Changes In Ribonuclease Research Articles

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.

Structural Changes in Ribonuclease P RNA in the Hyperthermophilic ArchaeonPyrococcus horikoshiiOT3 Induced on Interaction with Proteins

The activated structure of RNase P RNA (PhopRNA) in Pyrococcus horikoshii OT3 was characterized by circular dichroism (CD) and ultraviolet (UV) absorbance spectra. The results suggested that interaction of four RNase P proteins (PhoPop5, PhoRpp21, PhoRpp29, and PhoRpp30) with PhopRNA results in destabilization of base stacking in PhopRNA, whereas the addition of a fifth protein, PhoRpp38, increases base stacking in PhopRNA.

Tracking Local Conformational Changes of Ribonuclease A Using Picosecond Time-Resolved Fluorescence of the Six Tyrosine Residues

The six tyrosine residues of ribonuclease A (RNase A) are used as individual intrinsic probes for tracking local conformational changes during unfolding. The fluorescence decays of RNase A are well described by sums of three exponentials with decay times ( τ 1 = 1.7 ns, τ 2 = 180 ps, and τ 3 = 30 ps) and preexponential coefficients ( A 1 = 1, A 2 = 1, and A 3 = 4) at pH 7, 25°C. The decay times are controlled by photo-induced electron transfer from individual tyrosine residues to the nearest disulphide (–SS–), bridge, which is distance ( R) dependent. We assign τ 1 to Tyr-76 ( R = 12.8 Å), τ 2 to Tyr-115 ( R = 6.9 Å), and τ 3 to Tyr-25, Tyr-73, Tyr-92, and Tyr-97 (all four at R = 5.5 ± 0.3 Å) at 23°C. On the basis of this assignment, the results show that, upon thermal or chemical unfolding only Tyr-25, Tyr-92, and Tyr-76 undergo significant displacement from their nearest –SS– bridge. Despite reporting on different regions of the protein, the concordance between the transition temperatures, T m, obtained from Tyr-76 ( T m = 59.2°C) and Tyr-25 and Tyr-92 ( T m = 58.2°C) suggests a single unfolding event in this temperature range that affects all these regions similarly.

Changes in key enzymes of viral-RNA biosynthesis in chloroplasts from PVY and TMV infected tobacco plants

Effects of the infection with tobacco mosaic virus (TMV) and potato virus Y (PVY) on chloroplasts from susceptible tobacco plants were determined. Changes in ribonucleases (RNases), phosphomonoesterase (PME), phosphodiesterase (PDE), glucose-6-phosphate dehydrogenase (G6P DH), 6-phosphogluconate dehydrogenase (6PG DH), glucokinase (GK), and fructokinase (FK) activities in thylakoid/envelope and stroma fractions were studied. Slight increase in the activities of PME, PDE, G6P DH and 6PG DH of thylakoid/envelope fraction as well as of RNases, PME, PDE, G6P DH, 6PG DH, GK and FK of stroma fraction was found in chloroplasts isolated from leaf tissues infected with PVY. Infection with TMV produced higher increase in enzymes activities in chloroplasts; especially, PME, G6P DH and 6PG DH in fraction of thylakoid/envelope, and RNases, PME, PDE, G6P DH, 6PG DH, and GK in stroma fraction.

Changes in Composition of Soluble Intercellular Proteins Isolated from Healthy and TMV-Infected Nicotiana tabacum L. cv. Xanthi-nc

Changes in ribonucleases (RNases), phosphomonoesterase (PME), phosphodiesterase (PDE), glucose-6-phosphate dehydrogenase (G6P DH), polyphenoloxidases, peroxidases and proteases activity and PR-proteins composition in leaf tissue and intercellular fluid (ICF) isolated from leaf tissue of healthy and TMV-infected hypersensitive tobacco (Nicotiana tabacum L. cv. Xanthi-nc) plants (non-inoculated leaves) were studied. The amount of the proteins and the enzymes of intercellular space was less than 3 % of the total amount of proteins and the enzymes found in homogenate of healthy leaves. The TMV infection did not significantly change this observation. The great increase in the activities of the enzymes was observed in homogenates of the infected leaves, especially of the enzymes involved in biosynthesis of precursors needed for virus multiplication (G6P DH, RNase, PME, PDE). This is in contrast with the activities of the enzymes of ICF, which were only partly increased. The ICF proteins of infected plants were separated by means of ion exchange chromatography on DEAE cellulose. The isozymes of peroxidase, polyphenoloxidase, PME and PDE were identified. Using discontinuous nondenaturating polyacrylamide gel electrophoresis of DEAE cellulose fractions, the detection of isozymes of peroxidases and PR-proteins was performed. By means of SDS-PAGE the molecular masses of PR-proteins were identified: 15 - 16 kDa (group 1), 27 - 28 kDa (group 3: chitinases) and 36 - 40 kDa (group 2a: β-1,3-glucanases).

