Enzymatic Activity Of RNase Research Articles

Streptomyces coelicolor RraAS1의 Eschechia coli RNase E의 RNA 분해작용에 대한 활성제로서 기능 암시

RNase E는 대장균(Escherichia coli)에서 수많은 RNA의 가공 및 분해에 관여하는 필수적인 효소이다. RNase E의 효소 활성은 RraA와 RraB에 의해 조절된다. 그람양성균인 Streptomyces coelicolor는 RNase ES, RraAS1, RraAS2라고 명명되는 RNase E와 RraA의 동족체를 가지고 있다. 이 연구에서는 S. coelicolor 유래의 RraAS1이 E. coli에서 RNase E의 효소활성을 저해하는지 연구하였다. 대장균에서 RraAS1의 발현은 RNase E의 과발현에 의해 감소된 세포생장을 더욱 저하시켰으며, RNase E의 기질인 rpsO, ftsZ, rnhB mRNA의 양을 감소시키는 것을 확인 하였다. 이러한 RraAS1의 효과는 공동면역침전실험을 수행한 결과에서 유추할 수 있듯이, Rne 단백질과 RraAS1의 결합으로 유도되는 것으로 보인다. 이러한 결과는 RraAS1이 대장균에서 RNase E의 리보핵산 가수분해 활성을 유도함을 시사한다. RNase E (Rne) is an essential enzyme involved in the processing and degradation of a large portion of RNAs in Escherichia coli. The enzymatic activity of RNase E is controlled by regulators of ribonuclease activity, namely, RraA and RraB. Gram-positive bacterium Streptomyces coelicolor also contains homologs of Rne and RraA, designated as RNase ES (Rns), RraAS1, and RraAS2. In the present study, we investigated the effect of S. coelicolor RraAS1 on the ribonucleolytic activity of RNase E in E. coli. Coexpression of RraAS1 with Rne resulted in the decreased levels of rpsO, ftsZ, and rnhB mRNAs, which are RNase E substrates, and augmented the toxic effect of Rne overexpression on cell growth. These in vivo effects appeared to be induced by the binding of RraAS1 to Rne, as indicated by the results of co-immunoprecipitation analysis. These results suggested that RraAS1 induces ribonucleolytic activity of RNase E in E. coli.

Adsorption-induced changes in ribonuclease A structure and enzymatic activity on solid surfaces.

Ribonuclease A (RNase A) is a small globular enzyme that lyses RNA. The remarkable solution stability of its structure and enzymatic activity has led to its investigation to develop a new class of drugs for cancer chemotherapeutics. However, the successful clinical application of RNase A has been reported to be limited by insufficient stability and loss of enzymatic activity when it was coupled with a biomaterial carrier for drug delivery. The objective of this study was to characterize the structural stability and enzymatic activity of RNase A when it was adsorbed on different surface chemistries (represented by fused silica glass, high-density polyethylene, and poly(methyl-methacrylate)). Changes in protein structure were measured by circular dichroism, amino acid labeling with mass spectrometry, and in vitro assays of its enzymatic activity. Our results indicated that the process of adsorption caused RNase A to undergo a substantial degree of unfolding with significant differences in its adsorbed structure on each material surface. Adsorption caused RNase A to lose about 60% of its native-state enzymatic activity independent of the material on which it was adsorbed. These results indicate that the native-state structure of RNase A is greatly altered when it is adsorbed on a wide range of surface chemistries, especially at the catalytic site. Therefore, drug delivery systems must focus on retaining the native structure of RNase A in order to maintain a high level of enzymatic activity for applications such as antitumor chemotherapy.

