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PubMed2002

The ribbon of hydrogen bonds and the pseudomolecule in the three-dimensional structure of globular proteins. III. Bovine pancreatic ribonuclease A and bovine seminal ribonuclease.

The model of the three-dimensional structure of globular proteins, which is based on a ribbon of hydrogen bonds along the whole of the backbone, is now applied to the comparison between monomeric bovine pancreatic ribonuclease A and dimeric bovine seminal ribonuclease. Some waters are involved in the hydrogen bonding of the ribbon, and the protein molecule plus these waters forms a pseudomolecule. The conformations of the three backbones are essentially identical and the three ribbons of hydrogen bonds are conserved with greater than 90% accuracy. We suggest that the conservation of the backbone conformations of the two molecules is a consequence of the conservation of the ribbons of hydrogen bonds. There are 16 simple mutations between the two molecules, of which 15 involve only side-chain groups with no more than one hydrogen bond to the backbone. Such mutations are not sufficient to change the ribbon of hydrogen bonds and hence there is no change in the backbone conformation. Generalizing this result, we suggest that the conservation of the ribbon is the reason why single point mutations rarely change the conformation of the backbone of the globular proteins.

Animals↗

PubMed1999

Hydrogen exchange in ribonuclease A and ribonuclease S: evidence for residual structure in the unfolded state under native conditions.

Two-dimensional NMR spectroscopy has been used to monitor the exchange of backbone amide protons in ribonuclease A (RNase A) and its subtilisin-cleaved form, ribonuclease S (RNase S). Exchange measurements at two different pH values (5.4 and 6.0) show that the exchange process occurs according to the conditions of the EX2 limit. Differential scanning calorimetry measurements have been carried out in 2H2O under conditions analogous to those used in the NMR experiments in order to determine the values of DeltaCp, DeltaHu and Tm, corresponding to the thermal denaturation of both proteins. For the amide protons of a large number of residues in RNase A, the free energies at 25 degreesC for exchange competent unfolding processes are much lower than the calorimetric denaturation free energies, thus showing that exchange occurs through local fluctuations in the native state. For 20 other protons, the cleavage reaction had approximately the same effect on the exchange rate constants than on the equilibrium constant for unfolding, indicating that those protons exchange by global unfolding. There is a good agreement between the residues to which these protons belong and those involved in the putative folding nucleation site identified by quench-flow NMR studies. The unfolding free energies of the slowest exchanging protons, DeltaGex, as evaluated from exchange data, are much larger than the calorimetric free energies of unfolding, DeltaGu. Given the agreement between DeltaDeltaGex(A-S), the difference in free energy from exchange for a given proton of the two proteins, and DeltaDeltaGu(A-S), the difference in the calorimetric free energy of the two proteins, the discrepancy indicates that the intrinsic exchange rates in the unfolded state of those protons cannot be approximated by those measured in short unstructured peptides and, consequently, exchange for those protons in RNase A and S must occur through a rather structured denatured state.

Amino Acid Sequence↗

PubMed1993

Residues 287-301 of human ribonuclease inhibitor do not affect ribonuclease activity and inhibitor binding; a reply.

Residues 287-301 of human placental ribonuclease inhibitor have been reported to inhibit pancreatic ribonuclease A in a similar way as the entire inhibitor (Crevel-Thieffry, I., Cotterill, S. and Schuller, E. (1992) Biochim. Biophys. Acta 1122, 107-112). Using three different assays, we were unable to observe inhibition by the synthetic peptide. Moreover, the peptide did not compete with the entire inhibitor for binding to RNAase A.

Amino Acid Sequence↗

PubMed1991

Interaction of human placental ribonuclease with placental ribonuclease inhibitor.

The interactions of human placental ribonuclease inhibitor (PRI) with bovine pancreatic ribonuclease (RNase) A and human angiogenin, a plasma protein that induces blood vessel formation, have been characterized in detail in earlier studies. However, studies on the interaction of PRI with the RNase(s) indigenous to placenta have not been performed previously, nor have any placental RNases been identified. In the present work, the major human placental RNase (PR) was purified to homogeneity by a five-step procedure and was obtained in a yield of 110 micrograms/kg of tissue. The placental content of angiogenin was also examined and was found to be at least 10-fold lower than that of PR. On the basis of its amino acid composition, amino-terminal sequence, and catalytic properties, PR appears to be identical with an RNase previously isolated from eosinophils (eosinophil-derived neurotoxin), liver, and urine. The apparent second-order rate constant of association for the PR.PRI complex, measured by examining the competition between PR and angiogenin for PRI, is 1.9 X 10(8) M-1 s-1. The rate constant for dissociation of the complex, determined by HPLC measurement of the rate of release of PR from its complex with PRI in the presence of a scavenger for free PRI, is 1.8 X 10(-7) s-1. Thus the Ki value for the PR.PRI complex is 9 X 10(-16) M, similar to that obtained with angiogenin, and 40-fold lower than that measured with RNase A. Complex formation causes a small red shift in the protein fluorescence emission spectrum, with no significant change in overall intensity. The fluorescence quantum yield of PR and the Stern-Volmer constant for fluorescence quenching by acrylamide are both high, possibly due to the presence of an unusual posttranslationally modified tryptophan residue at position 7 in the primary sequence.

