Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The native state of a protein molecule in aqueous solutions represents one of the lowest states of Gibbs energy [Anfinsen, C.B. (1973) Science 181, 223-230]. Much progress has been made about the rules of protein folding [King, J. (1989) Chem. Eng. News 67, 32-54] and the dominant forces in protein folding [Dill, K.A. (1990) Biochemistry 29, 7133-7155]. However, the quantitative contributions of different Gibbs energy terms to protein stability remains a controversial issue [Moult, J., & Unger, R. (1991) Biochemistry 30, 3816-3824]. A molecular thermodynamic model has been proposed for the Gibbs energy of folding a residue in aqueous homopolypeptides from a random-coiled state to either the alpha-helix state or the beta-sheet state [Chen, C.-C., Zhu, Y., King, J.A., & Evans, L.B. (1992) Biopolymers 32, 1375-1392]. In this work, we present a generalization of the molecular thermodynamic model for the Gibbs energy of folding natural and synthetic heteropolypeptides from random-coiled conformations into alpha-helical conformations. The generalized model incorporates the intrinsic folding potential due to residue-solvent interactions, the cooperative folding effect due to residue-residue interactions, and the location and length of alpha-helices. The utility of the model was demonstrated by examining the stability of alpha-helical conformations of a number of natural polypeptides including C-peptide (residues 1-13) and S-peptide (residues 1-20) of RNase A (bovine pancreatic ribonuclease A), the P alpha fragment in BPTI (bovine pancreatic trypsin inhibitor), and synthetic polypeptides (the copolymers of different amino acid residues) including alanine-based peptides (16 or 17 residues long) in water. The computed Gibbs energies correspond well with the experimental data on helicity. The results also accounted for the effects of amino acid substitution and temperature on the stability of alpha-helical conformations of the test polypeptides.
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PMID:Molecular thermodynamic model to predict the alpha-helical secondary structure of polypeptide chains in solution. 142 Jan 74

Ribonuclease S (RNase-S) is a complex that consists of two proteolytic fragments of bovine pancreatic ribonuclease A (RNase-A): the S-peptide (residues 1-20) and S-protein (residues 21-124). We have refined the crystal structures of three RNase-S complexes. The first two contain the full-length 20-residue S-peptide and were studied at pHs of 4.75 and 5.5. The third one consists of a truncated form of S-peptide (residues 1-15) and was studied at pH 4.75 as the reference structure for a series of mutant peptide complexes to be reported separately. Excluding residues 16-23 which are either missing (in the S15 complex) or disordered (in both S20 complexes), all three structures refined at 1.6-A resolution are identical within the estimated errors in the coordinates (0.048 A for the backbone atoms). The R-values, residual error, range from 17.4% to 18.6%. The final model of S20, pH 4.75, includes 1 sulfate and 84 water molecules. The side chains of 11 residues were modeled in two discrete conformations. The final structures were independent of the particular RNase-A or RNase-S used as a starting model. An extensive comparison with refined crystal structures of RNase-A reveals that the core of the molecule which is held together with extensive hydrogen bonds is in identical pattern in all cases. However, the loop regions vary from one structure to another and are often characterized by high B-factors. The pattern of thermal parameters appears to be dependent on crystal packing and correlates well with the accessibility calculated in the crystal. Gln60 is a conserved residue in all sequences known to date for this class of ribonucleases. However, it is the only residue that is clearly defined in an unfavorable position (phi = -100 degrees, psi = -130 degrees) on the Ramachandran plot. The origin of the substantial differences between RNase-A and RNase-S in stability to both acid and temperature denaturation and in susceptibility to proteolysis at neutral pH is not obvious in our visual comparison of these two structures.
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PMID:Refinement of the crystal structure of ribonuclease S. Comparison with and between the various ribonuclease A structures. 146 19

Seven hydrophobic residues ranging in size from glycine to phenylalanine have been substituted for the wild-type methionine residue at position 13 in a 15-residue truncated version (S15) of S-peptide, the small component of ribonuclease S. Complexes of both S-15 and the seven variants with S-protein yielded isomorphous crystals. The structures of all eight complexes have been refined to final R-factors in the range of 17-19%. [See Kim, E. E. Varadarajan, R., Wyckoff, H. W., and Richards, F. M. (1992) Biochemistry (preceding paper in this issue) for the description of the reference S-15 complex.] Multiple side-chain conformations were seen for six residues in all of the complexes and for two to three additional residues in at least some of the complexes. Three of the complexes, Gly, Ala, and alpha-amino-n-butyric acid (ANB), contained a single water molecule in the cavity near residue 13 that makes three hydrogen bonds to protein atoms. Although space is available, no evidence for additional water in this region, ordered or disordered, was found. The atoms in the cavity wall tend to shrink the cavity by moving in on the small residues and to swell the cavity by moving out for the larger Phe substitution. A swelling seen with leucine was attributed to a shape effect since Leu, Ile, and Met all have the same volume. A slight volume contraction of the collection of interior residues outside of the region of position 13 was also noted. (All changes noted are in the direction to maintain a constant packing density averaged over the whole protein.) Leu51, a surface hydrophobic residue, moved considerably in the G, A, and ANB complexes in directionswhich would tend to decrease the cavity volume. The only other major change in position, 1.5 A, was the 66-69 loop, which is about 25 A from position 13. His12, Phe120, and Asp121 appear to be involved in this movement, but the connection with position 13 is not clear at all. The thermodynamic data on the association reaction for all of these complexes have been previously reported [Connelly, P. R., Varadarajan, R., Sturtevant, J. M., & Richards, F. M. (1990) Biochemistry 29, 6108-6114; Varadarajan, R., Connelly, P. R., Sturtevant, J. M., & Richards, F. M. (1992) Biochemistry 31, 1421-1426]. Some comments are offered on our initial attempts to correlate the structural changes with the changes in the thermodynamic parameters.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Crystallographic structures of ribonuclease S variants with nonpolar substitution at position 13: packing and cavities. 146 20