Changes in Ribonuclease and Glucose-6-Phosphate Dehydrogenase Activities Induced by Beet Necrotic Yellow Vein Virus in Sugar Beet

Activities of host ribonucleases and glucose-6-phosphate dehydrogenase were studied in three cultivars (Monosvalof, Steffi and Rimini) of sugar beet differing in their resistance to beet necrotic yellow vein virus (BNYVV). No differences were found in the susceptibility of cultivars to BNYVV between mechanically inoculated and Polymyxa betae (a natural fungal vector of the virus) infected plants, but the culmination of reproduction curves of BNYVV in mechanically inoculated plants was observed one week earlier than in plants inoculated by means of P. betae. The activities of ribonucleases corresponded with virus multiplication. In roots, activities of ribonucleases reached a maximum at day 7; in leaves, maximum activity was found at day 21 in cv. Monosvalof, and at day 14 in cv. Steffi. The relatively resistant cultivar Rimini showed much lower activities. The activity of glucose-6-phosphate dehydrogenase was only slightly increased at the time of culmination of the BNYVV reproduction curve in cvs. Monosvalof and Steffi.

Potato virus-Y multiplication in susceptible tobacco cultivar and transgenic breeding line producing coat protein mRNA

Changes in ribonucleases (RNases) and glucose-6-phosphate dehydrogenase (G6P DH) activities, their content and subcellular localisation were studied in relation to virus multiplication in susceptible (cv. Samsun) or resistant (transgenic breeding line NCTG 83) tobacco plants infected with the potato virus YN (necrotic strain of PVY). Activities of RNases and G6P DH from diseased susceptible tobacco plants were markedly increased during the experimental period and significantly correlated with the multiplication curve of the PVYN. In contrast, the activities of RNases and G6P DH were not changed after PVY inoculation of resistant breeding line NCTG 83 producing the CP mRNA of PVY. Changes in the content and in the subcellular localisation of RNases and G6P DH isozymes were also determined in mesophyll protoplasts isolated from healthy as well as PVYN infected plants of both cultivars by differential centrifugation of broken protoplasts on day eight post inoculation (the culmination of multiplication curve of PVY and enhanced activity of both enzymes). The chloroplasts fraction from infected protoplasts showed an enhanced content of RNases (192.4% when compared with that from healthy control ones), and of G6P DH (174.4 %). The cytosol fraction from infected protoplasts contained slightly enhanced levels of G6P DH (117.4 %) and considerably enhanced levels of RNases (141.7 %). No significant differences in the activities, contents and subcellular localisation of RNases and/or G6P DH isozymes were observed in the resistant line NCTG 83. This is in accordance with no detectable contents of PVY.

Probing subtle acid-induced conformational changes of ribonuclease A by electrospray mass spectrometry

The newly developed technique, electrospray mass spectrometry, was used to probe subtle conformational changes of bovine pancreatic RNase A during acid denaturation. In a dilute acid solution of pH 2.6, RNase A lost nearly all of its activity, whereas its intrinsic fluorescence intensity at 304 nm and its ellipticity at 222 nm were fairly resistant to denaturation by acetic acid. The observed maximum charged state of the enzyme in electrospray mass spectra was increased from 11 + (at pH 3.3) to 14 + (at pH 2.6). This could result from exposure of the buried basic amino acid residues R-10, K-41, H-12 and perhaps H-48.