Amino Acids↗

PubMed1987

Two histidine residues are essential for ribonuclease T1 activity as is the case for ribonuclease A.

Ribonuclease T1 (RNase T1, EC 3.1.27.3) is a guanosine-specific ribonuclease that cleaves the 3',5'-phosphodiester linkage of single-stranded RNA. It is assumed that the reaction is generated by concerted acid-base catalysis between residues Glu-58 and His-92 or His-40. From the results of chemical modification and NMR studies, it appeared that the residue Glu-58 was indispensable for nucleolytic activity. However, we have recently demonstrated that Glu-58 is an important but not an essential residue for catalytic activity, using the methods of genetic engineering to change Glu-58 to Gln-58 etc [Nishikawa, S., Morioka, H., Fuchimura, K., Tanaka, T., Uesugi, S., Ohtsuka, E., & Ikehara, M. (1986) Biochem. Biophys. Res. Commun. 138, 789-794]. In the present paper, we report that mutants of RNase T1 with residue Ala-40 or Ala-92 have almost no activity, while mutants that contain Ala-58 retain considerable activity. These results show that the two histidine residues, His-40 and His-92, but not Glu-58, are indispensable for the catalytic activity of the enzyme. We propose a revised reaction mechanism in which two histidine residues play a major role, as they do in the case of RNase A.

Aspergillus oryzae↗

PubMed1997

Kinetic and thermodynamic thermal stabilities of ribonuclease A and ribonuclease B.

The thermal stabilities of ribonuclease A (RNase A) and ribonuclease B (RNase B), which possess identical protein structures but differ by the presence of a carbohydrate chain attached to Asn34 in RNase B, were studied by proteolysis and UV spectroscopy at pH 8.0. Proteolysis was quantified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry. Increasing protease concentrations led to a hyperbolic increase of the rate constants of proteolysis. With thermolysin, which attacks the unfolded molecules only, the thermal unfolding constants were determined by extrapolating the rate constants of proteolysis to infinite concentration of protease. With trypsin, the unfolding constants of RNase A could be confirmed. Subtilisin attacked even the native RNases, where RNase B was more stable toward proteolytic degradation. Kinetic stabilities (deltaG++) calculated from the unfolding constants for temperatures between 52.5 and 65 degrees C revealed a higher kinetic stability of RNase B, which results from enthalpic effects only, whereas entropic effects counteract stabilization. delta deltaG++ at the transition temperature of RNase A (60.4 degrees C) was 2.2 +/- 0.3 kJ mol(-1). Thermodynamic stabilities (deltaG) were estimated from the thermal transition curves at 287 nm for the temperature range from 55 to 70 degrees C. For 17.5-25 degrees C, deltaG values were determined from transition curves of unfolding induced by guanidine hydrochloride and extrapolation of the free energy values to those in the absence of denaturant. At all temperatures, RNase B proved to be more stable than RNase A with essentially the same enthalpy and entropy of unfolding. delta deltaG was 2.5 +/- 0.2 kJ mol(-1) at 60.4 degrees C and 2.3 kJ mol(-1) at 25 degrees C.

Enzyme Stability↗

PubMed1992

Secretion of mammalian ribonucleases from Escherichia coli using the signal sequence of murine spleen ribonuclease.