Recognition of 2′,5′-linked oligoadenylates by human ribonuclease L: molecular dynamics study

The capability of current MD simulations to be used as a tool in rational design of agonists of medically interesting enzyme RNase L was tested. Dimerization and enzymatic activity of RNase L is stimulated by 2',5'-linked oligoadenylates (pA₂₅A₂₅A; 2-5A). First, it was necessary to ensure that a complex of monomeric human RNase L and 25A was stable in MD simulations. It turned out that Glu131 had to be protonated. The non-protonated Glu131 caused dissociation of 2-5A from RNase L. Because of the atypical 2'-5' internucleotide linkages and a specific spatial arrangement of the 25A trimer, when a single molecule carries all possible conformers of the glycosidic torsion angle, several versions of the AMBER force field were tested. One that best maintained functionally important interactions of 25A and RNase L was selected for subsequent MD simulations. Furthermore, we wonder whether powerful GPUs are able to produce MD trajectories long enough to convincingly demonstrate effects of subtle perturbations of interactions between 25A and RNase L. Detrimental impacts of various point mutations of RNase L (R155A, F126A, W60A, K89A) on 2-5A binding were observed on a time scale of 200 ns. Finally, 2-5A analogues with a bridged 3'--O,4'--C-alkylene linkage (B) introduced into the adenosine units (A) were used to assess ability of MD simulations to distinguish on the time scale of hundreds of nanoseconds between agonists of RNase L (pA₂₅A₂₅B, pB₂₅A₂₅A, pB₂₅A₂₅B) and inactive analogs (pA₂₅B₂₅A, pA₂₅B₂₅B, pB₂₅B₂₅A, pB₂₅B₂₅B). Agonists were potently bound to RNase L during 200 ns MD runs. For inactive 2-5A analogs, by contrast, significant disruptions of their interactions with RNase L already within 100 ns MD runs were found.

Structure and enzymatic activity of laser immobilized ribonuclease A

Ribonuclease A (RNase A) enzyme was immobilized on solid holders by matrix-assisted pulsed laser evaporation (MAPLE) technique. The experiments were performed inside a stainless steel irradiation chamber. A UV KrF* (λ = 248 nm, τFWHM ≅ 25 ns, ν = 10 Hz) excimer laser source was used for irradiations. Surface morphology, molecular structure, and enzymatic activity of laser transferred RNase A samples were investigated as a function of RNase A concentration in the frozen composite MAPLE targets. Surface morphology and thickness of the immobilized enzyme were investigated by atomic force microscopy, scanning electron microscopy, and surface profilometry. The molecular structure of the laser transferred RNase A was determined by Fourier transform infrared spectroscopy. The enzymatic activity of RNase A after immobilization was tested through ribonucleic acid removal from deoxyribonucleic acid (DNA) extract solutions isolated from plant and animal tissues. A molecular method based on polymerase chain reaction was used to investigate the functional properties of DNA extracts treated with laser immobilized RNase A.

Protein disulphide isomerase-induced refolding of sonochemically prepared Ribonuclease A microspheres

The present communication describes for the first time the development of Ribonuclease A (RNase A) microspheres using the sonochemical method followed by an enzymatic treatment with protein disulphide isomerase (PDI). Ultrasound application induced changes on the protein physicochemical and biological properties: the enzymatic activity of RNase A was decreased in 35% and the free thiol groups content was significantly increased, probably due to the breakage of protein disulphide bonds and assembly of RNase A monomers. The deconvolution of amide I band, from Fourier Transform Infrared Spectroscopy, showed that the secondary structure of RNase A was slightly changed after microspherization. The PDI application on microspheres promoted the recovery of RNase A biological activity and induced the release of active protein into solution in its native state. These results were promoted by different states of PDI active site: oxidized and reduced, respectively. The PDI aptitude to catalyze the refolding of a protein substrate in the form of spheres is here reported.

Thermal stability and enzymatic activity of RNase A in the presence of cationic gemini surfactants

The thermal stability and enzymatic activity of bovine pancreatic ribonuclease A (RNase A) have been investigated in the presence of a homologous series of cationic gemini surfactants (alkanediyl-α,ω-bis(hydroxyethyl methyl hexadecyl ammonium bromide)). UV, circular dichorism and fluorescence spectroscopies have been used for this study. The denaturation curves at various surfactant concentrations were analyzed on basis of a two-transition model to obtain values of Tm (temperature at the midpoint of denaturation) and ΔHm (enthalpy change at Tm) of each transition. The main conclusion of this study is that these cationic gemini surfactants slightly activate and stabilize RNase A below their critical micelle concentrations at pH 5.0. The cationic gemini surfactant with the shorter spacer interacts more efficiently with RNase A than those with longer spacers.