Pressure and Temperature Dependence Study of the Denaturation of Ribonuclease A Solutions

Abstract We have carried out molecular dynamics simulations on the Ribonuclease A solution to see the structural and energetic effects of the temperature and pressure on the protein denaturation and to compare the two effects. The simulations are done at 350 K for the thermal denaturation and at 6000 bar for the pressure denaturation. We used the consistent valence force field (CVFF) potential and the single point charge (SPC) water potential function to represent the interactions in the simulational systems. During 100 ps, both structures denatured by the temperature and pressure changes have retained the native-like framework. We have reported the structural changes of Ribonuclease A and water molecules through the root mean square displacement, the solvent accessible surface area including the contribution of each amino acid to the total solvent accessible surface area, and the hydrogen-bond pattern. We have examined the structure of water molecules in the systems by the distribution of small water clu...

Compactness of thermally and chemically denatured ribonuclease A as revealed by volume and compressibility.

The conformational changes of ribonuclease A due to thermal and guanidine hydrochloride denaturation were monitored by means of precise density and sound velocity measurements. It was found that the apparent molar volume decreased but the adiabatic compressibility increased on thermal denaturation under acidic conditions (pHs 1.60, 1.90, and 2.08). On the other hand, guanidine hydrochloride denaturation (pH 2.00) brought about large decreases in the compressibility and apparent molar volume. These results indicate that the conformation of the denatured protein is greatly different between the two types of denaturation: the thermally denatured state corresponds to the structure with enhanced thermal fluctuation having a residual secondary structure and a high local concentration of nonpolar groups exposed, but the guanidine hydrochloride denaturation leads to exposure of a large amount of amino acid residues, resulting in an increase in hydration and a decrease in the internal cavity. The compressibility changes due to both types of denaturation were not correlated to a loss of the secondary structure, as judged by means of circular dichroism. These findings suggest that the compactness and thermal fluctuation of the protein cannot be described by a two-state denaturation model and that there are some molten-globule-like intermediates in the denaturation processes.

Involvement of Ribonuclease and RNA Polymerase Activity in Chill-induced Increases of RNA Concentration in Figleaf Gourd Roots.

We previously found a large increase in RNAs in roots of chill-tolerant figleaf gourds (Cucurbita ficifolia Bouche) but not in those of less tolerant cucumbers (Cucumis sativus L.), when the roots were exposed to 14°C (Kanda et al., 1994). In this study, changes in ribonuclease and RNA polymerase activities in the roots were followed for 6 days after their exposure to 14° and 23°C. The root enzyme activities were assayed at a) the optimal temperatures for the enzymes and b) root-growing temperatures. Neither ribonuclease nor RNA polymerase activity accounted for the characteristic increase of RNA concentrations in chilled roots of figleaf gourds, irrespective of assay temperatures. However, the ratio of RNA polymerase activity to ribonuclease activity closely correlated with RNA concentrations in both species. This correlation was observed only when the enzyme activity was assayed at root-growing temperatures. In this case, ribonuclease activity 2 days after exposure to 14°C was much lower in figleaf gourd roots than that in cucumber roots. However, the activity changed little thereafter in either species, but the RNA polymerase activity in figleaf gourd roots did increase gradually during exposure to 14°C; it was still lower than the activity at 23°C after 6 days. This increase was not observed in the cucumber roots. These results strongly suggest that, 1) root RNA concentrations in figleaf gourds and cucumbers are largely regulated by RNA polymerase activity relative to ribonuclease activity, and 2) the marked increase of RNA concentrations in figleaf gourd roots grown at 14°C was caused mainly by low temperature-induced increases of RNA polymerase activity together with much reduced activity of ribonuclease.