A nucleotide sequence identical with that of the recently identified murine pancreatic ribonuclease (RNAase) was isolated from a murine spleen cDNA library. Active RNAase was expressed and secreted from Escherichia coli lon-htpr- transformed with a plasmid containing the E. coli trp promoter followed by the murine RNAase gene sequence, including the original eukaryotic 26-amino-acid signal sequence. Approx. 1 mg of properly matured RNAase protein/litre was secreted into the medium of a fermentor culture after the promotor was induced by tryptophan starvation. When the signal sequence was deleted from the plasmid, intracellular RNAase activity was very low and there was no significant supernatant RNAase activity. Even higher RNAase yields were obtained with a synthetic gene for bovine pancreatic ribonuclease cloned after the signal sequence of the murine gene. About 2 mg of correctly processed RNAase A/litre was isolated from the growth medium, and a further 8-10 mg of correctly processed RNAase/litre could be isolated from the soluble fraction of the cells. Thus this eukaryotic signal sequence is both recognized by the E. coli transport and processing apparatus and gives efficient secretion, as well as export, of active, mature mammalian RNAases.

Amino Acid Sequence↗

PubMed1988

The amino acid sequence of ribonuclease N1, a guanine-specific ribonuclease from the fungus Neurospora crassa.

The complete amino acid sequence of ribonuclease N1 (RNase N1), a guanine-specific ribonuclease from a fungus, Neurospora crassa, was determined by conventional protein sequencing, using peptide fragments obtained by tryptic digestion of cyanogen bromide-treated RNase N1 and by Staphylococcus aureus V8 protease digestion of heat-denatured RNase N1. The results showed that the protein is composed of a single polypeptide chain of 104 amino acid residues cross-linked by two disulfide bonds and has a molecular weight of 11,174: (sequence; see text) (Disulfide bonds: C2-C10, C6-C103) The amino acid sequence was homologous with those of RNase T1 (65% identity) and related microbial RNases.

Amino Acid Sequence↗

PubMed1976

The primary structure of non-initiator methionine transfer ribonucleic acid from Bakers' yeast. I. Purification and complete digestion with ribonuclease T1 and pancreatic ribonuclease A.

The methionine acceptor activity of a crude tRNA from bakers' yeast was resolved into two peaks (I and II) by column chromatography on DEAE-Sephadex A-25 with a 1 M phosphate system. Methionine tRNA from peak II was not formylated by E. coli methionyl-tRNA transformylase [EC 2.1.2.9.] after being charged with methionine, whereas that from peak I was formylatable under the same conditions. A substantial amount of unlabelled methionine tRNA, tRNAMetm, was highly purified from the peak II fraction by successive chromatographic procedures. The purified tRNAMetm was digested with pancreatic ribonuclease A [EC 3.1.4.22] and ribonuclease T1 [EC 3.1.4.8]. The digestion products were isolated into individual components and completely sequenced. The results of sequence analysis of the two RNase digests were in good agreement and indicated that the chain length of this tRNA is 76, including 13 modified nucleotides. These oligonucleotide fragments can be constructed into a unique total sequence, assuming a few conventional features of clover leaf structure for the tRNA was established by analyses of partial digestion products with RNase T1, as reported in the accompanying paper.

Base Sequence↗

PubMed1977

The structure and function of ribonuclease T1. XXII. Tryptic cleavages of the single lysyl and arginyl bonds in ribonuclease T1.

1. When ribonuclease T1 [EC 3.1.4.8] was treated with trypsin [EC 3.4.21.4] at pH 7.5 and 37 degrees, activity was lost fairly slowly. At higher temperatures, however, the rate of inactivation was markedly accelerated. The half life of the activity was about 2.5 h at 50 degrees and 1 h at 60 degrees. 3'-GMP and guanosine protected the enzyme significantly from tryptic inactivation. 2. Upon tryptic digestion at 50 degrees, the Lys-Tyr (41-42) and Arg-Val (77-78) bonds were cleaved fairly specifically, yielding two peptide fragments. One was a 36 residue peptide comprizing residues 42 to 77. The other was a 68 residue peptide composed of two peptide chains cross-linked by a disulfide bond between half-cystines -6 and -103, comprizing residues 1 to 41 and 78 to 104. 3. When the trinitrophenylated enzyme, in which the alpha-amino group of alanine-1 and the episolone-amino group of lysine 41 were selectively modified, was treated with trypsin at 37 degrees, the activity was lost fairly rapidly with a half life of about 4 h. In this case, tryptic hydrolysis occurred fairly selectively at the single Arg-Val bond. Thus the enzyme could be inactivated by cleavage of a single peptide bond in the molecule, an indication of the importance of the peptide region involving the single arginine residue at position 77 in the activity of ribonuclease T1.

Amino Acids↗

PubMed1977

Studies on the restoration of the activities of Ribonucleases by polyamines in the presence of various ribonuclease inhibitors.