Methodology has been developed to couple high-performance anion-exchange chromatography (HPAE) with mass spectrometry utilizing the ion spray liquid chromatography/mass spectrometry (LC/MS) interface. Anion micro-membrane suppression (AMMS) has been used to remove the high concentrations of NaOH and NaOAc (10-400 mM total [Na+]) necessary for the separation of mixtures of monosaccharides and oligosaccharides. Post-suppressor addition of CH3CN/H2O solutions containing NH4OAc or LiOAc provided low-nanomole detection of the monosaccharides by selected ion monitoring of the cationized adducts. Maltooligosaccharide mixtures (three to seven residues) were separated and detected by the HPAE/AMMS LC/MS system in the full-scan mode. Low declustering potentials (35 V) in the LC/MS API source afforded intact singly and doubly charge ammoniated and diammoniated adducts of the sugars. Higher declustering potentials (65 V) produced abundant fragmentation of the ammoniated adducts. The corresponding lithiated and dilithiated species produced intact molecule ion species at higher declustering potentials. The endo H-released oligomannose species from RNase B were determined by the HPAE/AMMS LC/MS system as ammoniated adducts and resulting fragment ions with a high declustering potential (95 V) in the full-scan mode.
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PMID:High-performance anion-exchange chromatography coupled with mass spectrometry for the determination of carbohydrates. 151 60

The crystal structures of ribonuclease from Streptomyces aureofaciens (RNase Sa) and its complex with 3'-guanylic acid (guanosine 3'-monophosphate, 3'-GMP) have been determined by the method of isomorphous replacement. The atomic parameters have been refined by restrained least-squares minimization using data in the resolution range 10.0-1.8 A. All protein atoms and more than 230 water atoms in the two crystal structures have been refined to crystallographic R factors of 0.172 and 0.175 respectively. The estimated r.m.s. error in the atomic positions ranges from 0.2 A for well-defined atoms to about 0.5 A for more poorly defined atoms. There are two enzyme molecules in the asymmetric unit, built independently, and referred to as molecules A and B. The value of the average B factor for protein atoms in both structures is about 19 A2 and for water molecules about 35 A2. Electron density for the substrate analogue 3'-GMP was found only at the active site of molecule A. The density was very clear and the positions of all 3'-GMP atoms were refined with precision comparable to that of the protein.
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PMID:Determination and restrained least-squares refinement of the structures of ribonuclease Sa and its complex with 3'-guanylic acid at 1.8 A resolution. 165 32

We have developed a novel plasmid isolation procedure and have adapted it for use on an automated nucleic acid extraction instrument. The protocol is based on the finding that phenol extraction of a 1 M guanidinium thiocyanate solution at pH 4.5 efficiently removes genomic DNA from the aqueous phase, while supercoiled plasmid DNA is retained in the aqueous phase. S1 nuclease digestion of the removed genomic DNA shows that it has been denatured, which presumably confers solubility in the organic phase. The complete automated protocol for plasmid isolation involves pretreatment of bacterial cells successively with lysozyme, RNase A, and proteinase K. Following these digestions, the solution is extracted twice with a phenol/chloroform/water mixture and once with chloroform. Purified plasmid is then collected by isopropanol precipitation. The purified plasmid is essentially free of genomic DNA, RNA, and protein and is a suitable substrate for DNA sequencing and other applications requiring highly pure supercoiled plasmid.
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PMID:Plasmid purification by phenol extraction from guanidinium thiocyanate solution: development of an automated protocol. 171 49

