosphate↗
PubMed1983

Studies on salivary gland ribonucleases. III. Purification and properties of three ribonucleases from bovine parotid gland.

Three alkaline ribonucleases [EC 3.1.4.22] were purified 4,500- to 7,850-fold from bovine parotid gland by repeated CM-Sephadex C-25 chromatography and Sephadex G-50 gel filtration, with a total recovery of about 33%. They were designated as RNase BP1, BP2, and BP3, based on their order of elution from a CM-Sephadex C-25 column. The molecular weights of these enzymes were estimated by gel electrophoresis to be 18,500, 18,000, and 14,000, respectively. These enzymes are very similar to RNase A in that they are inhibited by heparin, show preferential hydrolysis of C5'-O-P linkages adjacent to a cytosine nucleotide rather than a uracil nucleotide, and in their antigenic properties. Spermine was found to stimulate the activities of these enzymes; the degree of stimulation was in the order of RNase BP3 greater than BP2 greater than BP1. The stimulation by spermine is mainly due to the increased cleavage of C5'-O-P linkages adjacent to cytosine nucleotides. From the results of amino acid analysis, glycosidase digestion and amino-terminal sequencing, it was suggested that the differences in molecular weights of RNase BP1, BP2, and BP3 are due to the differences in carbohydrate contents. In addition, it was suggested that RNase BP3 is identical with RNase A.

Animals↗
PubMed1976

The structure and function of ribonuclease T1. XXI. Modification of histidine residues in ribonuclease T1 with iodoacetamide.

1. When ribonuclease T1 [EC 3.1.4.8] (0.125% solution) was treated with a 760-fold molar excess of iodoacetamide at pH 8.0 and 37 degrees, about 90% of the original activity was lost in 24 hr. The half-life of the activity was about 8 hr. The binding ability for 3'-GMP was lost simultaneously. Changes were detected only in histidine and the amino-terminal alanine residues upon amino acid analyses of the inactivated protein and its chymotryptic peptides. The inactivation occurred almost in parallel with the loss of two histidine residues in the enzyme. The pH dependences of the rate of inactivation and that of loss of histidine residues were similar and indicated the implication of a histidine residue or residues with pKa 7.5 to 8 in this reaction. 3'-GMP and guanosine showed some protective effect against loss of activity and of histidine residues. The reactivity of histidine residues was also reduced by prior modification of glutamic acid-58 with iodoacetate, of lysine-41 with maleic or cis-aconitic anhydride or 2,4,6-trinitrobenzenesulfonate or of arginine-77 with ninhydrin. 2. Analyses of the chymotryptic peptides from oxidized samples of the iodoacetamide-inactivated enzyme showed that histidine-92 and histidine-40 reacted with iodoacetamide most rapidly and at similar rates, whereas histidine-27 was least reactive. Alkylation of histidine-92 was markedly slowed down when the Glu58-carboxymethylated enzyme was treated with iodoacetamide. On the other hand, alkylation of histidine-40 was slowed down most in the presence of 3'-GMP. These results suggest that histidine-92 and histidine-40 are involved in the catalytic action, probably forming part of the catalytic site and part of the binding site, respectively, and that histidine-27 is partially buried in the enzyme molecule or interacts strongly with some other residue, thus becoming relatively unreactive.

Alanine↗
PubMed1977

The structure and function of ribonuclease T1. XXIII. Inactivation of ribonuclease T1 by reversible blocking of amino groups with cis-aconitic anhydride and related dicarboxylic acid anhydrides.

Ribonuclease T1 [EC 3.1.4.8] was inactivated rapidly by treatment at pH 8.0 and 0 degrees C with cis-aconitic anhydride and related dicabroxylic acid anhydrides, including citraconic, maleic, and succinic anhydrides. Under reaction conditions used, roughly 90% inactivation occurred within 30 min. Analyses of the inactivated enzymes indicated that the reaction took place fairly specifically at the alpha-amino group of the N-terminal alanine and the epsilon-amino group of lysine-41. Upon incubation of these inactivated enzymes at pH 3.6 and 37 degreeC, the activity was regenerated to various extents, depending on the nature of the introduced acyl groups. Under these conditions, the enzyme modified with cis-aconitc anhydride or citraconic anhydride recovered much of the origninal activity after 48 h whereas the enzyme modified with maleic anhydride recovered its activity only partially. Practically no activity was regenerated in the case of the enzyme modified with succinic anhydride under these conditions. The inactivation appears to be due mainly to the effect of the carboxyl group introduced at the epsilon-amino group of lysine-41. The results suggest the usefulness of cis-aconitic anhydride as a reversible blocking reagent for amino groups in proteins.

Aconitic Acid↗