The effect of polyamines on ribonucleases in the presence of various inhibitors (poly(G), heparin, and rat liver RNase inhibitor) has been studied. Bovine pancreatic RNas A and a ribonuclease from horse submaxillary gland (RNase HS) were inhibited by the inhibitors, but RNase T1 and RNase M were not inhibited. Polyamines were found to restore the activites of RNase A and RNase HS inhibited by poly(G) or heparin but not those activities inhibited by rat liver RNase inhibitor. When poly(U) and poly(C) were used as substrates, the inhibitory effects of poly(G) and heparin were greater with poly(U) than poly(C) as a substrate. However, when poly(C) was used as a substrate in the presence of either of the above inhibitors, the restoration of RNase activity by sperimine was more efficient. In fact, a stimulatory effect was observed. From the double-reciprocal plots, it was concluded that polyamines restored the activiities of RNases by increasing the availability of the substrate and enzyme to each other. The restoration of enzyme activity by polyamines occurred through the binding of the polyamines to the inhibitor and the subsequent release of enzyme from the inhibitor.

Animals↗

PubMed2003

Subtle functional collective motions in pancreatic-like ribonucleases: from ribonuclease A to angiogenin.

The analysis of the dynamic behavior of enzymes is fundamental to structural biology. A direct relationship between protein flexibility and biological function has been shown for bovine pancreatic ribonuclease (RNase A) (Rasmussen et al., Nature 1992;357:423-424). More recently, crystallographic studies have shown that functional motions in RNase A involve the enzyme beta-sheet regions that move concertedly on substrate binding and release (Vitagliano et al., Proteins 2002;46:97-104). These motions have been shown to correspond to intrinsic dynamic properties of the native enzyme by molecular dynamics (MD) simulations. To unveil the occurrence of these collective motions in other members of pancreatic-like superfamily, we carried out MD simulations on human angiogenin (Ang). Essential dynamics (ED) analyses performed on the trajectories reveal that Ang exhibits collective motions similar to RNase A, despite the limited sequence identity (33%) of the two proteins. Furthermore, we show that these collective motions are also present in ensembles of experimentally determined structures of both Ang and RNase A. Finally, these subtle concerted beta-sheet motions were also observed for other two members of the pancreatic-like superfamily by comparing the ligand-bound and ligand-free structures of these enzymes. Taken together, these findings suggest that pancreatic-like ribonucleases share an evolutionary conserved dynamic behavior consisting of subtle beta-sheet motions, which are essential for substrate binding and release.

Animals↗

PubMed2002

Fluorescence assay for the binding of ribonuclease A to the ribonuclease inhibitor protein.

Ribonuclease A (RNase A) and the ribonuclease inhibitor protein (RI) form one of the tightest known protein-protein complexes. RNase A variants and homologues, such as G88R RNase A, that retain ribonucleolytic activity in the presence of RI are toxic to cancer cells. Herein, a new and facile assay is described for measuring the equilibrium dissociation constant (K(d)) and dissociation rate constant (k(d)) for complexes of RI and RNase A. This assay is based on the decrease in fluorescence intensity that occurs when a fluorescein-labeled RNase A binds to RI. To allow time for equilibration, the assay is most readily applied to those complexes with K(d) values in the nanomolar range or higher. Using this assay, the value of K(d) for the complex of RI with fluorescein-labeled G88R RNase A was determined to be 0.55 +/- 0.03 nM. In addition, the value of K(d) was determined for the complex of RI with unlabeled G88R RNase A to be 0.57 +/- 0.05 nM by using a competition assay with fluorescein-labeled G88R RNase A. Finally, the value of k(d) for the complex of RI with fluorescein-labeled G88R RNase A was determined to be (7.5 +/- 0.4) x 10(-3) s(-1) by monitoring the increase in fluorescence intensity upon dissociation. This assay can be used to characterize complexes of RI with a wide variety of RNase A variants and homologues, including those with cytotoxic activity.

Amino Acid Substitution↗

PubMed1986

Comparison of the structure of turtle pancreatic ribonuclease with those of mammalian ribonucleases.

There are 33 invariant amino acid positions out of 132 positions in 42 investigated sequences of ribonucleases from a number of mammalian species and a reptile (snapping turtle, Chelydra serpentina). These invariant residues are unequally distributed over 3 different parts of the molecule. The lobe of the S-protein part of the molecule, which lacks one disulfide bridge and has two shortened loops in turtle ribonuclease, has the lowest percentage of invariant residues, although the active-site residue His 119 is located in this part.

Amino Acid Sequence↗

PubMed1997

Co-purification of a ribonuclease and human chorionic gonadotrophin beta-core protein from human urine and displacement of 125I-human luteinizing hormone from Candida albicans binding sites by ribonucleases.