Pressure‐induced secondary structure changes of ribonuclease A and ribonuclease S studied by FTIR spectroscopy

AbstractFourier transform infrared (FTIR) spectroscopy combined with a resolution‐enhancement technique has been used to characterize pressure‐induced changes in the secondary structure of ribonuclease A (RNase A) and ribonuclease S (RNase S) in D2O solution at 30°C. According to the observed changes in the amide I' band, both RNase A and RNase S are reversibly unfolded by application of high pressure. The pressure‐unfolded state of the two proteins does not have any secondary structure elements characteristic of their native state, such as α‐helix, β‐sheet, and reverse turn. However, there is a slight difference in the structural features between pressure‐unfolded RNase A and RNase S, which is distinguished by the formation of nonnative turns only for RNase S. The dramatic structural transition of RNase A occurs over the pressure range between 570 and 1030 MPa. The pressure stability of RNase S is decreased by about 300 MPa compared with RNase A, despite little difference in the two native secondary structural features identified by FTIR spectroscopy. The structural features upon the pressure‐induced unfolding are additionally characterized by the interesting behavior of hydrogen‐deuterium exchange at high pressure. Most of the backbone amide protons protected at atmospheric pressure, which are involved in the α‐helices and β‐sheet of both RNase A and RNase S, are exchanged with solvent deuterons in the pressure range where the two secondary structure elements are virtually identified as intact. It is suggested that the solvent accessibility to the internal regions is strongly correlated to the pressure stability. © 1995 John Wiley & Sons, Inc.

Cold-induced conformational changes of ribonuclease A as investigated by subzero transverse temperature gradient gel electrophoresis

Subzero temperature gradient gel electrophoresis is a new approach which allows to measure the transition temperature of low temperature-induced subtle conformational changes of proteins and to detect the different conformational states, including unfolded states. Using this technique under destabilizing conditions, i.e., in the presence of 4 M urea, bovine pancreas ribonuclease A exhibited two transitions: (i) a continuous transition with a midpoint temperature of −14°C corresponding to a rapid equilibrium between the initial enzyme state and a conformational state more compact than the initial one; (ii) a discontinuous transition at −22.5°C from intermediate to a non migrating species. Under reducing conditions this second transition was shifted toward high temperatures (−18.5°C). We attempted to detect these two transitions by differential scanning calorimetry, UV spectrophotometry and circular dichroism measurements. These transitions have been ascribed to subtle cold-induced conformational changes.

Changes in ribonuclease and glucose-6-phosphate dehydrogenase activities during PVY-RNA biosynthesis in potato leaf discs

Changes in fresh matter content, protein content, chlorophyl content, ribonuclease activity, and glucose-6-phosphate dehydrogenase activity, associated with potato Y-virus multiplication (common strain, PVY ordinary) were studied in discs cut from potato leaves. The results obtained showed that marked decreases in disc fresh matter, in protein content, and in chlorophyll content occurred during a 5-day-long cultivation period. The activity of glucose-6-phosphate dehydrogenase, that is of the rate limiting enzyme of the pentose phosphate pathway, and the activity of ribonucleases which characterize the rate and intensity of host RNA degradation were markedly enhanced in this period. The fact that activity curves of both these enzymes were in linear relationship with the PVY reproduction curve indicates that not only nucleotides produced in the reactions of the oxidative pentose phosphate pathway but also nucleotides released in the process of host RNA degradation were the main source of nucleotides necessary for PVY-RNA biosynthesis, in spite of a high photosynthetic rate.

Studies on the mechanism of glucocorticoid hormone induced alterations in rat thymic transcription—I. Evidence from reconstituted cross-over transcription assays that sequential increases and decreases in transcription are due to changes in the activity of rna polymerase ii rather than in the activity of chromatin template

In experiments to determine the mechanism of glucocorticoid induced decreases in thymic transcription, adrenalectomized rats were injected with hydrocortisone (50 mg/kg) or vehicle. Thymic nuclei were used to prepare chromatins and soluble nuclear extracts containing RNA polymerase II for cross-over experiments. With calf thymus DNA or rat thymic chromatins as templates limiting RNA polymerase II from rats treated with hydrocortisone 3 h previously had 130% of the [ 3H]UMP incorporating activity of RNA polymerase II from control vehicle treated rats. In contrast, limiting RNA polymerase II from rats treated with hydrocortisone 12 h previously had 40–50% of the [ 3H]UMP incorporating activity of RNA polymerase II from controls. When limiting calf thymus DNA or rat thymic chromatins were used in 12 h cross-over experiments. Individual RNA polymerases II produced equal [ 3H]UMP incorporations, but RNA polymerase II activity from hydrocortisone treated rats was again only 50% of control values. Thus with template saturation, RNA polymerase II from hydrocortisone treated rats could not transcribe rat thymic chromatin templates to the level achieved by RNA polymerase II from control rats. This suggests that the activity, rather than the amount, of RNA polymerase II from hydrocortisone treated rats is reduced. Double reciprocal plots of [ 3H]UMP incorporation on rat chromatins with increasing concentrations of RNA polymerases II were made at 12 h. The apparent K m for RNA polymerase II from animals treated with hydrocortisone was identical to that of RNA polymerase II from controls, but the V max of RNA polymerase II from hydrocortisone treated animals was reduced. These data suggest the presence of an inhibitor of transcription or an RNA polymerase II defective in its capacity to initiate and/or elongate RNA transcripts. Further experiments demonstrated that these effects were not due to steroid induced changes in ribonuclease or protease activities.