Improving the Heat Resistance of Ribonuclease A by the Addition of Poly(N,N-diethylaminoethyl methacrylate)-graft-poly(ethylene glycol) (PEAMA-g-PEG)

Poly(N,N-diethylaminoethyl methacrylate)-graft-poly(ethylene glycol) (PEAMA-g-PEG) has previously been used as a novel additive to improve the heat resistance of lysozyme, which has a positive net charge and a negatively charged active site. In the present study, we show that PEAMA-g-PEG prevents heat inactivation of ribonuclease A (RNase A), which has a positive net charge and a positively charged active site. After treatment at 98 °C for 10 min, the enzymatic activity of RNase A complexed with PEAMA-g-PEG was maintained at up to 75% of the level of the native RNase A. The extents of inactivation of RNase A and the complex of RNase A with PEAMA-g-PEG were strongly dependent upon the heating temperature and incubation time. Circular dichroism (CD) spectral analysis revealed that heat-induced irreversible inactivation was largely suppressed when RNase A was complexed with PEAMA-g-PEG. These findings suggest that the heat resistance of RNase A is improved by the external addition of PEAMA-g-PEG.

A single-label phenylpyrrolocytidine provides a molecular beacon-like response reporting HIV-1 RT RNase H activity.

6-Phenylpyrrolocytidine (PhpC), a structurally conservative and highly fluorescent cytidine analog, was incorporated into oligoribonucleotides. The PhpC-containing RNA formed native-like duplex structures with complementary DNA or RNA. The PhpC-modification was found to act as a sensitive reporter group being non-disruptive to structure and the enzymatic activity of RNase H. A RNA/DNA hybrid possessing a single PhpC insert was an excellent substrate for HIV-1 RT Ribonuclease H and rapidly reported cleavage of the RNA strand with a 14-fold increase in fluorescence intensity. The PhpC-based assay for RNase H was superior to the traditional molecular beacon approach in terms of responsiveness, rapidity and ease (single label versus dual). Furthermore, the PhpC-based assay is amenable to high-throughput microplate assay format and may form the basis for a new screen for inhibitors of HIV-RT RNase H.

Stability of ribonuclease A under hydrothermal conditions in relation to the origin-of-life hypothesis: verification with the hydrothermal micro-flow reactor system

The stability of ribonuclease A (RNase A) was quantitatively investigated with the hydrothermal micro-flow reactor system (HFRS) at temperatures of up to 275 °C from the viewpoint of the hydrothermal origin-of-life hypothesis. The enzymatic activity of RNase A was studied with regard to the catalytic degradation of polynucleotides with anion-exchange high-performance liquid chromatography, while the degradation of RNase A to shorter molecules was analyzed by size exclusion chromatography (SEC) and mass spectrometry (MS). The degradation of RNase A started within 10 s at 200 °C, and the enzymatic activity disappeared almost completely after 25 s. SEC and MS analyses indicated that RNase A was thermally degraded to 2 large fragments, which, along with RNase A, were further decomposed to smaller fragments. This study showed that RNase A is fairly stable under normal conditions, but its enzymatic activity disappears rapidly at extremely high temperatures. The half life of RNase A and its fragments under hydrothermal conditions is comparable to or longer than the enzymatic reaction time scale of modern enzymes. Furthermore, this study demonstrates that HFRS is reliable and useful for verifying the stability of several proteins in fundamental and applied research as well as for studying the origin-of-life problem.