The structures of two catalytically defective semi-synthetic RNases obtained by replacing aspartic acid 121 with asparagine or alanine have been determined and refined at a resolution of 2.0 A (R = 0.186 and 0.172, respectively). When these structures are compared with the refined 1.8-A structure (R = 0.204) of the fully active aspartic acid-containing enzyme (Martin, P.D., Doscher, M.S., and Edwards, B. F. P. (1987) J. Biol. Chem. 262, 15930-15938), numerous and widespread changes, much greater in number and magnitude than the small structural variations noted previously between the semisynthetic complex and RNase A, are found to have occurred. These changes include the movement of the loop containing residues 65-72 away from the active site, a more or less generalized relocation of crystallographically bound water molecules, and a number of rearrangements in the hydrogen bonding network at the active site. Most changes are far removed from the immediate site of the modifications and are distributed essentially throughout the molecule. The details of many of these changes are unique to each analog. In the asparagine analog, a destabilization in the positioning of active site residue His-119 also appears to have occurred.
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PMID:Structural changes that accompany the reduced catalytic efficiency of two semisynthetic ribonuclease analogs. 173 May 93

The steroid-binding capacity of the adrenocortical pregnenolone-binding protein (PBP) is effectively destroyed by extreme temperature (boiling water for 2-5 min); however, the boiled preparation contains a factor that potentiates ligand binding when readded to native PBP. Treatment of the boiled fraction with calf intestinal alkaline phosphatase at pH 9 reverses the stimulatory effect on PBP activity. Additionally, if native PBP is first incubated with alkaline phosphatase, which converts it to a nonbinding form, activity can be fully restored in a dose-dependent manner by the addition of the boiled preparation. The factor (itself devoid of binding capacity) can also be generated by exposing native PBP to acidic conditions (pH 4). The molecule is small (mol wt, less than 2000), as judged by Sephadex G-25 gel filtration and equilibrium dialysis. It is not retained on Concanavalin-A-Sepharose and is not extractable with a variety of organic solvents. The factor remains active after lyophilization and has a net negative charge at pH 7.4 (determined by DEAE-cellulose chromatography). While the binding capacity of native PBP is destroyed by a variety of proteases, the heat-stable factor is unaffected by similar treatment. Additionally, factor activity is not susceptible to RNase, DNase, or lipase digestion. Thus, the protein moiety of the PBP has an absolute requirement for a distinct phosphorylated heat-stable factor for expression of ligand-binding activity, and it may be through this factor that binding activity is regulated. It is not yet known whether the factor is acting allosterically or actually functions as part of the steroid-binding site.
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PMID:Adrenocortical pregnenolone-binding protein activity requires a small heat-stable factor: evidence that regulation by phosphorylation/dephosphorylation occurs at the level of the factor, not the protein. 177 Sep 49

A comparative study on the endotoxic effects of lipopolysaccharide (LPS) from Veillonella parvula ATCC 10790 and from Bacteroides intermedius BMH was performed using an in vivo approach in the C57BL/6 mouse. Phenol-water extracted LPS of such anaerobes was purified by ultracentrifugation and DNase/RNase digestion, and characterized by a metachromatic assay for endotoxins and by electrophoresis on SDS-polyacrylamide gel and silver staining. Mouse LD50 for V. parvula LPS was 1.479 mg and for B. intermedius greater than 3.160 mg. Sublethal amounts of the LPS from anaerobes as well as from facultative aerobes decreased daily water intake and body weight in the mouse. Endotoxin from Salmonella typhimurium SL1102, Escherichia coli 0128:B12 and V. parvula had a strong effect on water intake and body weight, whereas Bacteroides intermedius LPS activity was very weak. The results of the present report suggest that V. parvula LPS has a toxic in vivo activity on mouse, which is comparable to LPS from classic enteric organisms and stronger than B. intermedius LPS.
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PMID:Biological effects of Veillonella parvula and Bacteroides intermedius lipopolysaccharides. 177 87

A procedure for the purification of Neisseria meningitidis lipopolysaccharide (LPS) from outer membrane vesicles (OMV) in spent growth media was developed. Five different LPS strains of group A N. meningitidis were grown in tryptic soy broth with vigorous aeration for 36-48 h, and centrifuged to collect both cells and supernatants. The amount of LPS in the OMV in the supernatants was higher or at least equal to that in the cells. The OMV in each supernatant were concentrated, pelleted by ultracentrifugation, and treated with 2% sodium deoxycholate to dissociate LPS from OMV. The LPS was then separated from capsular polysaccharides, proteins and phospholipids by gel filtration on Sephacryl S-300 column in 1% sodium deoxycholate, and precipitated from the column fractions in 70% ethanol. In addition, LPS was also extracted from cells with hot phenol-water, ultracentrifuged once after treatment with ribonuclease, and purified on Sephacryl S-300. When compared with an improved phenol-water extraction method, the LPS obtained from either OMV or cells by the above methods gave a 40-180% increase in yield. The LPS also had much higher activities in limulus amebocyte lysate assay, rabbit pyrogenic test, and enzyme-linked immunosorbent assay. The LPS purified from cells and from OMV were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.
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PMID:Purification of rough-type lipopolysaccharides of Neisseria meningitidis from cells and outer membrane vesicles in spent media. 177 80


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