An 18 kDa pregnancy urine protein preparation, purified to apparent electrophoretic homogeneity as judged by silver-staining of polyacrylamide gels, inhibited binding of 125I-hLH (human luteinizing hormone) to Candida albicans microsomes, reacted with monoclonal and polyclonal antibodies raised against human chorionic gonadotrophin (hCG) beta-core protein and exhibited ribonuclease (RNase) activity. Eleven of the 12 amino acids at the N-terminus of a protein in this preparation were identical to those of the N-terminus of human non-secretory ribonuclease. These results indicate co-purification of hCG beta-core with a RNase. An 18 kDa RNase was also purified from a commercial hCG preparation (Chorulon). However, no RNase activity was detected in a highly purified commercial preparation (Profasi). Three commercial RNase preparations displaced 125I-hLH from C. albicans binders at extremely low concentrations (< 0.001 microg/ml RNase) whereas only slight displacement of 125I-hLH from sheep luteal binding sites was observed with very high concentrations of the RNases (100 microg/ml RNase). The co-purification of hCG beta-core and RNase from pregnancy urine and the displacement of 125I-hLH from C. albicans binding sites by RNases may be related to the close relationship that has been identified between mammalian RNase inhibitors and the extracellular domain of gonadotrophin receptors. The presence of RNase in commercial preparations of gonadotrophins should be borne in mind during any investigations that involve impure preparations of these hormones.

Amino Acid Sequence↗

PubMed2002

Genetic screen to dissect protein-protein interactions: ribonuclease inhibitor-ribonuclease A as a model system.

Protein-protein interactions are critical for the function of biological systems. Here, we describe a means to dissect a protein-protein interaction. Our method is based on the in vivo interaction between a target protein and the peptide epitopes derived from its partner. This interaction is detected by using hybrid proteins in which the target protein and peptide epitopes are fused to the DNA-binding domain of the lambda repressor protein. An interaction prevents the transcription of a reporter gene. The efficacy of this approach is demonstrated with the ribonuclease inhibitor protein and ribonuclease A, which form a complex with an equilibrium dissociation constant in the femtomolar range. Our method can enable the identification of residues important in a designated protein-protein interaction and the development of antagonists for that interaction.

Enzyme Inhibitors↗

PubMed1989

Tight-binding inhibition of angiogenin and ribonuclease A by placental ribonuclease inhibitor.

The dissociation rate constant of the angiogenin-placental ribonuclease inhibitor complex was determined by measuring the release of free angiogenin from the complex in the presence of scavenger for free placental ribonuclease inhibitor (PRI). In 0.1 M NaCl, pH 6, 25 degrees C, this value is 1.3 X 10(-7) s-1 (t1/2 congruent to 60 days). The Ki value for the binding of PRI to angiogenin, calculated from the association and dissociation rate constants, is 7.1 X 10(-16) M. The corresponding values for the interaction of RNase A with PRI, determined by similar means, are both considerably higher: the dissociation rate constant is 1.5 X 10(-5) s-1 (t1/2 = 13 h), and the Ki value is 4.4 X 10(-14) M. Thus, PRI binds about 60 times more tightly to angiogenin than to RNase A. The effect of increasing sodium chloride concentration on the binding of PRI to RNase A was explored by Henderson plots. The Ki value increases to 39 pM in 0.5 M NaCl and to 950 pM in 1 M NaCl, suggesting the importance of ionic interactions. The mode of inhibition of RNase A by PRI was determined by examining the effect of a competitive inhibitor of RNase A, cytidine 2'-phosphate, on the association rate of PRI with RNase A. Increasing concentrations of cytidine 2'-phosphate decrease the association rate in a manner consistent with a competitive mode of inhibition.

Angiogenesis Inducing Agents↗

PubMed1981

A procedure for the rapid preparation of 14C-aminoacyl-transfer RNA and its use in the assay of class I ribonucleases and ribonuclease inhibitor.

The presence of ribonuclease inhibitor and/or the activity of class I ribonucleases can conveniently be measured, at all stages of purification, by a highly sensitive assay based on the loss of radioactivity during the concomitant hydrolysis of tRNA and small amounts of 14C-labeled aminoacyl-tRNA. The rapid, economical assay, which is readily adaptable to homologous tRNA substrates, eliminates the necessity of filtration, centrifugation and ultraviolet spectroscopy measurements required by most other assays and is particularly suitable for multiple samples and kinetic measurements.

Animals↗