Irreversible thermoinactivation of ribonuclease‐A by soft‐hydrothermal processing

Ribonuclease (RNase), which often represents molecular biological contamination, is a thermostable enzyme. When RNase is heated at 121 degrees C by autoclave sterilization for 20 min, it does not lose its activity. However, the nature of the molecular events by which the irreversible denaturation occurs remains unknown. The purpose of this study was to elucidate the molecular mechanisms of irreversible thermal denaturation of RNase A and to develop an advanced sterilization method using soft-hydrothermal processing, which has the advantages of improved safety and cost-efficiency. The enzymatic activity of RNase was measured using polyacrylamide gel electrophoresis with torula yeast RNA. We evaluated the temperature and time course of irreversible thermoinactivation of RNase by normal autoclaving, hot-air sterilization, and soft-hydrothermal processing that had been controlled to the desired steam saturation ratio. The results indicated that RNase A was deactivated by autoclave sterilization (121 degrees C, 20 min) immediately after treatment, but was reactivated over time. Hot-air sterilization (180 degrees C, atmospheric pressure, 60 min) produced results similar to that of autoclave sterilization. In contrast, RNase A was irreversibly thermoinactivated by soft-hydrothermal processing (110 degrees C, 20 min) at 100% steam saturation ratio. We also determined that the mechanism of irreversible thermoinactivation of RNase A involved hydrolysis and deamidation under this condition at a steam saturation ratio of more than 100%.

Sequence-dependent Cleavage Site Selection by RNase Z from the Cyanobacterium Synechocystis sp. PCC 6803

Biosynthesis of transfer RNA requires processing from longer precursors at the 5'- and 3'-ends. In eukaryotes, in archaea, and in those bacteria where the 3'-terminal CCA sequence is not encoded, 3' processing is carried out by the endonuclease RNase Z, which cleaves after the discriminator nucleotide to generate a mature 3'-end ready for the addition of the CCA sequence. We have identified and cloned the gene coding for RNase Z in the cyanobacterium Synechocystis sp. PCC 6803. The gene has been expressed in Escherichia coli, and the recombinant protein was purified. The enzymatic activity of RNase Z from Synechocystis has been studied in vitro with a variety of substrates. The presence of C or CC after the discriminator nucleotide modifies the cleavage site of RNase Z so that it is displaced by one and two nucleotides to the 3'-side, respectively. The presence of the complete 3'-terminal CCA sequence in the precursor of the tRNA completely inhibits RNase Z activity. The inactive CCA-containing precursor binds to Synechocystis RNase Z with similar affinity than the mature tRNA. The properties of the enzyme described here could be related with the mechanism by which CCA is added in this organism, with the participation of two separate nucleotidyl transferases, one specific for the addition of C and another for the addition of A. This work is the first characterization of RNase Z from a cyanobacterium, and the first from an organism with two separate nucleotidyl transferases.

Direct Refolding of Inclusion Bodies Using Reversed Micelles

The protein refolding of inclusion bodies was investigated using reversed micelles formed by aerosol OT (AOT). Ribonuclease A (RNase A) was overexpressed in Escherichia coli and used as native inclusion bodies. The enzymatic activity of RNase A was completely regained from the inclusion bodies within 14 h by solubilization in reversed micelles. To further enhance the refolding rate, a molecular chaperone, GroEL, was incorporated into the refolding system. The resultant refolding system including GroEL showed better performance under optimized conditions for the refolding of RNase A inclusion bodies. The refolding rate was considerably improved by the addition of the molecular chaperone, and the refolding step was completed in 1 h. The protein refolding in the GroEL-containing refolding system was strongly dependent on the coexistence of ATP and Mg2+, suggesting that the GroEL hosted in the reversed micelles was biologically active and assisted in the renaturation of the inclusion bodies. The addition of cold acetone to the reversed micellar solution allowed over 90% recovery of the renatured RNase A.

Studies of the RNA Degradosome-organizing Domain of the Escherichia coli Ribonuclease RNase E

The hydrolytic endoribonuclease RNase E, which is widely distributed in bacteria and plants, plays key roles in mRNA degradation and RNA processing in Escherichia coli. The enzymatic activity of RNase E is contained within the conserved amino-terminal half of the 118 kDa protein, and the carboxy-terminal half organizes the RNA degradosome, a multi-enzyme complex that degrades mRNA co-operatively and processes ribosomal and other RNA. The study described herein demonstrates that the carboxy-terminal domain of RNase E has little structure under native conditions and is unlikely to be extensively folded within the degradosome. However, three isolated segments of 10–40 residues, and a larger fourth segment of 80 residues, are predicted to be regions of increased structural propensity. The larger of these segments appears to be a protein–RNA interaction site while the other segments possibly correspond to sites of self-recognition and interaction with the other degradosome proteins. The carboxy-terminal domain of RNase E may thus act as a flexible tether of the degradosome components. The implications of these and other observations for the organization of the RNA degradosome are discussed.