Free energy changes in ribonuclease A denaturation. Effect of urea, guanidine hydrochloride, and lithium salts.

The unfolding of ribonuclease A by urea, guanidine hydrochloride, lithium perchlorate, lithium chloride, and lithium bromide has been followed by circular dichroic and difference spectral measurements. All three abnormal tyrosyl residues are normalized in urea and guanidine hydrochloride (delta epsilon 287 = -2700), only two are normalized in lithium bromide and lithium perchlorate (delta epsilon 287 = -1700), and only one is exposed in lithium chloride solutions (delta epsilon 287 = -700). The Gibbs energies are 4.7 +/- 0.1 kcal mol-1 for urea- and guanidine hydrochloride-denaturation, 3.8 +/- 0.2 kcal mol-1 for lithium perchlorate-denaturation, and 12.7 +/- 0.2 kcal mol-1 for lithium chloride- and lithium bromide-denaturation of ribonuclease A. The latter results suggest that the mechanism of the unfolding process in urea and guanidine hydrochloride is quite different from that in lithium salts.

Changes in acid ribonuclease and other acid hydrolases during lymphocyte stimulation

Human peripheral blood lymphocytes were stimulated with phytohemagglutinin (PHA). The activities of several acid hydrolases were measured. The results indicate that the response of the individual enzymes varies widely. Most enzymes show an increase in total activity compared to controls and to time zero. However, acid ribonuclease activity exhibits a marked decrease below control values. This is consistent with the accumulation of RNA which occurs at this time. The possible significance of the changes in ribonuclease activity is discussed.

Simultaneous measurements of viscosity and density in solutions undergoing change

The magnetic suspension densimeter-viscometer has been modified so that the variations of viscosity and density in solutions undergoing change are determined continuously as a function of time. The changes in values for both of these properties are also obtained when solutions of varying composition are passed through the sample cell (<0.5 ml). The new method measures the torque on the magnetically suspended buoy which is held stationary in a stable velocity gradient; this procedure also facilitates the measurement of the density with greater accuracy. Preliminary results relating to the conformation changes of ribonuclease A in the presence of guanidinium chloride and a disulfide cleaving agent are presented.

The effect of age on protein synthesis and ribosome aggregation to messenger RNA in rat liver

The level of cell-free protein synthesis and ribosome aggregation to mRNA was determined in the livers of rats from 1 to 18 months of age. Protein synthesis by liver postmitochondrial supernatant fraction decreased rapidly in the first 9 months, with little change thereafter. The decrease in protein synthesis was associated with the ribosomal fraction of the postmitochondrial supernatant fraction. Ribosome aggregation to mRNA, as determined by sucrose density gradient centrifugation, decreased rapidly during the first 9 months and changed little thereafter. The decrease in protein synthesis with increasing age appears to be the result of reduced ribosome aggregation to mRNA. This decrease in aggregation was not due to changes in ribonuclease or the availability of amino acids for protein synthesis but appeared to be an age-related phenomenon.

Changes in ribonuclease with age in the fungus Rhizoctonia solani

The enzyme ribonuclease (RNase) which was isolated from Rhizoctonia solani Kuehn degraded ribonucleic acid from both Torula yeast and from Rhizoctonia solani mycelia. As the Rhizoctonia mycelia aged, there was an increase in both the total and the specific activities of the RNase present in the mycelia. This change is discussed in relation to the age dependent decrease in protein synthesis in this fungus.