Protein refolding by reversed micelles utilizing solid-liquid extraction technique

This article reports that a reversed micellar solution is useful for refolding proteins directly from a solid source. The solubilization of denatured RNase A, which had been prepared by reprecipitation from the denaturant protein solution, into reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) has been investigated by a solid-liquid extraction system. This method is an alternative to the ordinary protein extraction in reversed micelles based on the liquid-liquid extraction. The solid-liquid extraction method was found to facilitate the solubilization of denatured proteins more efficiently in the reversed micellar media than the ordinary phase transfer method of liquid extraction. The refolding of denatured RNase A entrapped in reversed micelles was attained by adding a redox reagent (reduced and oxidized glutathion). Enzymatic activity of RNase A was gradually recovered with time in the reversed micelles. The denatured RNase A was completely refolded within 30 h. In addition, the efficiency of protein refolding was enhanced when reversed micelles were applied to denatured RNase A containing a higher protein concentration that, in the case of aqueous media, would lead to protein aggregation. The solid-liquid extraction technique using reversed micelles affords better scale-up advantages in the direct refolding process of insoluble protein aggregates.

Protein refolding by reversed micelles utilizing solid‐liquid extraction technique

This article reports that a reversed micellar solution is useful for refolding proteins directly from a solid source. The solubilization of denatured RNase A, which had been prepared by reprecipitation from the denaturant protein solution, into reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) has been investigated by a solid–liquid extraction system. This method is an alternative to the ordinary protein extraction in reversed micelles based on the liquid–liquid extraction. The solid–liquid extraction method was found to facilitate the solubilization of denatured proteins more efficiently in the reversed micellar media than the ordinary phase transfer method of liquid extraction. The refolding of denatured RNase A entrapped in reversed micelles was attained by adding a redox reagent (reduced and oxidized glutathion). Enzymatic activity of RNase A was gradually recovered with time in the reversed micelles. The denatured RNase A was completely refolded within 30 h. In addition, the efficiency of protein refolding was enhanced when reversed micelles were applied to denatured RNase A containing a higher protein concentration that, in the case of aqueous media, would lead to protein aggregation. The solid–liquid extraction technique using reversed micelles affords better scale-up advantages in the direct refolding process of insoluble protein aggregates. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 620-623, 1998.

Trichloroacetic acid-induced unfolding of bovine pancreatic ribonuclease. Existence of molten globule-like state.

The exposure of ribonuclease A to trichloroacetic acid was earlier shown to alter the conformation of the protein resulting in reduced enzymatic activity (Sagar, A. J., Subbiah, V., and Pandit, M. W. (1989) Biochim. Biophys. Acta 995, 144-150). We have studied the structure and enzymatic activity of ribonuclease A treated with trichloroacetic acid over a wide range of acid concentrations (0-40%). The far ultraviolet circular dichroism spectra of ribonuclease A, on exposure to acid concentrations less than 10%, indicated an exceptionally high degree of chiral structure. Exposure of ribonuclease A to acid concentrations between 10 and 30% resulted in the formation of a molecule with significant chiral structure (conventionally assigned to residual secondary structure) but reduced tertiary structure (characteristics very similar to those of molten globule). Increased binding of the hydrophobic probe 1-anilinonaphthalene-8-sulfonate to the enzyme treated with 15-30% acid, as compared with the untreated or completely unfolded protein, supported the existence of a state having characteristics of molten globule. Reversed phase high performance liquid chromatography corroborated the data obtained by circular dichroism as well as 1-anilino-naphthalene-8-sulfonate-binding studies. Beyond acid concentrations of 30%, the ribonuclease is completely denatured. The trichloroacetic acid-induced unfolding is shown to be completely reversible.

Carboxamidomethylation of a ribonuclease from Aspergillus saitoi.

1) The inactivation of a RNase from Aspergillus saitoi (RNase Ms) was studied to obtain information on its active site. 2) Inactivation of RNase Ms by iodoacetamide was greater at an alkaline pH, and was protected more by 2',(3')-AMP than by 2',(3')-GMP. 3) Analysis of the hydrolysis products with 6 N HCl and alkaline treatment of carboxamidomethylated RNase Ms showed that the sites of reaction were one carboxyl group and one histidine residue. 4) Since the incorporation of a carboxamidomethyl group into carboxylic acid was not protected by 2',(3')-AMP, it was concluded that the formation of N1-carboxamidomethylhistidine was responsible for the loss of enzymatic activity of RNase Ms.

Effects of Gamma Irradiation on the Function and Conformation of Ribonuclease A in Dilute Solution

Bovine pancreatic ribonuclease A (EC 2.7.7.16) in aqueous solutions has been exposed to gamma irradiation, and several properties of the irradiated enzyme molecule were examined. Kinetic and thermodynamic parameters of RNase A were not affected even when the enzymatic activity was considerably lost by gamma irradiation. While the structural changes of the irradiated RNase A were detected, STAm'!/sub 233/ values gradually decreased with increasing dose. The accessibility of tyrosyl and histidyl residues in the enzyme molecule for a chemical reagent was greatly increased in relatively lower dose ranges. All six tyrosyl residues were diazotized with DHT when 70% of enzymatic activity was still retained. 2'-Cytidilate-binding capacity and enzymatic activity of RNase A were reduced depending on the increase of dose at somewhat different rates. Decrease of the heat of denaturation also indicates that the local conformation change of thc enzymes through gamma irradiation precedes the disappearance of the enzymatic activity. (auth)

Zur Strahleninaktivierung von Ribonuclease

Dry ribonuclease was irradiated with 60Co gamma radiation in vacuo, under oxygen atmosphere, and at 77 °K. By chromatography on Sephadex G-50 active ribonuclease was separated from inactive radiation products. From the elution pattern and by ultracentrifugation it was shown that mainly unfolded dimers are formed by gamma irradiation of dry ribonuclease. Amino acid analysis of these various products shows that in all components cystine, methionine, tyrosine, phenylalanine, lysine, and histidine are destroyed with increasing dose whereas glycine shows a small increase. Thus, in ribonuclease irradiated in the dry state the same amino acids are changed as was found after irradiation in aqueous solutions. The radiosensitivity of dry ribonuclease shows an increase by the presence of oxygen of about 2 and a decrease at low temperature in vacuo of about 5. The same factors were also found for the alteration of amino acids, which means that under various experimental conditions amino acid destruction is proportional to loss of enzymatic activity of ribonuclease. The observed selectivity of amino acid destruction may be explained by energy migration or by the attack of atomic hydrogen liberated at random from the molecule. The total number of amino acids destroyed per ribonuclease molecule increases with dose. In enzymatically inactive products this value is always higher by one amino acid residue than in the active components. From this result and from the increase with dose it is concluded that after destruction of one amino acid residue the ribonuclease molecule has a probability (not depending on dose of irradiation) of 0.45 to become inactivated whereas in 55 per cent of all cases the molecule maintains its enzymatic activity.

Zur biologischen Wirksamkeit elastischer Kernstöße

Extremely thin layers of ribonuclease were irradiated with slow protons and the differential inactivation cross section determined for various proton energies in the range from 0.8 to 60 keV. At higher proton energies the inactivation cross section is not strongly dependent on energy but with decreasing proton energy it decreases rapidly, reaches a sharp minimum at 1.2 keV and increases again at still smaller energies. By comparing the experimentally determined inactivation cross sections with the cross sections for energy loss in elastic nuclear collisions and in ionizations, respectively, elastic collisions were demonstrated to destroy, in fact, the enzymatic activity of ribonuclease. The energy required for an inactivation by nuclear collision is only one fourth of the energy necessary for an inactivation by ionization.